We are searching data for your request:
Upon completion, a link will appear to access the found materials.
I have been baffled with this question approx. for a dozen of years for now. There is a default "lock & key" mechanism of action assumed for the interaction between a signalling molecule and its receptor, but while applying a bit of combinatorics and biophysics to it I find the following inconsistencies.
Suppose a long-lasting neuropeptide is circulating in a CSF or blood macrocirculation. Or suppose a norepinephrine or other neurohormones are released by hypothalamic and pituitary cells into the macrocirculation for sympathetic or parasympathetic signalling. Accroding to the logic of "lock & key" mechanism, in order for a "key" signalling molecule to activate its "lock" receptor an active site of this molecule - the molecule partition which directly reacts with the receptor as it is generally not the whole molecule which reacts with the receptor - should come in direct contact with the relevant active site of its in-membrane receptor. So by such combinatorial logic, if there are thousands of receptors on outer membranes of cells in CSF or vessel endotelium along the way of which this "key"-molecule is travelling, this molecule should try to "lock into" every such potential "lock" receptor in order to "check" if this is the right "lock". In other words, while travelling across long distances such "key" signalling molecules should not just float in CSF or blood vessels as is generally depicted, but slide across all outer cell membranes in CSF or across vascular endotelium in blood or lymph circulation in order to "check" every potential "lock" receptor for finding their relevant receptors for eventual activation. It is definitely not the case, but it the result of applying combinatorial "lock&key" logic.
Somehow it seems (for myself, at least) that it is just assumed that a "key" molecule released at distances which are millions> of times larger than the molecule size should just "lock" for granted into its appropiate receptors throughout the organism. But suppose a "key" molecule travels in CSF or interstitial fluid in the vicinity of its appropriate membrane "lock" receptor, but with the molecule's reaction (active) site pointed away from its receptor or at an angle. Should the "key" molecule not then react with its "lock" receptor?? Is there a minimum effective reaction distance established between a "key" molecule and its "lock" receptor in order to effectively activate the receptor?
The latter argument holds true also for small distance 20-40 nm synapses with molecules released in it whose size would be minimum 1000 times smaller than the synapse size. Should a neurotransmitter molecule not react with its receptor in a synapse if the molecule's active site would be pointed 180 degrees away from the receptor's active site?
Yes, any individual ligand may have the wrong orientation to interact effectively with an individual receptor, but so what? There are typically thousands of each type of receptor on a single cell1 and there will be many molecules of a given ligand present as well.
For your synapse example, I suggest looking up:
- How many neurotransmitter molecules will be present in a synapse
- How many receptors will present in a synapse
- How many of those receptors need to be bound to get a signal
I think that will help you make sense of this phenomenon and I encourage you to post a detailed answer to your own question!
You might also want to look into molecular dynamics simulations…
1: Milo, R., & Phillips, R. (2015). Cell biology by the numbers. Garland Science.
Types of Receptors
A cell within a multicellular organism may need to signal to other cells that are at various distances from the original cell (Figure 1). Not all cells are affected by the same signals. Different types of signaling are used for different purposes.
Figure 1 In chemical signaling, a cell may target itself (autocrine signaling), a cell connected by gap junctions, a nearby cell (paracrine signaling), or a distant cell (endocrine signaling). Paracrine signaling acts on nearby cells, endocrine signaling uses the circulatory system to transport ligands, and autocrine signaling acts on the signaling cell. Signaling via gap junctions involves signaling molecules moving directly between adjacent cells. Receptors are protein molecules inside the target cell or on its surface that receive a chemical signal. Chemical signals are released by signaling cells in the form of small, usually volatile or soluble molecules called ligands. A ligand is a molecule that binds another specific molecule, in some cases, delivering a signal in the process. Ligands can thus be thought of as signaling molecules. Ligands and receptors exist in several varieties however, a specific ligand will have a specific receptor that typically binds only that ligand.
45 Signaling Molecules and Cellular Receptors
By the end of this section, you will be able to do the following:
- Describe four types of signaling mechanisms found in multicellular organisms
- Compare internal receptors with cell-surface receptors
- Recognize the relationship between a ligand’s structure and its mechanism of action
There are two kinds of communication in the world of living cells. Communication between cells is called intercellular signaling , and communication within a cell is called intracellular signaling . An easy way to remember the distinction is by understanding the Latin origin of the prefixes: inter- means “between” (for example, intersecting lines are those that cross each other) and intra- means “inside” (as in intravenous).
Chemical signals are released by signaling cells in the form of small, usually volatile or soluble molecules called ligands. A ligand is a molecule that binds another specific molecule, in some cases, delivering a signal in the process. Ligands can thus be thought of as signaling molecules. Ligands interact with proteins in target cells , which are cells that are affected by chemical signals these proteins are also called receptors . Ligands and receptors exist in several varieties however, a specific ligand will have a specific receptor that typically binds only that ligand.
Forms of Signaling
There are four categories of chemical signaling found in multicellular organisms: paracrine signaling, endocrine signaling, autocrine signaling, and direct signaling across gap junctions ((Figure)). The main difference between the different categories of signaling is the distance that the signal travels through the organism to reach the target cell. We should note here that not all cells are affected by the same signals.
Signals that act locally between cells that are close together are called paracrine signals . Paracrine signals move by diffusion through the extracellular matrix. These types of signals usually elicit quick responses that last only a short period of time. In order to keep the response localized, paracrine ligand molecules are normally quickly degraded by enzymes or removed by neighboring cells. Removing the signals will reestablish the concentration gradient for the signal, allowing them to quickly diffuse through the intracellular space if released again.
One example of paracrine signaling is the transfer of signals across synapses between nerve cells. A nerve cell consists of a cell body, several short, branched extensions called dendrites that receive stimuli, and a long extension called an axon, which transmits signals to other nerve cells or muscle cells. The junction between nerve cells where signal transmission occurs is called a synapse. A synaptic signal is a chemical signal that travels between nerve cells. Signals within the nerve cells are propagated by fast-moving electrical impulses. When these impulses reach the end of the axon, the signal continues on to a dendrite of the next cell by the release of chemical ligands called neurotransmitters from the presynaptic cell (the cell emitting the signal). The neurotransmitters are transported across the very small distances (20–40 nanometers) between nerve cells, which are called chemical synapses ((Figure)). The small distance between nerve cells allows the signal to travel quickly this enables an immediate response, such as, “Take your hand off the stove!”
When the neurotransmitter binds the receptor on the surface of the postsynaptic cell, the electrochemical potential of the target cell changes, and the next electrical impulse is launched. The neurotransmitters that are released into the chemical synapse are degraded quickly or get reabsorbed by the presynaptic cell so that the recipient nerve cell can recover quickly and be prepared to respond rapidly to the next synaptic signal.
Signals from distant cells are called endocrine signals , and they originate from endocrine cells . (In the body, many endocrine cells are located in endocrine glands, such as the thyroid gland, the hypothalamus, and the pituitary gland.) These types of signals usually produce a slower response but have a longer-lasting effect. The ligands released in endocrine signaling are called hormones, signaling molecules that are produced in one part of the body but affect other body regions some distance away.
Hormones travel the large distances between endocrine cells and their target cells via the bloodstream, which is a relatively slow way to move throughout the body. Because of their form of transport, hormones become diluted and are present in low concentrations when they act on their target cells. This is different from paracrine signaling, in which local concentrations of ligands can be very high.
Autocrine signals are produced by signaling cells that can also bind to the ligand that is released. This means the signaling cell and the target cell can be the same or a similar cell (the prefix auto- means self, a reminder that the signaling cell sends a signal to itself). This type of signaling often occurs during the early development of an organism to ensure that cells develop into the correct tissues and take on the proper function. Autocrine signaling also regulates pain sensation and inflammatory responses. Further, if a cell is infected with a virus, the cell can signal itself to undergo programmed cell death, killing the virus in the process. In some cases, neighboring cells of the same type are also influenced by the released ligand. In embryological development, this process of stimulating a group of neighboring cells may help to direct the differentiation of identical cells into the same cell type, thus ensuring the proper developmental outcome.
Direct Signaling Across Gap Junctions
Gap junctions in animals and plasmodesmata in plants are connections between the plasma membranes of neighboring cells. These fluid-filled channels allow small signaling molecules, called intracellular mediators , to diffuse between the two cells. Small molecules, such as calcium ions (Ca 2+ ), are able to move between cells, but large molecules like proteins and DNA cannot fit through the channels. The specificity of the channels ensures that the cells remain independent but can quickly and easily transmit signals. The transfer of signaling molecules communicates the current state of the cell that is directly next to the target cell this allows a group of cells to coordinate their response to a signal that only one of them may have received. In plants, plasmodesmata are ubiquitous, making the entire plant into a giant communication network.
Types of Receptors
Receptors are protein molecules in the target cell or on its surface that bind ligand. There are two types of receptors, internal receptors and cell-surface receptors.
Internal receptors , also known as intracellular or cytoplasmic receptors, are found in the cytoplasm of the cell and respond to hydrophobic ligand molecules that are able to travel across the plasma membrane. Once inside the cell, many of these molecules bind to proteins that act as regulators of mRNA synthesis (transcription) to mediate gene expression. Gene expression is the cellular process of transforming the information in a cell’s DNA into a sequence of amino acids, which ultimately forms a protein. When the ligand binds to the internal receptor, a conformational change is triggered that exposes a DNA-binding site on the protein. The ligand-receptor complex moves into the nucleus, then binds to specific regulatory regions of the chromosomal DNA and promotes the initiation of transcription ((Figure)). Transcription is the process of copying the information in a cell’s DNA into a special form of RNA called messenger RNA (mRNA) the cell uses information in the mRNA (which moves out into the cytoplasm and associates with ribosomes) to link specific amino acids in the correct order, producing a protein. Internal receptors can directly influence gene expression without having to pass the signal on to other receptors or messengers.
