Introduction and Goals
This tutorial describes how cells communicate with each other. In a previous tutorial you learned about a very specialized case of cell communication, neurons receiving and sending chemical signals. Now you will learn about some of the more general mechanisms that cells use to receive and respond to signals. Reception of a signal occurs through the binding of a signal molecule to a specific receptor; examples will be described and their mechanisms of action explained. In addition, you will learn how the binding of signal molecules to receptors triggers intracellular changes in the receiving cell, affecting many cellular processes. You will learn how protein modification by phosphorylation and induced conformational change by protein:protein interactions are used to mediate intracellular changes in response to a signal.
By the end of this tutorial you should know:
- the meaning of paracrine and endocrine signaling
- the mechanism of intracellular nuclear receptors
- the mechanism of G-protein-linked receptors, and how they activate a G-protein
- how protein kinases and G-proteins act as molecular switches
- the role of second messengers such as cAMP, InsP3 and calcium ions
- the mechanism of tyrosine kinase receptors, and how they recruit and activate multiple proteins
- how the activity of Ras is regulated
- the general difference between cell signaling in plants and animals
A hallmark of any multicellular organism is the ability of its cells to communicate with each other. This communication, referred to as cell signaling, regulates almost all cellular processes, including growth, metabolism, gene expression and cytoskeletal organization. Cell signaling is the release of a signal molecule, a chemical message, from one cell and the detection of this signal by another cell, the target cell. The signal molecule is detected by the target cell through its binding to a specific receptor. Once the receptor has bound the signal molecule, a series of changes is initiated in the target cell that affects many cellular processes. You have already learned about a rather specialized type of cell signaling in the nervous system, chemical transmission at synapses that alter membrane potential. We will now consider cell signaling that is more widespread among many cell types. Cell signaling occurs in both animals and plants; however, signaling in plants is not as well understood, so most of the material presented in this tutorial relates to animal cells.
Signal molecules encompass a variety of molecules, including proteins, peptides and small organic molecules. There are two distinct types of cell signaling: paracrine signaling, which is mediated by signal molecules that are local mediators and affect neighboring cells; and endocrine signaling, which is mediated by signal molecules called hormones (secreted by specialized cells into the bloodstream) and affect cells distributed throughout the organism. The molecular mechanisms of paracrine and endocrine signaling are similar and depend on the molecular structure of the signal itself. Most signal molecules are extracellular and bind to receptors located in the plasma membrane of the target cell. However, some are small lipid-soluble molecules that readily diffuse across the plasma membrane of the target cell. Most notable of this type of signal molecule are the steroid hormones estrogen and testosterone.
Intracellular nuclear receptors
Lipid-soluble hormones, such as the steroid hormones, diffuse across the plasma membrane and bind to intracellular receptors termed nuclear receptors (illustrated in Figure 1). Members of this family of receptors are composed of two distinct domains, a ligand binding domain and a transcription activation domain. In the inactive state, many nuclear receptors are located in the cytoplasm, bound to inhibitor proteins. Once a lipid-soluble hormone has entered the cell it binds to the receptor, which is released from the inhibitor protein, and the receptor/hormone complex enters the nucleus. In the nucleus, the receptor/hormone complex will bind to specific regions of DNA and activate transcription of a subset of genes. The expression of these genes is the primary response of the cell to the hormone, however, these genes encode a variety of proteins that will affect many aspects of the cell's physiology.
Extracellular Receptors and Signal Transduction
Many signal molecules are too large or insoluble to pass through the plasma membrane. These signal molecules bind to specific cell surface, or extracellular, receptors in the plasma membrane of target cells. Extracellular receptorsare transmembrane proteins that have two distinct regions of activity, an extracellular ligand-binding domain and an intracellular signaling domain. There are many different members of this family of extracellular receptors, some of which will be described in detail below; however, as a class, extracellular receptors function by binding ligand on the outside of the cell and activating the receptor. Activation of the receptor results in a conformational change in the intracellular portion of the receptor, which, in turn, will recruit and activate a variety of intracellular proteins that regulate many cellular processes. Thereby, the extracellular signal is transmitted into the cell and affects change without actually entering the cell through a process referred to as signal transduction. Unlike the nuclear receptors that directly regulate transcription in the target cell, activated extracellular receptors recruit and activate a variety of proteins in a signal transduction pathway. Central to these signal transduction pathways are two classes of protein, protein kinases and GTP-binding proteins, both of which act as molecular switches to regulate the activity (either "on" or "off") of many other proteins. So the binding of a ligand to a receptor on the outside of the cell triggers receptor activation, which, in turn, recruits one or more intracellular molecular switches, and these activate (or inactivate) a host of other proteins in the cell. Ultimately this leads to changes in gene expression, metabolism and many other cellular processes. The beauty of signal transduction pathways is that they are very rapid, reversible and versatile, affecting many aspects of the cell's physiology simultaneously. The remainder of this tutorial will describe several examples of specific signal transduction pathways mediated by two distinct groups of extracellular receptors: G-protein-linked receptors and enzyme-linked receptors.
