Once a hormone or other extracellular "first messenger" binds to its receptor on a cell surface, a signal is transmitted or "transduced" to the cell interior, thus setting in motion a series of events that produce a biological response. Such "events" include both chemical reactions and physical reactions, such as conformational changes in protein molecules. The biological responses include cell differentiation, altered metabolism and cell growth and division.
We will look at 3 signaling pathways that share many of the same intracellular events. Each pathway is characterized by its receptor and by the cascade of intracellular events that lead to a biological response. Each receptor has an extracellular, transmembrane, and intracellular component and the binding of a ligand to the receptor represents the "primary message". The term "secondary messenger" is used for those mediators that diffuse from one part of the intracellular space to its spatially removed target. Among these secondary messengers are :
gases (NO in eukaryoyes, ethylene in plants)
Each pathway can also be thought of as having 3 domains: membrane-bound, cytoplasmic and nucleus-bound.
G Protein Coupled Receptors and the Adenylate Cyclase Signaling System
Many integral membrane glycoprotein membranes share a seven transmembrane alpha-helix motif. The ß-adrenergic receptor, whose natural ligands are epinephrine and norepinephrine, is an example of such receptors. The binding of a ligand to the receptor presumably initiates a conformational change in the membrane protein that is transmitted to the cell interior. This physical reaction can then facilitate other physical or chemical reactions. You have already studied ion channels, whose conformational change in response to receptor stimulation causes transmembrane ion flow. It's the same idea.
The transduction of the signals from many external messengers, including epinephrine and norepinephrine, involves intracellular heterotrimeric G-proteins, which are bound to the inner cell (plasma) membrane, a secondary messenger system, involving cyclic AMP, and a target response. Let us look at each of these in detail:
As the name implies, these proteins are trimers, consisting of ,ß, and subunits.They are bound to the inner membrane at the and subunits and the subunit can bind the guanine nucleotides, GTP and GDP. G-proteins are involved in vision, smell, cognition, hormone secretion and muscle contraction in humans, and in mating in yeast. There are more than 100 receptors (not including odor receptors) that utilize G-proteins, and there are at least 20 members of the G-protein family, with each member having its characteristic ,ß, and subunit.While the subunit is different for each G-protein, the beta-gamma pair can be the same. However, all of the G-proteins share a similar structure. We will specifically look at how G-proteins transmit the signal from the intracellular part of the receptor to the effector, adenylyl cyclase, which is an inner membrane-bound enzyme that regulates the production of the secondary messenger, adenylyl cyclase. Other effectors that are G-protein-dependent include additional enzymes, like cyclic GMP phosphodiesterase, and transmembrane ion channels.
In its resting conformation, the G-protein consists of a complex of the 3 subunit chains and a GDP molecule bound to the alpha subunit. The alpha subunit in close proximity to the intracellular part of the transmembrane receptor and, when a ligand binds to the receptor, the change in its conformation causes it to bind to the G-protein at the alpha subunit. This results in an exchange of bound GDP for GTP, which is more abundant in the cell than GDP. GTP causes a conformational change in the alpha subunit, thus "activating" it so that the alpha subunit dissociates from the beta-gamma pair. The alpha subunit diffuses along the membrane until it binds to an effector, thereby activating it. The alpha subunit is also a GTPase, so the signal transduction is regulated at this level by hydrolysis of GTP to GDP and inorganic phosphate.Such hydrolysis can occur spontaneously or upon interaction with a GTPase activating protien, "GAP". The GDP-alpha subunit complex then binds to the beta-gamma complex to reform the original trimeric protein.
Since the stimulation of the external receptor can activate a number of G-proteins, signal amplification can occur. While this is a desired response in many instances, control at this level is needed to modulate it. G-proteins, then, are nano-switches when they turn on the effector by binding of the alpha subunit and turning it off when the GTP is hydrolyzed. The duration of production of secondary messenger, like cyclic AMP, is determined by the rate of hydrolysis. In this sense, the G-protein acts as a nano-timer.
Although there is controversy over the role of the beta-gamma subunits in modulation of signals, it is likely that there are both inhibitory and stimulatory effects. If different receptors act on the same G-protein, or if different G-proteins act on the same effector, the potential exists for a "graded" response to an extracellular signal. If the same receptor acts on many G-proteins, or if one G-protein acts on many effectors, then there may be many simultaneous responses to the primary messenger.
The following is a summary of various physiologic effects that are mediated by G-proteins. It appears in the article entitled "G Proteins" (Sci. Am.,July 1992,pages 56 - 65) which was authored by Linder and Gilman.