Cell-surface receptors , also known as transmembrane receptors, are cell surface, membrane-anchored (integral) proteins that bind to external ligand molecules. This type of receptor spans the plasma membrane and performs signal transduction, through which an extracellular signal is converted into an intracellular signal. Ligands that interact with cell-surface receptors do not have to enter the cell that they affect. Cell-surface receptors are also called cell-specific proteins or markers because they are specific to individual cell types.
Because cell-surface receptor proteins are fundamental to normal cell functioning, it should come as no surprise that a malfunction in any one of these proteins could have severe consequences. Errors in the protein structures of certain receptor molecules have been shown to play a role in hypertension (high blood pressure), asthma, heart disease, and cancer.
Each cell-surface receptor has three main components: an external ligand-binding domain, a hydrophobic membrane-spanning region called a transmembrane domain, and an intracellular domain inside the cell. The ligand-binding domain is also called the extracellular domain . The size and extent of each of these domains vary widely, depending on the type of receptor.
How Viruses Recognize a Host Unlike living cells, many viruses do not have a plasma membrane or any of the structures necessary to sustain metabolic life. Some viruses are simply composed of an inert protein shell enclosing DNA or RNA. To reproduce, viruses must invade a living cell, which serves as a host, and then take over the hosts cellular apparatus. But how does a virus recognize its host?
Viruses often bind to cell-surface receptors on the host cell. For example, the virus that causes human influenza (flu) binds specifically to receptors on membranes of cells of the respiratory system. Chemical differences in the cell-surface receptors among hosts mean that a virus that infects a specific species (for example, humans) often cannot infect another species (for example, chickens).
However, viruses have very small amounts of DNA or RNA compared to humans, and, as a result, viral reproduction can occur rapidly. Viral reproduction invariably produces errors that can lead to changes in newly produced viruses these changes mean that the viral proteins that interact with cell-surface receptors may evolve in such a way that they can bind to receptors in a new host. Such changes happen randomly and quite often in the reproductive cycle of a virus, but the changes only matter if a virus with new binding properties comes into contact with a suitable host. In the case of influenza, this situation can occur in settings where animals and people are in close contact, such as poultry and swine farms. 1 Once a virus jumps the former “species barrier” to a new host, it can spread quickly. Scientists watch newly appearing viruses (called emerging viruses) closely in the hope that such monitoring can reduce the likelihood of global viral epidemics.
Cell-surface receptors are involved in most of the signaling in multicellular organisms. There are three general categories of cell-surface receptors: ion channel-linked receptors, G-protein-linked receptors, and enzyme-linked receptors.
Ion channel-linked receptors bind a ligand and open a channel through the membrane that allows specific ions to pass through. To form a channel, this type of cell-surface receptor has an extensive membrane-spanning region. In order to interact with the double layer of phospholipid fatty acid tails that form the center of the plasma membrane, many of the amino acids in the membrane-spanning region are hydrophobic in nature. Conversely, the amino acids that line the inside of the channel are hydrophilic to allow for the passage of water or ions. When a ligand binds to the extracellular region of the channel, there is a conformational change in the protein’s structure that allows ions such as sodium, calcium, magnesium, and hydrogen to pass through ((Figure)).
G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein. The activated G-protein then interacts with either an ion channel or an enzyme in the membrane ((Figure)). All G-protein-linked receptors have seven transmembrane domains, but each receptor has its own specific extracellular domain and G-protein-binding site.
Cell signaling using G-protein-linked receptors occurs as a cyclic series of events. Before the ligand binds, the inactive G-protein can bind to a newly revealed site on the receptor specific for its binding. Once the G-protein binds to the receptor, the resulting change in shape activates the G-protein, which releases guanosine diposphate (GDP) and picks up guanosine 3-phosphate (GTP). The subunits of the G-protein then split into the α subunit and the βγ subunit. One or both of these G-protein fragments may be able to activate other proteins as a result. After awhile, the GTP on the active α subunit of the G-protein is hydrolyzed to GDP and the βγ subunit is deactivated. The subunits reassociate to form the inactive G-protein and the cycle begins anew.
G-protein-linked receptors have been extensively studied and much has been learned about their roles in maintaining health. Bacteria that are pathogenic to humans can release poisons that interrupt specific G-protein-linked receptor function, leading to illnesses such as pertussis, botulism, and cholera. In cholera ((Figure)), for example, the water-borne bacterium Vibrio cholerae produces a toxin, choleragen, that binds to cells lining the small intestine. The toxin then enters these intestinal cells, where it modifies a G-protein that controls the opening of a chloride channel and causes it to remain continuously active, resulting in large losses of fluids from the body and potentially fatal dehydration as a result.
Enzyme-linked receptors are cell-surface receptors with intracellular domains that are associated with an enzyme. In some cases, the intracellular domain of the receptor itself is an enzyme. Other enzyme-linked receptors have a small intracellular domain that interacts directly with an enzyme. The enzyme-linked receptors normally have large extracellular and intracellular domains, but the membrane-spanning region consists of a single alpha-helical region of the peptide strand. When a ligand binds to the extracellular domain, a signal is transferred through the membrane, activating the enzyme. Activation of the enzyme sets off a chain of events within the cell that eventually leads to a response. One example of this type of enzyme-linked receptor is the tyrosine kinase receptor ((Figure)). A kinase is an enzyme that transfers phosphate groups from ATP to another protein. The tyrosine kinase receptor transfers phosphate groups to tyrosine molecules (tyrosine residues). First, signaling molecules bind to the extracellular domain of two nearby tyrosine kinase receptors. The two neighboring receptors then bond together, or dimerize. Phosphates are then added to tyrosine residues on the intracellular domain of the receptors (phosphorylation). The phosphorylated residues can then transmit the signal to the next messenger within the cytoplasm.
HER2 is a receptor tyrosine kinase. In 30 percent of human breast cancers, HER2 is permanently activated, resulting in unregulated cell division. Lapatinib, a drug used to treat breast cancer, inhibits HER2 receptor tyrosine kinase autophosphorylation (the process by which the receptor adds phosphates onto itself), thus reducing tumor growth by 50 percent. Besides autophosphorylation, which of the following steps would be inhibited by Lapatinib?
- Signaling molecule binding, dimerization, and the downstream cellular response
- Dimerization, and the downstream cellular response
- The downstream cellular response
- Phosphatase activity, dimerization, and the downsteam cellular response
Produced by signaling cells and the subsequent binding to receptors in target cells, ligands act as chemical signals that travel to the target cells to coordinate responses. The types of molecules that serve as ligands are incredibly varied and range from small proteins to small ions like calcium (Ca 2+ ).
Small Hydrophobic Ligands
Small hydrophobic ligands can directly diffuse through the plasma membrane and interact with internal receptors. Important members of this class of ligands are the steroid hormones. Steroids are lipids that have a hydrocarbon skeleton with four fused rings different steroids have different functional groups attached to the carbon skeleton. Steroid hormones include the female sex hormone, estradiol, which is a type of estrogen the male sex hormone, testosterone and cholesterol, which is an important structural component of biological membranes and a precursor of steriod hormones ((Figure)). Other hydrophobic hormones include thyroid hormones and vitamin D. In order to be soluble in blood, hydrophobic ligands must bind to carrier proteins while they are being transported through the bloodstream.
Water-soluble ligands are polar and, therefore, cannot pass through the plasma membrane unaided sometimes, they are too large to pass through the membrane at all. Instead, most water-soluble ligands bind to the extracellular domain of cell-surface receptors. This group of ligands is quite diverse and includes small molecules, peptides, and proteins.
Nitric oxide (NO) is a gas that also acts as a ligand. It is able to diffuse directly across the plasma membrane, and one of its roles is to interact with receptors in smooth muscle and induce relaxation of the tissue. NO has a very short half-life and, therefore, only functions over short distances. Nitroglycerin, a treatment for heart disease, acts by triggering the release of NO, which causes blood vessels to dilate (expand), thus restoring blood flow to the heart. NO has become better known recently because the pathway that it affects is targeted by prescription medications for erectile dysfunction, such as Viagra (erection involves dilated blood vessels).
Cells communicate by both inter- and intracellular signaling. Signaling cells secrete ligands that bind to target cells and initiate a chain of events within the target cell. The four categories of signaling in multicellular organisms are paracrine signaling, endocrine signaling, autocrine signaling, and direct signaling across gap junctions. Paracrine signaling takes place over short distances. Endocrine signals are carried long distances through the bloodstream by hormones, and autocrine signals are received by the same cell that sent the signal or other nearby cells of the same kind. Gap junctions allow small molecules, including signaling molecules, to flow between neighboring cells.