Molecular Switches: Protein Kinases and GTP-Binding Proteins
Before we can discuss specific signaling pathways, you need to understand the activity and regulation of protein kinases and GTP-binding proteins. Protein kinases are a class of enzymes that phosphorylate other proteins; they hydrolyze ATP to ADP and add a phosphate to specific amino acids (see Enzyme Kinetics and Catalysis tutorial). Phosphorylation will alter the conformation of the target protein, and in some cases, activate it, or in other cases, inactivate it. Serine/threonine protein kinases phosphorylate those two residues, tyrosine protein kinases phosphorylate the residue tyrosine, and a few protein kinases phosphorylate all three residues. Phosphorylation is a reversible modification of a protein, and there are a large number of phosphatases (enzymes that remove phosphates from proteins that are phosphorylated). Therefore, the process of phosphorylation/dephosphorylation is a molecular switch; phosphorylation by a specific protein kinase activates (turns "on") that protein, and dephosphorylation by a specific phosphatase inactivates (turns "off") that protein (see Figure 2- panel A). Protein kinases are an abundant and diverse class of enzymes. It has been estimated that in a typical eukaryotic cell there are several hundred different protein kinases, and about one-third of the total protein of the cell is phosphorylated. All protein kinases have a distinct subset of proteins that they can phosphorylate, however, some are more limited in their substrates than others. Any given protein can be phosphorylated by multiple kinases at a variety of sites. All kinases are regulated between the active and inactive state through a variety of mechanisms. Some kinases are themselves activated by phosphorylation, which initiates signal transduction cascades of one kinase activating another kinase, which, in turn, activates yet another kinase in the sequence, and so on.
GTP-binding proteins are another class of molecular switches. All GTP-binding proteins (G-proteins) are characterized by their ability to bind to and hydrolyze GTP, hence their name. G-proteins are active when bound to GTP but inactive when bound to GDP (see Figure 2- panel B). When active, G-proteins can physically interact with and activate (or in some cases, inactivate) many other proteins. When bound to GTP, G-proteins remain active only briefly. The G-protein rapidly hydrolyzes GTP to GDP, then returns to its inactive state. Although both protein kinase and G-proteins hydrolyze nucleotides (ATP and GTP, respectively), recall, G-proteins do not transfer a phosphate onto the proteins they activate. A GTP-bound G-protein activates its target protein by physical interaction and an induced conformational change.
The first signal transduction pathway that we will consider is triggered by G-protein-linked receptors. G-protein-linked receptorsare the largest group in the family of extracellular receptors found in animals, with as many as 2000 genes encoding G-protein-linked receptors in the human genome. This diverse group of receptors binds to a variety of signal molecules, including neurotransmitters (e.g. acetylcholine) and hormones (e.g. adrenaline). The signaling pathways that they initiate regulate cellular processes, including metabolism and transcription. Our senses of sight and smell are mediated by G-protein-linked receptors. A large number of olfactory G-protein-linked receptors are located in specific neurons of the nose. They bind odorant molecules and trigger a signaling pathway, which, in turn, triggers a nerve impulse along the axons to the brain. Our ability to perceive light is mediated by light-activated G-protein-linked receptors in the photoreceptors of the eye.
Although quite diverse, members of this group of extracellular receptors share some common structural features. G-protein-linked receptors are a single, transmembrane polypeptide threaded through the lipid bilayer seven times (see animation). There is an extracellular ligand-binding domain and an intracellular domain that interacts with and activates G-proteins. When the receptor is activated by binding to the ligand on the extracellular surface of the plasma membrane, it undergoes a conformational change. The activated receptor can now, in turn, activate a trimeric G-protein.