As we have seen, Gs proteins activate the membrane-bound effector, adenylyl cyclase. This enzyme catalyzes the synthesis of cyclic AMP:
ATP cAMP + PPi
Because this molecule is freely diffusing through the cytoplasm, it is a "secondary messenger". The reverse reaction, the formation of ATP from cAMP and pyrophosphate, is catalyzed by a specific phosphodiesterase. cAMP is involved in a number of physiologic processes, as indicated in the above chart. For the breakdown of glycogen, stimulation of the ß-adrenergic receptor involves activation of adenylyl cyclase and synthesis of cyclic AMP. The activity of cAMP-dependent protein kinase (cAPK) requires cAMP in order to phosphorylate Ser and Thr residues on other cellular proteins. Glycogen phosphorylase is activated by cAPK, making glucose-6-phosphate available for glycolysis.
Adenylyl cyclase activity is regulated at a number of levels, including modulation of GTPase activity of Ga, phosphodiesterase activity, and protein phosphatases. Inhibitory G proteins, Gi , are analogous to the stimulatory G proteins, Gs , except for the exchange of GTP by GDP by the a-subunit and the subsequent inhibitory action of Gia on adenylyl cyclase.
Signaling systems that rely upon G proteins are implicated in several disease states, including cholera and pertussis. In cholera, a toxin secreted by Vibrio cholerae enters intestinal cells where it interferes with the hydrolysis of GTP to GDP on the alpha subunit of the G protein, thus impeding the autoregulation of GTP activity. The result is excessive intracellular cAMP accumulation, causing secretion of electrolytes and water into the gut lumen, which causes an osmotic diarrhea with subsequent dehydration and often death. In pertussis, a toxin produced by Bordetella pertussis prevents the inhibitory receptor, Ri , from activating Gi , again causing overstimulation of adenylyl cyclase.
Mutations of G proteins are implicated in the development of some cancers. Referring again to the table of physiologic effects mediated by G proteins, one can speculate about the possible roles of these proteins in other disease states.
Receptor Tyrosine Kinases
Most of the activities in cells are controlled by kinases and phosphatases. The intracellular C-terminal domains of many receptors have tyrosine kinase activity. Such receptors are usually monomers in their unliganded states, and contain only a single transmembrane segment. Ligand binding to these receptors stimulates tyrosine kinase catalytic activity in the intracellular domain of the receptor, and such intracellular protein phosphorylation events are now well-established as a means of transmembrane signal transduction. Structurally, though, it is unlikely that the signal from bound receptor to the kinase domain is mediated by a conformational change, as there is only a single transmembrane segment. Rather, it has been determined that ligand induced dimerization is the mechanism through which the receptor PTKs are activated. This dimerization brings the tyrosine kinase catalytic domain on each receptor into close enough arrangement so that each kinase can phosphorylate Tyr residues in the other's tyrosine kinase domain. Such activated catalytic domains can then phosphorylate tyrosines outside of the catalytic domains, which can then modify other intracytoplasmic proteins, either by phosphorylation or by other means.
Many proteins that bind to the autophosphorylated receptors have what are known as Src homology 2 domains (SH2) which have a high affinity for phosphorylated Tyr residues. At the bottom of a cleft lined by basic residues in the SH2 domain is a positively charged Arg residue that interacts with two phosphate oxygens on the negatively charged phospho-Tyr via hydrogen bonds, leading either to a direct alteration of the protein's activity or to its own phosphorylation. While all SH2 domains bind to phospho-Tyr, they have virtually no affinity for unphosphorylated Tyrs. The specificity of different SH2s is a result of their recognition of different sequences of 4 or 5 residues that lie next to the phosphorylated tyrosine in the C-terminal direction. The control mechanism for this signal is a "switch" provided by protein tyrosine phosphatases, causing dephosphorylation of the phospho-Tyr residues. Some of the PTB domains also bind to target proteins in a phosphorylation-dependent manner.
Src-homology-3 (SH3) and pleckstrin homology (PH) are related to SH2 in that all three have similar 50-100 amino acid length sequences that fold into domains which function independently of surrounding sequences. SH2 recognizes short peptide motifs containing a phosphoTyr residue,while SH3 recognizes motifs containing one or more Pro residues. PH domains are probably associated with phospholipids, and may promote association of signalling proteins with membranes. The high affinity and high specificity of binding of SH2 and SH3 domains are conferred by variable amino acid sequences surrounding the domains. The 3 domains are often found in the same polypeptide, in which case the SH2 domain is involved in the tyrosine kinase signal pathway while the other two provide pathway diversity downstream of PTKs, as well as helping in the overall regulation of the signalling process.
Proteins exhibiting the SH2 homology are involved in various biochemical pathways involving:
activation of Ras-like GTPases
The next step in the signaling pathway involves activation of an inner membrane-bound monomeric G protein known as Ras, which initiates a series of kinase reactions that ultimately carry the signal to the transcriptional apparatus of the nucleus. Ras, being a G protein, is activated when its bound GDP in the resting state is replaced by GTP. It, too, has GTPase activity, but the half life is too slow to allow for effective regulation of a signal. Another GTPase activating protein, GAP, increases the rate of GTP hydrolysis by Ras. A "kinase cascade" ensues, involving Raf (a Ser/Thr kinase), MAP kinase kinase (also known as MEK, which is both a Tyr kinase and Ser/Thr kinase)), and a family of proteins known as MAPKs or ERKs.