Internal receptors are found in the cell cytoplasm. Here, they bind ligand molecules that cross the plasma membrane these receptor-ligand complexes move to the nucleus and interact directly with cellular DNA. Cell-surface receptors transmit a signal from outside the cell to the cytoplasm. Ion channel-linked receptors, when bound to their ligands, form a pore through the plasma membrane through which certain ions can pass. G-protein-linked receptors interact with a G-protein on the cytoplasmic side of the plasma membrane, promoting the exchange of bound GDP for GTP and interacting with other enzymes or ion channels to transmit a signal. Enzyme-linked receptors transmit a signal from outside the cell to an intracellular domain of a membrane-bound enzyme. Ligand binding causes activation of the enzyme. Small hydrophobic ligands (like steroids) are able to penetrate the plasma membrane and bind to internal receptors. Water-soluble hydrophilic ligands are unable to pass through the membrane instead, they bind to cell-surface receptors, which transmit the signal to the inside of the cell.
Visual Connection Questions
(Figure) HER2 is a receptor tyrosine kinase. In 30 percent of human breast cancers, HER2 is permanently activated, resulting in unregulated cell division. Lapatinib, a drug used to treat breast cancer, inhibits HER2 receptor tyrosine kinase autophosphorylation (the process by which the receptor adds phosphates onto itself), thus reducing tumor growth by 50 percent. Besides autophosphorylation, which of the following steps would be inhibited by Lapatinib?
- Signaling molecule binding, dimerization, and the downstream cellular response.
- Dimerization, and the downstream cellular response.
- The downstream cellular response.
- Phosphatase activity, dimerization, and the downsteam cellular response.
(Figure) C. The downstream cellular response would be inhibited.
What property prevents the ligands of cell-surface receptors from entering the cell?
- The molecules bind to the extracellular domain.
- The molecules are hydrophilic and cannot penetrate the hydrophobic interior of the plasma membrane.
- The molecules are attached to transport proteins that deliver them through the bloodstream to target cells.
- The ligands are able to penetrate the membrane and directly influence gene expression upon receptor binding.
The secretion of hormones by the pituitary gland is an example of _______________.
- autocrine signaling
- paracrine signaling
- endocrine signaling
- direct signaling across gap junctions
Why are ion channels necessary to transport ions into or out of a cell?
- Ions are too large to diffuse through the membrane.
- Ions are charged particles and cannot diffuse through the hydrophobic interior of the membrane.
- Ions do not need ion channels to move through the membrane.
- Ions bind to carrier proteins in the bloodstream, which must be removed before transport into the cell.
Endocrine signals are transmitted more slowly than paracrine signals because ___________.
- the ligands are transported through the bloodstream and travel greater distances
- the target and signaling cells are close together
- the ligands are degraded rapidly
- the ligands don’t bind to carrier proteins during transport
A scientist notices that when she adds a small, water-soluble molecule to a dish of cells, the cells turn off transcription of a gene. She hypothesizes that the ligand she added binds to a(n) ______ receptor.
Critical Thinking Questions
What is the difference between intracellular signaling and intercellular signaling?
Intracellular signaling occurs within a cell, and intercellular signaling occurs between cells.
How are the effects of paracrine signaling limited to an area near the signaling cells?
The secreted ligands are quickly removed by degradation or reabsorption into the cell so that they cannot travel far.
What are the differences between internal receptors and cell-surface receptors?
Internal receptors are located inside the cell, and their ligands enter the cell to bind the receptor. The complex formed by the internal receptor and the ligand then enters the nucleus and directly affects protein production by binding to the chromosomal DNA and initiating the making of mRNA that codes for proteins. Cell-surface receptors, however, are embedded in the plasma membrane, and their ligands do not enter the cell. Binding of the ligand to the cell-surface receptor initiates a cell signaling cascade and does not directly influence the making of proteins however, it may involve the activation of intracellular proteins.
Cells grown in the laboratory are mixed with a dye molecule that is unable to pass through the plasma membrane. If a ligand is added to the cells, observations show that the dye enters the cells. What type of receptor did the ligand bind to on the cell surface?
An ion channel receptor opened up a pore in the membrane, which allowed the ionic dye to move into the cell.
Insulin is a hormone that regulates blood sugar by binding to its receptor, insulin receptor tyrosine kinase. How does insulin’s behavior differ from steroid hormone signaling, and what can you infer about its structure?
Insulin’s receptor is an enzyme-linked transmembrane receptor, as can be determined from the “tyrosine kinase” in its name. This receptor is embedded in the plasma membrane, and insulin binds to its extracellular (outer) surface to initiate intracellular signaling cascades.
Normally, steroid hormones cross the plasma membrane to bind with intracellular receptors. These intracellular hormone-receptor complexes then interact directly with DNA to regulate transcription. This limits steroid hormones to be small, non-polar molecules so they can cross the plasma membrane. However, since insulin does not have to cross into the cell it could be large or polar (it is a small, polar molecule).
- A. B. Sigalov, The School of Nature. IV. Learning from Viruses, Self/Nonself 1, no. 4 (2010): 282-298. Y. Cao, X. Koh, L. Dong, X. Du, A. Wu, X. Ding, H. Deng, Y. Shu, J. Chen, T. Jiang, Rapid Estimation of Binding Activity of Influenza Virus Hemagglutinin to Human and Avian Receptors, PLoS One 6, no. 4 (2011): e18664.
RLP-type receptors rely on others to communicate the message
The number of RLP-type receptors predicted from genomic sequences varies according to the plant species studied. Arabidopsis has 57 while rice has more than 90 ( Fritz-Laylin et al., 2005 Wang G et al., 2008). Some of these receptors also contribute to development or defence. For example, Arabidopsis CLAVATA2 (CLV2, AtRLP10) and Too Many Mouths (TMM, AtRLP17) proteins play a significant role in meristem and stomatal development, respectively ( Jeong et al., 1999 Nadeau and Sack, 2002). Conversely, in the tomato, the RLP-encoding Cf and Ve genes confer race specific resistance to Cladosporium fulvum and Verticillium spp isolates, respectively ( Kawchuk et al., 2001 Kruijt et al., 2005). Recently, in collaboration with several other laboratories, homozygous T-DNA insertion lines have been identified for all the Arabidopsis RLP-encoding genes. These were subjected to a wide range of stress inducers including adapted and non-adapted pathogens, MAMPs, and abiotic stimuli. It has also been investigated if the mutation in these RLP-type receptors causes altered plant growth or development (Wang G et al., 2008). A number of novel developmental phenotypes were observed for the clv2 and tmm insertion mutants. These were slow growth, more rosette leaves, shorter stems, and late flowering for the Atrlp10-1 T-DNA insertion line, and chlorosis and reduced growth for the Atrlp17-1 and tmm-1 mutants upon abscisic acid (ABA) treatment (Wang G et al., 2008). Atrlp30 and, in addition, Atrlp18 were found to be more susceptible to the non-adapted bacterial bean pathogen Pseudomonas syringae pv. phaseolicola. Similarly, it was confirmed that AtRLP52 confers resistance to the non-adapted fungal pathogen Erysiphe cichoracearum ( Ramonell et al., 2005). Mutation in the AtRLP41 gene leads to enhanced sensitivity to ABA, the plant hormone that integrates and fine-tunes abiotic and biotic stress-response signalling networks both in plants and animals ( Asselbergh et al., 2008 Nagamune et al., 2008).
It is surprising that a biological role has been found for only a few of the defined AtRLP genes. This may be attributed to several factors (i) the approach taken may have been biased towards the pathogens and mainly race-specific resistance may have been investigated, (ii) no insects or nematodes were included in our screen, (iii) the assay used may not have been sensitive enough to discover some of the roles that these proteins may play, (iv) these receptors may be involved in the recognition of DAMPs, which were not addressed in our study, or (v) there may be functional redundancy. In many ways, this is similar to the abundance of NLRs in the animal genome without known functions. The Arabidopsis genome harbours 24 loci containing a single AtRLP gene and 13 loci comprising multiple AtRLP genes ( Fritz-Laylin et al., 2005 Wang G et al., 2008). Most homologous AtRLP genes reside at the same locus and the identification of a T-DNA insertion mutation in one gene may, because of the functional redundancy, not be enough to uncover the role of those genes. In addition, generation of double mutants by crossing individual T-DNA lines would be impossible. In order to overcome the problem of functional redundancy and further investigate the role of RLP-type proteins in Arabidopsis, Ellendorf et al. (2008) used an RNA interference (RNAi) approach and confirmed some of the phenotypes observed before. However, no new phenotype has been identified.
Since RLP-type receptors lack a cytoplasmic catalytic domain, one of the intriguing questions concerning RLP-mediated signalling is how the message is transmitted from the extracellular matrix to the intracellular space. Although RLP-type receptors in tomato recognize some pathogen effectors indirectly, it is not known how this message is internalized. The simplest explanation could be similar to that suggested for CLV2 and TMM where these RLPs may function in combination with RLK-type receptors CLAVATA1 and ERECTA, respectively, thus relaying the message ( Waites and Simon, 2000 Shpak et al., 2005). Although it has not been reported, it is tempting to speculate that AtRLP41 may also interact with an RLK such as RPK1 ( Osakabe et al., 2005) to regulate abscisic acid signalling in Arabidopsis.
Biophysicists find a way to take a peek at how membrane receptors work
General appearance of the G protein-coupled receptor. Credit: Anastasia Gusach et al./Current Opinion in Structural Biology
In a study published in Current Opinion in Structural Biology, MIPT biophysicists explained ways to visualize membrane receptors in their different states. Detailed information on the structure and dynamics of these proteins will enable developing effective and safe drugs to treat many sorts of conditions.
Every second, living cells receive myriads of signals from their environment, which are usually transmitted through dedicated signaling molecules such as hormones. Most of these molecules are incapable of penetrating the cell membrane so for the most part, such signals are identified at the membrane. For that purpose, the cell membrane is equipped with cell-surface receptors.