Trimeric G-proteins are composed of three subunits (alpha, beta and gamma) and are attached to the inner face of the plasma membrane through a covalent lipid modification. In the inactive state, the alpha subunit is bound to GDP and the three subunits form a complex. Upon ligand binding to the G-protein-linked receptor, the receptor is activated to bind the G-protein and induce a conformational change in the alpha subunit of the G-protein, so that GDP is exchanged for GTP and the three subunits dissociate to release the GTP-alpha subunit and the beta-gamma complex. The activated GTP-alpha subunit can induce a conformational change in several target proteins, and, in turn, activate (or inhibit) them. The alpha subunit also contains the GTPase activity, so it remains active for a brief time period, but then it hydrolyses the GTP to GDP. The GDP-bound alpha subunit reassociates with the beta-gamma complex and the G-protein returns to its inactive state, ready to be activated once more in response to ligand binding and receptor activation. There are several different activating and inhibitory G-proteins in most animal cells, some with a specific tissue distribution and others with a wide tissue distribution.
The Cyclic AMP Signaling Pathway
In many signaling pathways, including the olfactory G-protein signaling pathways, G-proteins stimulate production of cyclic AMP (cAMP), a small intracellular molecule that regulates many proteins in the cell. Cyclic AMP is synthesized from ATP by the enzyme adenylyl cyclase, which is a transmembrane protein. The GTP-bound alpha subunit of an active trimeric G-protein activates adenylyl cyclase, resulting in a rapid increase in the intracellular levels of cAMP (see previous animation). Cyclic AMP is referred to as a second messenger, with the first messenger being the ligand that binds to and activates the receptor.
A second messenger is characterized as a small intracellular molecule that is an effector of other proteins. In response to receptor activation, there is a pronounced increase in the level of second messengers, thus amplifying the signal and transmitting it into the interior of the cell. G-protein-linked receptors and trimeric G-proteins are fixed at the plasma membrane; however, the production of cAMP, which is small and diffusable, allows the signal to be broadcast to the interior of the cell, regulating the activity of cytoplasmic and nuclear proteins. In addition, the signal is amplified so that one molecule of ligand binds to its receptor. This activates several molecules of trimeric G-protein, which, in turn, activate adenylyl cyclase to synthesize many molecules of cAMP. Thus, one molecule of ligand binding to its receptor results in hundreds to thousands of molecules of cAMP becoming available in the cell.
In some neurons, including the olfactory neurons, cAMP regulates cAMP-gated ion channels to trigger an axon potential and a nerve impulse. In many other cells, however, the primary role of cAMP is allosteric regulation of the serine/threonine protein kinase cAMP-dependent kinase (protein kinase A). In its inactive state, cAMP-dependent kinase is composed of four subunits: two catalytic subunits that encode kinase activity and two regulatory subunits that inhibit kinase activity. Cyclic AMP binds to the regulatory subunits and alters their conformation, which causes them to dissociate from the complex and release the catalytic subunits (Figure 3). Active cAMP-dependent kinase can phosphorylate and regulate a variety of proteins. In muscle cells, cAMP-dependent kinase phosphorylates and activates the enzymes involved in glycogen breakdown and simultaneously inactivates the enzymes involved in glycogen synthesis. In other cells, cAMP-dependent kinase phosphorylates and regulates a DNA binding protein that regulates the transcription of a subset of genes.
The Inositol Phospholipid Signaling Pathway
Not all signaling pathways initiated by G-protein-linked receptors activate adenylyl cyclase and employ cAMP as a second messenger. In some signaling pathways, the trimeric G-proteins activate another transmembrane protein, phospholipase C. This enzyme cleaves a lipid found on the inner surface of the lipid bilayer, phosphatidylinositol 4,5-biphosphate, to generate two products: diacylglycerol and inositol 1,4,5-triphosphate (InsP3) (Figure 4). Diacylglycerol remains associated with the plasma membrane, and activates the serine/threonine protein kinase known as protein kinase C. Activation of protein kinase C also requires calcium (see below), and once the kinase is active, it will phosphorylate and regulate a variety of different proteins, including other kinases.
The second cleavage product, InsP3, is a small, water-soluble molecule that rapidly diffuses through the cytoplasm. When it reaches the endoplasmic reticulum, InsP3 binds to InsP3-gated Ca2+ channels in the ER membrane. The lumen of the smooth ER has a large store of calcium ions, and when the InsP3-gated Ca2+ channels are open, there is a large influx of calcium ions into the cytoplasm, raising the calcium concentration by 10- to 20-fold. Calcium ions act as second messengers. Protein kinase C binds to calcium, and both calcium and diacylglycerol are required for the activation of this kinase. However, many of the effects of intracellular calcium ions are mediated by calcium-binding proteins. The most common of these is^ calmodulin, which is active when it is bound to calcium. Calmodulin has no enzymatic activity. It functions by binding to and activating other enzymes (see Figure 5). Calmodulin also has indirect effects by activating serine/threonine protein kinases, the family of Ca2+/calmodulin-dependent kinases. These kinases have a broad specificity and mediate many of the effects of calcium in animal cells.