The activated MAPKs diffuse to the nucleus where they can activate transcription factors, also by phosphorylating them. Many extracellular growth factors bind to RTKs , thus regulating cell growth and differentiation.
Variations on the theme of transmembrane phosphorylation as the first step in signal transduction include protein serine kinases in plants and vertebtrates, and the formation of "binary" receptors by the binding of an intracellular protein with PTK activity to the intracellular domain of a receptor without PTK activity, like the cytokine receptors.
It has become apparent that many of the interactions between proteins in a signaling pathway are "modular", rather than enzymatic. In such instances, the chemical activity of the protein domains is not altered by binding to phosphorylated receptors. Rather, the signal is transduced through a series of linked modules. For example, the interaction between the membrane-bound Ras protein and the activated RTK for platelet-derived growth factor, occurs via a linkage of a protein called Grb2, which consists of an SH2 domain flanked by 2 SH3 domains, and an enzyme-containing protein called Sos. It is the Sos protein that activates the Ras protein enzymatically. The Grb2 just acts as a mechanical adaptor. Furthermore, the scaffolding seen in some signal pathways can serve to assure that the right kinase or phosphatase acts on the appropriate target protein.
The Insulin Receptor
This receptor is a transmembrane glycoprotein that has intrinsic tyrosine kinase activity, but it is a dimer in its unliganded state. Each polypeptide chain of the dimer has an a and b subunit, with the a subunits being extracellular and the b subunits spanning the membrane and having both an extracellular and intracellular component. The a subunits are connected by a disulfide bond and each a subunit is also linked to a b subunit by a disulfide bond. The insulin binding site is part of the a subunit. The Tyr kinase domain is part of the intracellular component.
Insulin binds to the a subunit(s), initiating a signal which is mediated by conformational change in the intracellular domains. Autophosphorylation occurs when the binding of insulin to the a subunit of one ab subunit phosphorylates its adjacent b subunit,thereby increasing tyrosine kinase activity in the cytosol. There are a number of Tyr residues that can be phosphorylated, including 3 that are in a regulatory region (Tyrs 1146,1150 and 1151). Maximum biological activity occurs when all 3 regulatory Tyrs are phosphorylated, with a progressive reduction of activity as the number of stimulated regulatory sites decreases. There is a direct relationship between the serum concentration of insulin and the level of tyrosine kinase activity.
There are also some Ser and Thr phosphorylation sites on the insulin receptor, and these are phosphorylated in the basal state and also in response to insulin. Some of these are inhibitory to tyrosine kinase activity, representing another control mechanism (two others are autophosphorylation and ligand binding) in the transduction of signals.
Although there is no direct association with SH2- containing proteins, other proteins (e.g.,insulin receptor substrate 1) are phosphorylated and these, in turn, can bind to SH2-containing proteins. IRS-1 is at the proximal end of a phosphorylation cascade that ultimately affects fatty acid and glycogen metabolism. The two other members of the insulin receptor family are IGF-1 and the "insulin receptor-related receptor". There has not been a ligand identified for the latter receptor. There is about 80% sequence homology in the kinase domain but low sequence homology in the extracellular domain.
Between one and two insulin molecules are bound per receptor, and there is negative cooperativity between the sites. An unoccupied a receptor inhibits the tyrosine kinase activity of its bound b subunit. Removal of the a subunit removes the inhibition.
The mechanism of signal transduction for most other autophosphorylated tyrosine residues involves the binding of SH2-proteins, which in turn is dictated by the amino acid sequence in the area surrounding the autophosphorylation site. The RTKs EGF and PDGF, require such associations. The modular nature of the combination of such proteins to such RTK determines the specific biologic response. It is this modularity that allows for the wide spectrum of potential responses that might be needed in the stimulated cell.
Allthough the insulin family of receptors differs from many other RTKs in not binding to SH2-proteins, there is similarity in their ligand response and regulation. In an experiment in which an EGF binding domain is linked to an insulin receptor kinase domain, the receptor kinase is stimulated by EGF.
IRS1 is the principal substrate for the insulin receptor, and it is probably essential for all of insulin's biological responses.. There are 21 potential Tyr phosphorylation sites and over 30 potential Ser/Thr phosphorylation sites. At least 8 of the Tyrs are phosphorylated when the insulin receptor is activated. A large number of signaling pathways are potentially mediated through the activated insulin receptor in complex with the phosphorylated IRS1 molecule. The following cartoon shows this network, and appears in an article by White and Kahn in the Journal of Biological Chemistry, Vol. 269, No. 1, pp. 1-4, January 7,1994.