These receptors receive outside signals and 'interpret' them into the language the cell can understand. Cell surface receptors are vital for proper functioning of cells and the organism as a whole. Should the receptors stop working as intended, the communication between cells becomes disrupted, which can lead to the organism developing a medical condition.
GPCRs (G protein-coupled receptors) are a large family of membrane receptors that share a common structure they all have seven protein spirals that cross the membrane and couple the receptor to a G protein situated on the inside of the cell. Interaction of the signal molecule with the receptor triggers a change in the 3-D structure, or conformation, of the receptor, which activates the G protein. The activated G protein, in turn, triggers a signal cascade inside the cell, which results in a response to the signal.Key GPCR family receptors that are related to diseases. Credit: Anastasia Gusach et al./Current Opinion in Structural Biology
GPCR family membrane proteins have been linked to a lot of neurodegenerative and cardiovascular diseases and certain types of cancer. GPCR proteins have also been proven to contribute to conditions such as obesity, diabetes, mental disorders, and others. As a result, GPCRs have become a popular drug target, with a large number of drugs currently on the market targeting this particular family of receptors.
One of the modern approaches to drug development involves analyzing 3-D structures of GPCR molecules. But membrane receptor analysis is a slow and extremely laborious process and even when successful, it does not completely reveal the molecule's behavior inside the cell.
"Currently, scientists have two options when it comes to studying proteins. They can either 'freeze' a protein and have its precise static snapshot, or study its dynamics at the cost of losing details. The former approach uses methods such as crystallography and cryogenic electron microscopy the latter uses spectroscopic techniques," comments Anastasia Gusach, a research fellow at the MIPT Laboratory of Structural Biology of G-protein Coupled Receptors.
The authors of the study demonstrated how combining both the structural and the spectroscopic approaches result in "the best of both worlds" in terms of obtaining precise information on functioning of GPCRs. For instance, the double electron-electron resonance (DEER) and the Förster resonance energy transfer (FRET) techniques act as an "atomic ruler," ensuring precise measurements of distances between separate atoms and their groups within the protein. The nuclear magnetic resonance method enables visualizing the overall shape of the receptor molecule while modified mass spectrometry methods (MRF-MS, HDX-MS) help trace the susceptibility of separate groups of atoms constituting the protein to the solvent, thus indicating which parts of the molecule face outwards.Sequence of processes triggered by activation of an GPCR receptor. Credit: Anastasia Gusach et al./Current Opinion in Structural Biology
"Studying the GPCR dynamics uses cutting-edge methods of experimental biophysical analysis such as nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and advanced fluorescence microscopy techniques including single-molecule microscopy," says Alexey Mishin, deputy head of the MIPT Laboratory for Structural Biology of G-protein Coupled Receptors.
"Biophysicists that use different methods to study GPCRs have been widely organizing collaborations that already bore some fruitful results. We hope that this review will help scientists specializing in different methods to find some new common ground and work together to obtain a better understanding of receptors' functioning," adds Anastasia Gusach.
The precise information on how the membrane receptors function and transfer between states will greatly expand the capabilities for structure-based drug design.
Modulation of Death Receptor signaling
There are several levels of modulation of DR signaling (French and Tschopp, 2003). As mentioned above, decoy receptors can compete with DRs for ligand binding, and FLIP blocks procaspase-8 activation at the DISC. Further downstream, IAPs (inhibitors of apoptosis) inhibit effector caspase activation. Our current understanding of DR signaling has opened possibilities for the design of new therapeutic strategies for targeting death receptor pathways. This would allow the treatment of a number of diseases potentially associated with defects in DR signaling, such as multiple sclerosis and Alzheimer's disease.
Lecture 20: Cell Signaling 1—Overview
After completing the topic of protein trafficking, Professor Imperiali introduces cell signaling. In the first of two lectures on this topic, she covers the paradigms and mechanics of cell signaling.
Instructor: Barbara Imperiali
Lecture 1: Welcome Introdu.
Lecture 2: Chemical Bonding.
Lecture 3: Structures of Am.
Lecture 4: Enzymes and Meta.
Lecture 5: Carbohydrates an.
Lecture 9: Chromatin Remode.
Lecture 11:Cells, The Simpl.
Lecture 16: Recombinant DNA.
Lecture 17: Genomes and DNA.
Lecture 18: SNPs and Human .
Lecture 19: Cell Traffickin.
Lecture 20: Cell Signaling .
Lecture 21: Cell Signaling .
Lecture 22: Neurons, Action.
Lecture 23: Cell Cycle and .
Lecture 24: Stem Cells, Apo.
Lecture 27: Visualizing Lif.
Lecture 28: Visualizing Lif.
Lecture 29: Cell Imaging Te.
Lecture 32: Infectious Dise.
Lecture 33: Bacteria and An.
Lecture 34: Viruses and Ant.
Lecture 35: Reproductive Cl.
BARBARA IMPERIALI: Now, I want to talk today about one small thing before we move on to signaling, because it really kind of completes the work that we talked about with respect to trafficking. So I popped this question up last time, and it seemed like there weren't quite enough sort of people leaping to give me an answer. But let's just take a look at the big picture of things, as it's always good to do. Because this will also get me to one other topic, which is protein misfolding.
So at the end of the day, what really defines where a protein is, what it does, is defined by its sequence. But you always want to remember that a protein sequence is defined by its messenger. The messenger is defined by the pre-messenger. Yes, there may be splicing events that really cause changes in localization. But the pre-messenger includes the content. And then what defines that is the DNA.
There's certain aspects of regulation at the epigenetic level that we don't talk about barely in this course. But I want you to make sure that you realize at the end of the day, what the protein is, how it folds, is defined originally by the sequence of the DNA, although a long way along here. The post-translational modifications that we started talking about last time are defined by the protein sequence, which all the way is defined by the DNA-- so, so much of protein function.
And there's one more aspect of proteins that's defined by the DNA sequence, and that's whether a protein folds well, or perhaps, in some cases, misfolds. And that's the thing I want to talk about very briefly today. Because I think that this captures the picture.
So let's just go over here and write misfolded proteins, which, just like everything else, largely end up being dictated by the DNA. Because whether a protein folds faithfully into a good structure or misfolds can be a function of the protein sequence.
So there could be mutations in the protein that ultimately end up that the protein misfolds and forms either a misfolded tertiary structure, or even worse, adopts an aggregated form that causes a lot of damage within cells and outsides of cells. So I want to talk just briefly about the processes that we have-- it's just one slide-- to deal with misfolded proteins.
So when a protein is translated, it almost starts folding straight away, especially large proteins. A fair amount of a protein may have already emerged from the ribosome and started folding, even when the whole protein isn't made. The sequence ultimately ends you up with a well-folded protein.
But if the protein does not fold fast enough, or there is a mistake in this, which might be caused intrinsically by the primary sequence, if there's a mistake in that-- so slow folding or incorrect folding-- then you will end up with a protein that's partially folded within the context of a cell.
We especially encounter misfolded proteins when we are overexpressing proteins in cells, because you're just making one of a type of protein really quickly, and it doesn't have a chance to adopt its faithful structure.
So there are proteins within the cell that helped sort of protect the folding process early on, to allow the protein to have enough time in singular, not with a lot of copies of itself around that are misfolded, to adopt a folded structure. And these proteins are called chaperones.
I don't know if you guys are familiar with the term chaperone. It was a term that was heavily used in the sort of 18th and 19th century. A chaperone used to be an aunt or someone who you would send out with your beautiful young daughter to chaperone her, to protect her so she didn't get bothered by those mean men out there. So chaperones were-- that was the original definition of the chaperone. And it's kind of interesting that the chaperones are now proteins that help folding or protect against misfolding.
How do they do this? Generally, a protein will fold poorly if it's very, very-- if it's quite hydrophobic. And hydrophobic patches are exposed in aggregate.
So let's say, you have a protein, and there's a lot of copies, but they're not folded. If you have things that are hydrophobic that would normally end up tucked inside the protein, if the protein hasn't folded in its good time, these will just start to form aggregates, sort of associating with each other. It's just a physical phenomenon.
If you put something that's got a lot of hydrophobic faces on the outside, this will start forming an aggregated bundle, and not a nicely folded protein at all. What the chaperones may do is in part hold the partially folded protein. So let's just think of this big jelly bean as a chaperone until things start to adopt a favorable state.
But sometimes it's just too much. The chaperone cannot handle the flux of protein. So the protein ends up being recognized as misfolded. And then it gets tagged as a misfolded protein, and it gets taken to a place in the cell for disposal. So if you are unable to fold, there is a tagging process. And I mentioned it last time. It's a process known as ubiquitination.
This is also a post-translational modification, but it's one that occurs on poorly folded proteins. And I'm going to describe to you that system, because the ubiquitination is the flag, the signal, or the tag, to take this protein to the great shredder, basically. And so what does the-- what's a paper shredder-- I like the analogy with a paper shredder.
So here's a fellow who's got too much on in his inbox. So he just sends it straight to the shredder. It's a little bit about too much misfolded protein being made. So instead of sort of waiting to deal with the paperwork, you just send it straight to the shredder. And the proteasome is the cellular shredder that actually breaks proteins up into small chunks, and then digests them out.
So think of the proteasome as a shredder, which chops up proteins into small pieces, mostly into short peptides that are 8 to 14 amino acids in length-- fairly small. Short peptides won't cause a problem in aggregation, and will then be further digested.