The predominant type of enzyme-linked receptors found in animals is the family of the tyrosine kinase receptors, also referred to as receptor tyrosine kinases (illustrated in Figure 6). These receptors have intrinsic kinase activity encoded by the intracellular domain, also referred to as the cytoplasmic tail. Tyrosine kinase receptors bind extracellular ligands as a dimer (a pair of identical proteins). Once ligand is bound, the receptors are activated and the cytoplasmic kinase domains are brought into close proximity where they can cross-phosphorylate on multiple tyrosines. The process by which one molecule of the receptor dimer phosphorylates its partner is referred to as autophosphorylation.
How does autophosphorylation broadcast the signal to the interior of the cell? Once the cytoplasmic tails of tyrosine kinase receptors are phosphorylated, they act as docking sites that recruit and activate many different types of signaling molecules, including a form of phospholipase C, protein kinases and G-proteins. Although the recruited proteins are not similar in overall structure or function, they all share the ability to directly, or indirectly, bind to the phosphorylated tyrosines of the activated receptors via a shared protein domain. A distinct, relatively short protein motif termed the SH2 domain, found in many unrelated signaling molecules, encodes the phosphotyrosine binding activity. Therefore, proteins that contain SH2 domains will be recruited to the activated tyrosine kinase receptors via binding to the phosphotyrosines, and, in turn, will be activated either by phosphorylation or simply by binding. For instance, phospholipase C is recruited to the activated receptors by the binding of its SH2 domian to the phosphotyrosines of the receptors. Upon binding, it is activated to generate diacylglycerol and InsP3and to trigger the inositol phospholipid signaling pathway. An activated pair of tyrosine kinase receptors can recruit many different proteins simultaneously, each protein binding to a specific phosphotyrosine in the cytoplasmic tails of the receptors.
Among the proteins that are commonly recruited to activate tyrosine kinase receptors is the G-protein Ras. Ras is a member of a large family of monomeric (single subunit) G-proteins. Ras has a covalently attached lipid group and is associated with the intracellular face of the lipid bilayer. Similar to trimeric G-proteins, Ras is active when bound to GTP and inactive when bound to GDP. It is also a GTPase and it hydrolyzes GTP to inactivate itself. There is reciprocal regulation of Ras GTPase activity by GTPase-activating proteins (GAPs) that promote hydrolysis of GTP, and guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP (see Figure 7). Ras is recruited to the activated tyrosine kinase receptors indirectly through its association with a particular GEF known as SOS. SOS also does not bind phosphotyrosines, and is itself recruited to activated tyrosine kinase receptors via a small adapter protein known as GRB2, which contains an SH2 domain and can bind phosphotyrosine. To recapitulate, activated tyrosine kinase receptors recruit the adapter protein GRB2 via its SH2 domain that binds phosphotyrosines, and GRB2 is bound to SOS. Once SOS is recruited to the receptor complex, it can activate Ras by promoting the release of GDP and the binding of GTP. Ras-GTP will activate a variety of proteins by induced conformational change. Ultimately, activated Ras leads to changes in the pattern of gene expression. This effect is largely mediated by Ras activation of a protein kinase pathway referred to as the mitogen-activated protein (MAP) kinasepathway. Ras activates a kinase, which, in turn, phosphorylates and activates another kinase that activates the last kinase is this cascade (see Figure 8). MAP kinase is the last in the cascade, and when it is activated by phosphorylation, it will phosphorylate a number of nuclear proteins that regulate transcription.
Integration of Signaling Pathways
You have learned about several signaling pathways involving G-protein-linked receptors or enzyme-linked receptors, however, there are many more signaling pathways that have not been discussed herein. Signal transduction is a rapidly growing field of biology, and new signaling pathways are revealed regularly. Unfortunately there is not enough time to explore all of the different signaling pathways in an introductory course. This tutorial presents a few examples that are well-established paradigms for how cells integrate and respond to extracellular signals.
Although each pathway has been presented as distinct and separate, this is not true in a cell. Any given animal cell responds to multiple signals simultaneously. Some signals trigger G-protein-linked receptor pathways, and others trigger enzyme-linked receptor pathways. There is also cross talk between pathways. For example, recall that phospholipase C and the inositol phospholipid signaling pathway can be activated via G-protein-linked receptors and tyrosine kinase receptors.