Now, if you've got this shredder sitting around in the cell, it's like having a paper shredder on all the time. You've got a-- there's a risk things may end up in there without meaning to be. So for things to be tagged for shredding, they go through what's known as the ubiquitin system.
So the first thing to get into that is-- and it's only then that proteins are tagged for shredding up or chopping up by the proteasome. So as you can sort of tell by its name, it's got protease function, but it's a large, macromolecular protease, with lots and lots of subunits that are important to cut that polypeptide into smaller pieces.
But because many of your proteins may be partially folded or misfolded, they first have to be unfolded. So the ubiquitin is the signal to send proteins to the proteasome, where the second action is protease activity, and the first action is unfolding.
So what I show you on this picture is the barrel structure of a proteasome. Let me explain the components of it. The red component of the proteasome is a multimeric ring that uses ATP and starts tugging apart the protein that you need to destroy. But it will only do that if the protein becomes labeled for destruction by the ubiquitin system.
And I am showing you here a massively simplified version. Let's say this is a misfolded protein. It gets tagged with another protein. It's a really little protein known as ubiquitin. I've shown you the three-dimensional structure here. And using ATP, you end up managing to put a ubiquitin chain on the protein that's going to be destroyed. That is a post-translational modification that is a tagging for destruction.
If the protein is not tagged, then it's not going to be chewed up. That makes sense. You don't want to be chopping up proteins in a cell with wild abandon. Once the ubiquitin chain is on here, the protein will bind to the unfoldase part of the proteasome, and with ATP, it will just stop tugging the rest of the residual structure apart to thread the protein down into the blue part of the barrel.
It's a little hard to see it like this, but it's literally, the proteasome, are four concentric rings. Let me see. I hope my artwork is going to be good enough. Well, that's an unfoldase. And so is that. And then in the center, there is a protease. And each of these components is multimeric, having six or seven subunits.
So it's a huge structure. It has a sedimentation coefficient of 20S, that entire structure. I don't know if you remember when I talked about ribosomes, they were so big we didn't tend to talk about them by size. We talked about them by sedimentation coefficient. And the large and small subunits of the ribosome, the eukaryotic one, just to remind you, were 40S and 60S.
So just remember that S stands for Svedberg. It's a sedimentation coefficient unit. It describes how fast approaching precipitates. So once the protein has been labeled with ubiquitin, it binds to the unfoldase. And then the single strand feeds into the center core, which is two sections of protease. So it's feeding in here. It sees the protease activity. And then it's just short pieces of protein are spit out of the proteasome.
Once these are really little pieces of peptide, they're readily digested by proteases within the cell. And you can recycle the amino acids, or you can do other things with these small pieces of peptide. They actually end up sometimes being sent for presentation on the surface of the cell by the immune system. And you may hear a little bit more about that later.
So the proteasome-- oh, I apologize-- this should have been 26S-- has a molecular weight that's very large-- 2,000 kilodaltons. That's why we refer to it by its sedimentation coefficient. So this machinery is very important to get rid of misfolded or aggregated proteins to destroy them.
Now, does-- are people aware of the sorts of diseases that can result from misfolded proteins? Has anyone been reading the news much about certain types of diseases, particularly in neurobiology? Anyone aware of those? Yeah.
AUDIENCE: Was it mad cow disease?
BARBARA IMPERIALI: Which one?
BARBARA IMPERIALI: Yes. Mad cow. So there are a variety of neurological disorders, and mad cow is one of them. Creutz U-T-Z feldt-Jakob. But Alzheimer's disease is another one. Pick's disease is another.
There are a wide variety of neurological disorders that result from misfolded proteins, both inside the cell and in the extracellular matrix, forming these tangles that are toxic to the neurons, causing them to no longer function, and then resulting in many of these neurological disorders.
The ones I've described to you, I've mentioned to you here. I know many of you are familiar with Alzheimer's disease. Mad cow disease is a variant of a particular protein misfolding disease that was first noted in cattle. And they basically just fell down, dropped down. And it was in some cases ascribed to-- the contagion with the disease is ascribed not to a virus or to a microorganism, but literally, to misfolded proteins causing the formation of more misfolded proteins.
So these are all collectively designated as prion diseases. I think you'll have read that term. And it's a particular kind of disease that the infectious agent isn't a living system-- not a virus, not a microbe, a fungus, a protozoan, but rather a protein, where it's misfolded structure nucleates the formation of more misfolded structure that leads to the disease.
So I grew up in England during the years where there was a lot of mad cow disease in England. And even though I'm a vegetarian in the US for 30 years, I can't give blood in the US, because I lived in England during the time when there was a lot of mad cow disease. And this can be dormant for a long, long time before it suddenly takes over.
So there's restrictions on blood donation in certain cases. And it's because it's not something you can treat with an antibiotic, you can treat with an antiviral. It's literally traces of badly folded protein that can nucleate the formation of more badly folded protein, that can lead to the diseases.
These were-- there's particular instances of some of these diseases in tribes where there's pretty serious cannibalism, and eating your sort of senior relative's brains was considered to be something-- an important act of respect. And there was this transfer of some of these prion-type diseases through cannibalism as well.
So eating contaminated meat, be it a cow, be it your grandparents, whatever, it's something that actually is-- it's a serious transmissible disease. And it's really-- in the situations where it can be sort of related back to contaminated meat are one thing. But there are variations in the case of Alzheimer's, where the sequence of proteins may dictate that they don't fold well, or they're not post-translationally modified properly, so they end up as misfolded proteins.
So these are often genetically linked disorders, some of the things like Alzheimer's. And once again, remember that goes all the way back to the DNA, which might, in some cases, trigger the misfolded disease.
So it's a fascinating area, and there's a tremendous amount to be studied. Because of the aging population, these diseases are piling up, and we need to mitigate the causes of the disease, and find ways, for example, to slow down. If there are these fibrils of protein that are misfolded, can we maybe inhibit that formation with some kind of small molecule inhibitor to mitigate the symptoms of the disease? So it's a very, very active area, because almost every-- many, many neurological disorders seem to be coming down to misfolded proteins.
So let's move on now to signaling. All right. So we're going to spend two lectures on-- the remainder of this lecture plus the next lecture. And what I want to do in this lecture is introduce you to some of the paradigms, the nuts and bolts, the mechanics of protein signaling. And then in the next lecture, I'm going to show you examples of how all the characteristics that we define signaling by get represented in signaling pathways within cells.
So I'm going to give you all the moving parts, and then we'll move forward to see how the moving parts might function in a physiological action, such as a response to something particularly scary, or as a trigger to do-- for the cells to do something different.
So let me take you, first of all, to a cartoon-like image of a cell. And we're going to just take from the very simplest beginning. But then this topic will get quite complex, as you see. But that's why I think it's important to reduce the process of protein signaling down to simple aspects of it that we can really recognize, even in much more complicated pathways.
So in protein cellular signaling, this is a complex system of communication that governs all basic activities of the cell. There are no cells that don't do signaling. Bacteria and eukaryotic cells may do signaling slightly differently. But they still do have an integrated correlated system that's responsible for triggering functions of the cell through a series of discrete steps.
So protein signaling can be dissected into three basic steps, where you, first of all, receive a signal. And we're going to talk about what that signal is. What's the nature of that signal, is it small molecule, large molecule? Where is the signal? Where does it act?
Then the next step is to transduce the signal. And finally, you have an outcome, which is a response. So we're going to talk about each of these components in order to understand flux through cellular signaling pathways, and how they work to give you a rapid response to a necessary signal.
All right. So in this cartoon, let's just, for example, think about what if we want to trigger cell division? We might have a signal, which is the yellow molecule-- a small molecule, large molecule. We'll get to that later.
There's a cell here, where on the surface of the cell is a receptor. And that would be the entity that receives the signal. So in the first step in the process, there's a buildup of a concentration of a signal. And it occupies the receptors on the surface of the cell, and in some cases, inside the cell. We'll talk about a bifurcation there. But really, a lot of cellular signaling is dominated by cells coming-- by signals coming from outside the cell.
What happens upon this binding event is the transduction. If you bind to something on the outside of the cell, as a consequence, you might have a change on that structure. If it crosses the membrane, you might have a change on that same molecule structure that's on the inside of the cell.
So that's why it's called transduction. You're transducing a soluble signal from outside, binding that signal to the cell surface receptor. And the cell surface receptor is responding in some way.
And there are two principle ways in which we respond to extracellular signals, and we'll cover them both. The next event that might happen is through the change that happens to the intracellular component of the receptor. There might be a change, a binding event, another step occur within the cell. And as a function of that, you get a response. All right?
So it's really-- thinking it in these three components is a good way to kind of dissect out the beginnings of the complication. And then what we'll be able to do is really start to see what kinds of molecules come in? How are they received? How is the signal transduced? And what's the ultimate outcome with respect to a response? Everyone OK with that? All right.
Now this is what you have to look forward to. So we give you something with three moving parts, and suddenly we show you something with sort of, you know, 100 moving parts. And cell biologists very, very frequently look at these maps of cells, where what they're looking at with each of these sort of little acronyms or names, all of these are proteins, where they have been mapped out through cell biology and cellular biochemistry to be existing in certain components of the cell.
And what has also been mapped out very frequently is, who talks to who? So the fact that JAK S might interact with STAT 35 and so on. So much of this was worked out through cell biology and biochemistry, and also by genetics.
So Professor Martin has talked to you about identifying a player in a complex system by genetics. Let's say you have a cell that fails to divide. You might perhaps screen or divides unevenly, or has some defect in cell division. You might be able to pick out a particular player.
Now, the key thing I want to point out to you with this cell is what's on the outside of the cell and runs across the membrane, and might have the chance, the opportunity, to have both an extracellular receptor and an intracellular function. And those key proteins are things like receptor tyrosine kinases. And we're going to talk about all of these in a moment.
G protein-coupled receptors, and various other cell surface receptors-- so all of these-- anything that spans a membrane has the opportunity to be an important component of a signaling pathway. Because what you're routinely trying to do is have your signal recognized on the outside of the cell by something that spans the membrane. The signal will bind to that. And then you will have an intracellular response.
So that's breaking it down. That's why proteins that are made through the secretory pathway that we talked about in the last lecture, that go through that endomembrane system, and end up being parked in the plasma membrane are so important.
Other proteins that actually get secreted through that pathway are also important. What do you think they may be important for? Let's say you've made a protein within the cell. It goes through all the system. It doesn't stay parked in the cell membrane. It actually gets released from the cell. What might that be doing?
BARBARA IMPERIALI: Yeah. Exactly. So that endomembrane system that I described to you, that pathway is great for making receivers. And it's great for making signals. And that's really what can sort of fuel the functions of cells. OK.
So in systems biology, you may have heard this term quite frequently. Systems biology is research that helps us understand the underlying structure of signaling networks. So a lot of people who have common interests in engineering, computational analysis and cell biology, might bring in data to allow them to make models of cellular systems, to understand flux through signaling pathways.
So they may make fundamental measurements about the concentrations of some components within the cell. And then try to say, OK, I know based on everything I've measured that this is a dominant pathway for gene regulation. And I could control this pathway by different-- by sort of different types of interactions.
In this cellular system, I also show you another component, which is the nucleus. And when we discuss and describe specific cell signaling networks, in some cases the signaling network may involve receiving a signal, undergoing a variety of changes in the cytoplasm, but then a change that eventually results in a protein going to the nucleus. And oftentimes, those proteins that run into the nucleus are transcription factors that then trigger DNA replication or transcription. And then promote activities.
So this is how you think about it. When you think of cellular signaling, it's really about, what does the signal need to do? And what's the pathway that I follow to get there? So all of those are membrane proteins.
So now let's look at the canonical aspects of signal transduction. So the first-- and I'm going to rely on these little cartoons. But I want, both in this lecture and the next, to really show you where these recur in so many systems. So to that purpose, I want to talk about the characteristics.
So the first critical characteristic is a signal and it's specificity. So a signal will be something that comes from outside of the cell. It could be a hormone that's produced in the hypothalamus and sent to another organ. But the most important thing about the signal is that that signal, which binds to a receptor in a cell membrane, is specific for a particular receptor, and the different signal won't blind to the same receptor. You have to have faithful signal specificity to trigger the right function.
So if it's a hormone, it's got to be the hormone that you want to trigger the receptor, not a related but different-looking structure. If it's a small protein, you want it to be the exact one that binds with high specificity to a receptor.
So what that means is if something is binding-- if a small molecule is binding to a protein on the surface of a cell with high specificity and high affinity, it means that even at a low concentration, it will make that binding contact. But all the other small molecules that are around won't crosstalk into that triggering that interaction. So we have high specificity, and we gain that specificity through macromolecular interactions, just like the ones we talked about in biochemistry.
So if we have a small molecule or a protein bind to the receptor, it's making all those hydrogen bonding, electrostatic, noncovalent types of interactions with high specificity, so that a low concentration of the signal molecule is efficient for binding to the receptor to trigger the function.
The next characteristic is amplification. Now let's put some lines between these guys. Now, with all the signaling pathways that you're going to see, we're going to be looking where in a pathway you get amplification. Very commonly, you might have a response that's just the result of a single molecule binding a single receptor.
But at the end of the day, you might want a large response. You might want to make a lot of ATP. Or you might want to replicate all of the genome. So you need some kind of amplification where in a sense you're turning up the volume on your signal. And you need to do that rapidly.
So frequently in signaling pathways, you go through a cascade of reactions where the signal might affect an enzyme. But once you make that enzyme active, it might work on many, many copies of another enzyme. And then each of those may work on even more copies.
So that's what I mean by amplification, where at some stage you've generated a molecule that can result in the cascade of a reaction. So we often refer to these as cascades. So if you're Spanish-speaking, cascada. You want to think about a waterfall coming from just a single molecule of water. You're getting a large increase in your signal as a result of amplification.
The next feature or characteristic of signaling is feedback. At the end of the day, if you're signaling, I got to make some ATP. I got to run out of the woods. I'm getting chased. At a certain stage, you need to stop all of the process occurring. So feedback is just a negative feedback loop that might slow down some of those steps that are involved in amplification.
So for a pathway, you only want the pathway turned on for a prescribed amount of time. And then you want to be able to say, I'm done with that whole pathway. I don't need to keep churning through all those enzymes. It's time to stop that. And that usually occurs through negative feedback.
And remember, we talked about negative feedback when we were talking about enzyme catalyzed pathways. So feedback is very often some kind of negative feedback, which suppresses a series of transformations, perhaps through a product of those transformations acting as an inhibitor on an early step.
And then finally, the other component of a signaling network-- if you think of signaling networks as electronic structures, you have integration. So that's the last characteristic feature. And let me go back to that big circuit diagram quickly to show you an example of integration.
So if you look at this signaling pathway, all these signaling steps are not single. You just have a signal come in, and you end up, for example, in the nucleus. But rather, other components may have crosstalk within one pathway, and start out either amplifying or turning down a particular signaling pathway. So these are networks. They're not pathways. They're networks that interact and communicate, all to amplify signals or turn down signals.
So integration is an important part of signaling, because you're often dealing with the integrated function of a number of pathways to get a particular response. And that actually ends up being one of the situations where sometimes a particular enzyme may look like a perfect target for a therapeutic agent.
But if you don't take into account the integration steps, you may be trying to deal with a-- you may think you're dealing with a single pathway, but you're, rather, dealing with crosstalk with a lot of other pathways. And what often happens in a cell is there's compensation from other pathways. Is everybody following? Any questions here about this?
So what I want you to think about is that it's just amazing what is orchestrated to have even the simplest functions in the cell, how many interacting components there may be.
Specificity, amplification, feedback, and integration-- all right, so let's talk briefly about types of signals and how we name them, where they come from, in order to make sure we're all on the same page with respect to the language that's used.
So now, signals may take different molecular forms. They may be small molecules, for example, an amino acid or a phospholipid-- just something little. Alternatively, they may be proteins. They may be carbohydrates. They might take different forms in terms of their molecular structure. But we tend to describe signals by where they come from.
So what I've shown you here is a picture from the book that just describes how we refer to certain signals. So there are four different terms-- autocrine, juxtacrine-- and I'm going to just give you a little hint to how to remember these terms-- paracrine, endocrine. OK. So these don't tell you anything about the molecule. They tell you about where it's come from.
So an autocrine signal is a signal that may come from a cell, but it's signaling to itself. So it may produce a component that's released. And so it's producing this through a secretory pathway. It's a release, and it stays in the vicinity of the cell. So the self is self-signaling. So whenever you see something auto, you just want to say, oh, that means it's coming from the same cell where the signal occurs.
Let's move to the next one, which is paracrine. I'm going to talk about jux-- and that's usually from a nearby cell, not a cell that's in contact-- definitely a different cell. So paracrine is-- we would always call nearby. And endocrine is completely from somewhere else, so perhaps coming through the circulatory system. One cell may release an endocrine signal. It may weave its way through the vascular system, and then target a cell. So endocrine is always from a distance.
And juxtacrine is the only one that's a little odd. It's really from cells that actually are in contact with each other. So it's not self-signaling within a cell. It's not a cell that's nearby but pretty close. It's actually physically making a contact. And so that's the last terminology there.
So hopefully, I can get this calcium wave to show you. This is just a video of juxtacrine to signaling. I just want you to sort of keep an eye on things. It's usually a cell. What you're observing here is a dye that lights up in the presence of calcium flux. It's called Fura-2.
And so when you stare at these for long enough, what you can notice is that when one signal will often come from an adjacent cell right near it-- so there are long prostheses. You're not looking at the entire cell, but they're definitely-- for example, this little duo down here, they keep signaling to each other. And that's just a juxtacrine signaling, because the cells are in the contact.
So that just shows you the difference there. If it was autocrine, you just have a single cell responding. If it's paracrine, they would be at more of a distance to each other. I hope that imagery-- this is from a website in the Smith Lab at Stanford.
OK. And then the last thing I want to give you an example, there are many, many hormones in the body that undergo endocrine signaling, and so one example I thought I would tell you about, you all know that insulin is made in the pancreas. It's an important hormone for regulating glucose levels. And it's actually-- functions at the muscle level. So insulin is an example of an endocrine signal, because it travels a distance from where it's made in the body to where it functions in the body. All right.
Now-- so we've talked about the types of signals. Let's now move to the types of receptors. Now, we cover both the intracellular and the cell surface receptors. But we really will focus a lot on the cell surface receptors.
I just want to give you a clue that not all signaling is cell surface. So what I've shown you here is a cartoon where you see signaling, where a signal comes from outside the cell. It goes into the cell and triggers a change. And then the majority of the time we'll talk about these receptors that are in the plasma membrane. And they have an outside place where the signal binds, and they trigger a response inside.