Sometimes distinct signals can act synergistically to reinforce the same changes in a cell, whereas other times they can act antagonistically and have opposing effects in a cell. For instance, a cell may receive two signals that both induce proliferation, or one signal that is proliferative and the other a cue to stop dividing. Signals can also result in different outcomes in different cell types, depending on the receptors and other signaling molecules expressed in the cell. In fact, there is a whole network of signaling pathways in cells that run parallel, that intersect, and that act either synergistically or antagonistically.
Cell Signaling in Plants
The signaling pathways that have been described so far are specific for animal cells. Plant cells are capable of cell signaling and do employ signal transduction pathways, although they appear to be quite distinct. Also, signaling pathways in plants have not been as extensively studied as those in animals and until recently have been poorly understood. The recent determination of the genomes of several plant species has expanded our knowledge of the type of signaling molecules plants possess. There are no known intracellular nuclear receptors in plants. There are a few putative G-protein-linked receptors and a relatively small number of trimeric G-proteins, but it remains unclear if cAMP is involved in signaling pathways in plants. The predominant type of receptor in plants is the enzyme-linked receptor, specifically the serine/threonine kinase receptors. This type of receptor binds to ligands and has intrinsic serine/threonine kinase activity. Mechanistically, they function in a manner similar to the tyrosine kinase receptors in animals, although the proteins and their specific interactions are distinct. In plants, the serine/threonine kinase receptors autophosphorylate and recruit a variety of signaling proteins via binding to phosphoserine or phosphothreonine, whereupon they are phosphorylated and activated. Interestingly, there do not appear to be any tyrosine kinase receptors or Ras monomeric G-proteins in plants. However, there is good evidence for the MAP kinase signaling pathway.
Cells communicate through the actions of chemical signals. These can be locally acting paracrine signals or globally acting endocrine signals that circulate in the bloodstream and affect cells far from the source. The signal itself can be a protein, peptide or small organic molecule. All signals bind to a specific receptor and trigger a set of intracellular events that alter the state of the cell. Lipid-soluble signals, such as the sex hormones, pass directly through the plasma membrane and bind to intracellular nuclear receptors that are in an inactive state in the cytoplasm. When the hormone binds to the receptor, the complex enters the nucleus and directly binds to DNA to regulate gene expression. Extracellular signals are more common. These molecules cannot enter the cell by passive diffusion; instead, they bind to receptors on the cell surface. Once the extracellular receptor is bound, it is activated and will recruit and activate a variety of proteins, resulting in a signal transduction pathway.
Two distinct groups of extracellular receptors that are common in animals are G-protein-linked receptors and enzyme-linked receptors. G-protein-linked receptors bind extracellular ligand (a messenger) and activate an intracellular trimeric G-protein by stimulating the alpha subunit to bind GTP, in favor of GDP, and dissociate from the beta-gamma subunits. The GTP-bound alpha subunit is active for a period of time and then hydrolyzes the GTP to return to the inactive GDP-bound state. During the period that the G-protein is active, it will trigger the release of second messengers through a variety of mechanisms. Some G-proteins activate adenylyl cyclase to synthesize cAMP. cAMP, in turn, activates the cAMP-dependent kinases. Other G-proteins stimulate phospholipase C, an enzyme that generates InsP3 and diacylglycerol. Diaclyglycerol and calcium activate protein kinase C. InsP3 triggers the opening of InsP3-gated calcium channels in the ER and results in a rapid influx of calcium ions. Calcium ions have multiple effects in a cell. Many of these are mediated by the calcium-binding protein calmodulin, which, in turn, activates other proteins (including the Ca^+2^/calmodulin-dependent kinases).
Tyrosine kinase receptors bind ligand as a dimer and undergo autophosphorylation of specific tyrosines in the cytoplasmic tails of the proteins. Once phosphorylated, the receptor complex recruits a variety of different signaling molecules. Recruitment is mediated by binding to the phosphotyrosines via a conserved protein domain, the SH2 domain, within the signaling molecules, or within adapter proteins associated with the signaling molecules. Among the proteins recruited to an activated tyrosine kinase receptor is the G-protein Ras. Ras is recruited and activated by the guanine exchange factor SOS, which is itself recruited to the activated receptor by the adapter protein GRB2. Once RAS is delivered to the activated receptor complex, it will activate the MAP kinase cascade, resulting in active MAP kinase that will phosphorylate a variety of transcription factors.
Plants have distinct signaling pathways that share some common features with the signaling pathways in animals. The predominant type of receptor in plants is the serine/threonine kinase receptor. These receptors undergo autophosphorylation when active and recruit other proteins via binding to phosphoserines and phosphothreonines.