And it's only very specific signals that are able to signal intracellularly, that is, to cross the membrane to get inside the cytoplasm to do the triggering. What kinds of molecules can cross the membrane easily? We talked about that before, when we talked about getting across that barrier. Yeah? Nonpolar. OK.
So you can look at a-- you can stare at a molecule, and if it's very polar or pretty large, it's not going to be able to sneak through a membrane. So something like a steroid molecule, a large, greasy molecule, can definitely make that transition. And so those are the only types of signals that we can really do inside the cell, because they can get across the cell. Many, many other signals have to go through this-- bind to the outside of a cell, and transduce a signal to the inside of the cell.
So one very typical signal that can bind to an intracellular receptor is a steroid. So remember when I talked to you about these lipidic molecules, things like testosterone and cortisol. These are very hydrophobic molecules. So they literally can cross from the outside of the cell without a transporter. So for example, the hormone cortisol.
And when that functions, it just-- an amount of it becomes available, for example, in the bloodstream. It crosses the cell, and it binds to an intracellular receptor. Once it binds to that intracellular receptor, this disengages a different kind of chaperone protein that's keeping it stable. Once it's found, it can then go into the nucleus and trigger transcription. So this is the one example of an intracellular receptor that we'll talk about.
I just wanted to show you a little bit about the steroid receptors. These are molecules that are very-- these are macromolecules, proteins that are very-- quite a complex structure. But they can literally-- and I'll show you the picture at the beginning of the talk next time-- they can literally engulf these proteins. So once the steroid is bound to that, it completely changes shape. And that's what enables the change for it to be triggered and sent to the nucleus.
Now, the key types of receptors that we'll focus on, though, are the cell surface receptors. And there are three basic classes of molecules that occur in the plasma membrane that are critical for cellular signaling. They are the G protein-coupled receptors, the receptor tyrosine kinases, and then you will talk in the lecture 22 about ion channels, and how they perform a receptor function.
So the membrane proteins, first of all, I want to underscore their importance. 50%-- they comprise 50% of drug targets, the receptor tyrosine kinases and the G protein-coupled receptors. The G protein-coupled receptors have this 7 transmembrane helix structure, which spans a membrane. This would be the outside of the cell, and the inside of the cells-- so there's signals going across there.
The receptor tyrosine kinases are another important type of receptor. They are dimeric proteins that in the presence of a ligand dimerize, and then cause intracellular signaling. Once again, they cross the plasma membrane from the outside to the cytosol. And then lastly, there are the ion channels, which also may cross the plasma membrane.
And when you think about these classes of proteins, there's a tremendous amount to be learned with respect to their functions. And they are so important to understand their physical functions in the body, because they really represent the place, the nexus, where signaling happens in the cell.
So I want to briefly show you a picture of a GPCR. It's a 7 transmembrane helix structure. You can see it here. There are about 30% of modern drugs actually target the GPCRs. And here, I'm just going to show you the structure of a GPCR. Those are the 7 transmembrane helices. If you stretch them out, that's about the width of a membrane. That's typical of a signal that would bind to that kind of receptor.
This is a chemokine. It's a small protein receptor. So you can see that structure and how it would go from one side of the membrane to the other. In it's bound state, the chemokine binds to the 7 transmembrane helix receptor through kind of a clamping action. The magenta is the chemokine. The blue and the green space filled parts are actually what holds the chemokine.
And if you look at it where the membrane would be, you can see how you can transduce a signal from one side of the membrane to the other, by the binding of the magenta molecule to the outside of the cell, to those loops outside the cell. That would have a significant perturbation to the biology and chemistry of what's going on on the inside.
So next class, we'll talk about pathways that are initiated by these G protein-coupled receptors, and what that terminology means. OK.
Responding to the signal
The information below was adapted from OpenStax Biology 9.3, and Khan Academy Signal relay pathways. All Khan Academy content is available for free at www.khanacademy.org
Step 4: signal response: There are many different types of cellular responses to a hormone, including:
- changes in gene expression
- changes in cell metabolism
- cell growth and division
The overall response to a hormone signal can be to amplify the signal and increase the response (positive feedback), or to decrease the signal and decrease the response (negative feedback). The key is that in positive feedback, the response to the stimulus causes the stimulus to continue in the same direction while in negative feedback, the response to the stimulus causes the stimulus to change direction. So in a positive feedback loop, if the stimulus is increasing, then the response to the stimulus causes it to increase even more. In a negative feedback loop, if the stimulus is increasing, then the response will cause the stimulus to decrease. Positive and negative feedback loops are important mechanisms of cellular and organismal regulation.
This video (beginning at 1:13) provides an overview of positive vs negative feedback loops (watch through at least 5:58):
The response to a particular signal can be very straightforward, but there can be differences between different cell types and in different conditions for a number of reasons:
- The same ligand can cause different responses in different cell types due to differences in protein expression in the different cells, where the same signal activates different signaling pathways, leading to a different response in each cell type.
- The same ligand can cause different responses in different cell types due to different receptors in the two different cell types, which then activate different signaling pathways, leading to different responses in each cell type.
- Often multiple signaling pathways interact with each other because the same signaling proteins are involved in each pathway. As a result, if two different signals come in at the same time, the outcome can be different than if they came in separately. The phenomenon where the outcome can change based on interactions between different signaling pathways is called signaling crosstalk.
Protein Targeting (With Diagram) | Molecular Biology
Let us make an in-depth study of the protein targeting. After reading this article you will learn about: 1. Introduction to Protein Targeting 2. Signal Sequence 3. Transport of Proteins into ER 4. Signal Sequence Recognition Mechanism 5. Role of Golgi Complex in Protein Transportation 6. Transport of Proteins from Golgi to Lysosomes 7. Targeting of Proteins to Mitochondria and Chloroplasts 8. Protein Targeting to Chloroplasts 9. Protein Targeting into Nucleus and 10. Membrane Proteins.
Introduction to Protein Targeting:
A typical mammalian cell may contain numerous kinds of proteins and numerous individual protein molecules. The eukaryotic cell is a multi-compartmental structure. Its many organelles each requires different proteins. Except a few of them which are synthesized in mitochondria and chloroplasts all other proteins necessary for the cell and the ones to be secreted by the cell are synthesized in the cytosol on free ribosomes and on ribosomes bound to the endoplasmic reticulum.
Most proteins are coded by the nuclear genome and synthesized in the cytoplasm. The proteins are present in the ER, mitochondria, chloroplasts, Golgi, peroxisomes, nucleus, in the cytosol and in the membranes of all these organelles. They are selectively transported into their appropriate organelles inside the cell and across the plasma membrane to be secreted outside the cell.
Some of them are carried into membrane bound vesicles which bud off from one organelle and transported in definite pathways. Different destinations of different proteins require sophisticated system for labelling and sorting newly synthesized proteins and ensuring that they reach their proper places. This transportation of proteins to their final destinations is called protein targeting.
Proteins destined for cytoplasm and those to be incorporated into mitochondria, chloroplasts and nuclei are synthesized on free ribosomes in the cytoplasm. Proteins destined for cellular membranes, lysosomes and extracellular transport, use a special distribution system. The main structures in this system are the rough endoplasmic reticulum (RER) and Golgi complex.
The RER is a network of interconnected membrane enclosed vesicles or vacuoles. The endoplasmic reticulum is coated with polyribosomes to give it a rough appearance. The golgi complex is also a stack of membrane bound sacs but they are not interconnected. The golgi complex acts as a switching center for proteins to various destinations.
Proteins to be directed to their destinations via Golgi complex are synthesized by ribosomes associated with endoplasmic reticulum.
Protein sorting requires proper address labels which are in the form of peptide signal sequences. A signal sequence that directs the protein to its target is present in the form of 13-35 amino acids in the newly synthesized protein itself. It is the first to be synthesized and is mostly present at the amino N-terminal, sometimes at the carboxyl C- terminal.
It is known as signal sequence or leader sequence. Some proteins are further sorted to a sub-compartment within the target organelle. For this purpose, a second signal sequence is present behind the first signal sequence which is cleaved.
Proteins carried inside the membrane bound vesicles are called cargo proteins. An embedded or integrated protein is carried in the membrane of the vesicle, while secretory protein is carried within the lumen of the vesicle. The vesicle buds off from the donor surface and fuses with the target surface releasing its contents into the target organelle and the membrane protein is incorporated into the membrane of the target organelle. The process is repeated during the passage of protein from ER to Golgi to lysosomes and from Golgi to plasma membrane.
Transport of Proteins into ER:
A short N-terminus signal sequence at the beginning of the growing nascent protein chain’ determines whether a ribosome synthesizing the proteins binds to ER or not. The protein synthesis always begins on free ribosomes. As the signal sequence emerges out of the ribosome, the large ribosomal sub-unit binds to ER membrane.
This is decided by the type of signal sequence. This is the first sorting as the ribosome binds to ER, forming rough ER. Translocation takes place into the ER while growing chain is still bound to the ribosome. This is called co-translational translocation. The process is facilitated by the signal sequence recognition mechanism.
Signal Sequence Recognition Mechanism:
It consists of a signal recognition particle (SRP) present in the cytosol. SRP binds to the signal sequence of the nascent protein as soon as it emerges out of ribosome and directs it towards the ER membrane. The binding of SRP stops further synthesis of protein chain when it is about 70 amino acids long.
This prevents it from folding. The SRP-ribosome complex binds to the SAP receptor, which is an integral membrane protein in the wall of ER and is a docking protein of the ER. At this point GTP hydrolysis hydrolyses frees SRP which is ready for the next round of directing next nascent protein of ER.
Now lengthening of nascent polypeptide restarts which enters ER lumen. Ribosome is aligned to a channel in the wall of ER. This channel is called translocon. It allows the elongating chain to enter the translocon into the ER lumen.
As the growing polypeptide chain emerges into the ER lumen, the signal sequence is cleaved by a peptide called signal peptidase. Inside the lumen, the protein may become folded into its final active form or may be carried into its secretary pathway or may be embedded in the ER membrane.
Once inside the lumen of ER, the protein undergoes folding and several modifications for which the ER lumen contains a number of enzymes and chaprone proteins. The most common processing is glycosylation which involves addition of carbohydrates to the protein chain. Glycosylation generally occurs in the ER lumen but sometimes in Golgi also.
Most oligosachharides or glycons are attached to the amino group NH3 and the proteins are called N-linked glycoprotiens e.g. oligosachharide attached to aspargine. A preformed oligosachharide is added to the proteins. This structure is Man 9 (Glc NAC)2 called high mannose structure.
This contains mannose, glucose and N-acetylglucosamine). All nascent proteins start the sorting pathway by addition of the same pre-formed oligosachharide in plants and animals. Almost all proteins that enter the secretary pathway are glycosylated.
In ER lumen, after glycosylation, many protiens are folded and stabilized by disulphide proteins bonds (-S-S-). This reaction is catalyzed by an enzyme, protein disulphide isomerase (PDI). Most of human proteins are stabilized by disulphide bonds.
Role of Golgi Complex in Protein Transportation:
The role of Golgi complex is to act as a switching center for proteins to various destinations. Both ER and Golgi apparatus are flattened cisternae. Transport of proteins from one compartment (donor) to the next one (target) is carried out in transport vesicles. The vesicles contain cargo proteins in their lumen and integral membrane proteins in their membranes.
The vesicles bud off from ER and fuse with the cis-compartment or receiving compartment of Golgi. In this process cargo proteins are delivered into the lumen of Golgi and membrane proteins become part of the membrane of the target vesicles. The proteins are glycosylated, folded, modified and sorted in ER. This process of glycosylation, modification and sorting of proteins continues in successive Golgi cisternae.
Starting from the cis-compartment to medial compartment and lastly to trans-Golgi network proteins are exported to the end target. In trans-golgi network (TGN) proteins are further sorted to be delivered to lysosomes, for secretion outside the cell and to plasma membrane according to signals present in the nascent proteins.
Transport of Proteins from Golgi to Lysosomes:
The lysosomal enzymes and lysosomal membrane proteins are synthesized in rough ER and transported to Golgi cisternae and ultimately to lysosomes. The sorting signal that directs the lysosomal enzymes from the trans- Golgi network (TGN) to lysosomes is mannose 6-phosphate (M6P). The attachment of M6P to lysosomal enzymes prevents their further modification.
Separation of M6P bearing lysosomal enzymes from other proteins takes place in TGN. The wall of TGN contains M6P receptors. These M6P receptors bind to lysosomal proteins. The vesicles containing these receptor bearing proteins bud off from TGN. These vesicles are called lysosomes. Later these vesicles fuse with vesicles which have arisen by pinacocytosis and phagocysis to form secondary lysosomes. Low pH of Lysosomes triggers the dissociation of enzymes from the receptors.
The M6P receptors are recycled back to trans-golgi network in vesicles. Lysosomes contain hydrolyzing proteolytic enzyme, which digests proteins meant for degradation. A protein named ubiquitin marks the proteins meant for destruction. Ubiquitin is present in all eukaryotic cells. This mechanism degrades only those proteins which are meant for destruction and not the proteins which are to be left alone.
The proteins meant for secretion travel to plasma membrane from trans-golgi network.
All this transportation of vesicles from the RER to the cis face of golgi to successive levels of golgi and on to their final destinations requires the high levels of specificity in targeting. Transport of vesicles to wrong destinations would lead to cellular chaos.
Targeting of Proteins to Mitochondria and Chloroplasts:
Mitochondria and chloroplasts possess their own DNA, ribosomes, mRNA and synthesize a few proteins. But most of the proteins required for mitochondria and chloroplasts are synthesized in cytosol by nuclear DNA and then imported into these organelles. Both these organelles are covered by double membranes. The proteins are translocated into these organelles after they are fully synthesized. This is known as post-translational translocation.
There are four mitochondrial locations where the proteins are targeted. These are outer membrane, inner membrane, intermembranal space and mitochondrial matrix. The proteins are released in unfolded state and they bind to a family of chaprones. These chaprones are cytosolic hsp 70 proteins (heat shock proteins) that deliver the proteins to an import receptor on the outer mitochondrial membrane.
The import receptor then slides to a site where inner and otuer mitochondrial membrane form a channel through which the unfolded protein enters into mitochondria leaving out cytosolic hsp 70 protiens. As the protein reaches the matrix, mitochondrial heat shock protein, mitochondrial hsp 70 binds to it. A protease cleaves the signal sequence.
These proteins have more than one successive N-terminal targeting signal sequence. The first signal sequence imports the protein into matrix and the second signal re-directs the protein into membranes or inter membranal space.
Mitochondria processes machinery for cellular respiration. Each membrane and each compartment of mitochondria has its unique proteins. Enzymes of electron transport chain lie in the inner membrane while most enzymes of citric acid cycle are found in the matrix.
Protein Targeting to Chloroplasts:
The newly synthesized proteins by free ribosomes are impored into chloroplasts as in mitochondria. Calvin cycle enzymes fix atmospheric CO2 into carbohydrates during photosynthesis.
Protein Targeting into Nucleus:
The nuclear envelope consists of outer and inner membranes and has inter membranous space between them. The outer membrane is continuous with ER and has ribosomes on it. Proteins for the nucleus are synthesized on free ribosomes in the cytosol and imported into nucleus through 3000-4000 nuclear pores known as nuclear pore complexes which are special gates.
The proteins that are imported into nucleus are in fully folded state and do not require any chaprones. Protiens imported into nucleus have targeting signal sequences on them which are called nuclear localization signals (NLS). Each one has 4-8 amino acids and they are internal sequences and not terminal. NLS is not cleaved from the protein. Due to this feature proteins can re-enter the nucleus whenever the nuclear envelope is lost during cell division.
The proteins embedded in different membranes may have single trans-membrane domain which is a segment of 20-25 amino acids. Other proteins may have many trans-membrane domains connected by loops on both sides of the membrane. These proteins are called multi-pass orientation proteins. In photosynthetic bacteria a protein called bacterio-rodospin spans 12-14 times across the lipid bilayer membrane of bacteria. It traps energy from sunlight and uses it to pump protons across the bacterial membrane.
The plasma membrane contains molecules other than phospholipids, primarily other lipids and proteins. The green molecules in Figure below, for example, are the lipid cholesterol. Molecules of cholesterol help the plasma membrane keep its shape. Many of the proteins in the plasma membrane assist other substances in crossing the membrane.
The plasma membranes also contain certain types of proteins. A membrane protein is a protein molecule that is attached to, or associated with, the membrane of a cell or an organelle. Membrane proteins can be put into two groups based on how the protein is associated with the membrane.
Integral membrane proteins are permanently embedded within the plasma membrane. They have a range of important functions. Such functions include channeling or transporting molecules across the membrane. Other integral proteins act as cell receptors. Integral membrane proteins can be classified according to their relationship with the bilayer:
- Transmembrane proteins span the entire plasma membrane. Transmembrane proteins are found in all types of biological membranes.
- Integral monotopic proteins are permanently attached to the membrane from only one side.
Some integral membrane proteins are responsible for cell adhesion (sticking of a cell to another cell or surface). On the outside of cell membranes and attached to some of the proteins are carbohydrate chains that act as labels that identify the cell type. Shown in Figure below are two different types of membrane proteins and associated molecules.
Peripheral membrane proteins are proteins that are only temporarily associated with the membrane. They can be easily removed, which allows them to be involved in cell signaling. Peripheral proteins can also be attached to integral membrane proteins, or they can stick into a small portion of the lipid bilayer by themselves. Peripheral membrane proteins are often associated with ion channels and transmembrane receptors. Most peripheral membrane proteins are hydrophilic.
Some of the membrane proteins make up a major transport system that moves molecules and ions through the polar phospholipid bilayer.
The Fluid Mosaic Model
In 1972 S.J. Singer and G.L. Nicolson proposed the now widely accepted Fluid Mosaic Modelof the structure of cell membranes. The model proposes that integral membrane proteins are embedded in the phospholipid bilayer, as seen in Figure above. Some of these proteins extend all the way through the bilayer, and some only partially across it. These membrane proteins act as transport proteins and receptors proteins.
Their model also proposed that the membrane behaves like a fluid, rather than a solid. The proteins and lipids of the membrane move around the membrane, much like buoys in water. Such movement causes a constant change in the "mosaic pattern" of the plasma membrane.
Extensions of the Plasma Membrane
The plasma membrane may have extensions, such as whip-like flagella or brush-like cilia. In single-celled organisms, like those shown in Figure below, the membrane extensions may help the organisms move. In multicellular organisms, the extensions have other functions. For example, the cilia on human lung cells sweep foreign particles and mucus toward the mouth and nose.
Flagella and Cilia. Cilia and flagella are extensions of the plasma membrane of many cells.