The refined rhodopsin structure is from Ref. See Refs. The rate-limiting step in G protein activation is the release of GDP from the nucleotide-binding pocket. Higashijima et al. GDP release is greatly facilitated by receptor activation of the G protein Iiri et al. Posner et al. For a thorough review of specific sites on heptahelical receptors, which direct receptor-G protein coupling specificity, the reader is referred to Refs.
Many examples of mutations in this region that alter receptor-G protein specificity have been also reported 36 — In addition, several investigators have generated sequence-specific C-terminal peptides or antibodies targeting the C-terminal domain to study receptor-G protein interaction.
Instead, sequence-specific C-terminal synthetic peptides either stabilize the active agonist-bound form of the receptor mimicking the G protein 40 — 42 or serve as competitive inhibitors of receptor-G protein interface Although blocking peptides are commonly interpreted as evidence of a direct receptor-G protein contact site, peptides may also stabilize or disrupt regions of the protein that transmit conformational changes to the guanine nucleotide binding motif and thereby indirectly affect receptor-mediated G protein activation.
The C terminus is not the only region directing receptor-G protein interactions. As suggested by Blahos et al. A novel class of signaling proteins, termed AGS proteins, has been identified 55 , AGS proteins activate heterotrimeric G proteins independently of receptor activation.
The mechanism for AGS activation differs among members of this family. AGS3 contains a G protein-regulatory motif. The physiological role of these proteins in vivo remains to be determined, but one possible role for these proteins may be in the regulation of G proteins that do not reside near the plasma membrane and cannot be activated directly by receptors, e. Little is known about the role of this pool of G proteins, and the discovery of AGS proteins may stimulate research into a new dimension of heterotrimeric G protein signaling.
Traditionally, the extracellular surface and transmembrane domains of G protein-coupled receptors have served as a target for the development of drugs that can selectively activate or inactivate specific cellular pathways. However, some receptor isoforms, such as the dopamine D 2L and D 2S receptors, and the D 4 receptor variants differ only on the intracellular surface of the protein 59 , 60 and cannot be readily distinguished by targeting the ligand-binding site.
Moreover, many receptors promiscuously couple to several G protein subtypes in what may be a tissue- or cell-specific phenomenon. Therefore, additional therapeutic targets will certainly be required to more specifically influence intracellular signaling events.
One avenue being explored by our laboratory and others is the use of peptide inhibitors that target the receptor-G protein interface 43 , 61 , In the short term, these peptides may provide useful tools for exploring specificity of G protein-mediated signaling. The delivery of peptide inhibitors represents a challenge to the therapeutic use of these tools.
Possible delivery systems include the use of inducible retroviral minigene vectors 64 , incorporation of peptides into liposomes 65 , or the fusion of peptides to a viral peptide sequence that carries the C-terminal peptide into the cell Alternatively, peptidomimetics may prove to be more stable and bioavailable. Selective targeting to specific organs is likely to prove beneficial, because Akhter et al. Despite the significant hurdles, targeting the receptor-G protein interface will clarify the complex coordination of players in signaling cascades and may prove therapeutically useful in the future.
The molecular basis for this divergence has not been completely elucidated. Sunahara et al. Using a yeast-two-hybrid screen, Jordan et al. However, receptor-mediated activation of these proteins was not demonstrated. These findings reveal a novel mechanism of G protein function that is dependent on GDP-liganded G proteins. However, the generalization of these results to other G q family members remains to be determined. GIRK, G protein-activated inwardly rectifying potassium channel;?
Isolation of the structural features responsible for effector variation remains to be completely determined. Recently, Mirshahi et al. Signaling specificity could be brought about by factors such as discrete subcellular localization of effectors, compartmentalization of scaffolding components, and cell type-specific expression of signaling molecules The formation of signaling networks that bring together specific receptors, G proteins, regulatory proteins, enzymes, and substrates is a hot area of research and will likely reveal key factors regulating signaling specificity.
These effector interactions can be independent, synergistic, or antagonistic. These novel effector interactions expand the role of G proteins in the regulation of various cellular processes and are briefly discussed below.
However, the mechanism responsible for this exclusivity remains to be elucidated. Using a yeast-two-hybrid screen with the protein kinase KSR-1, Bell et al. This Arg residue plays a key role in GTP hydrolysis. The mechanisms responsible for variations in GTP hydrolysis rates have not been studied in detail.
Some of these key studies are discussed below. For the interested reader, detailed descriptions of the mechanism of GTP hydrolysis can be found elsewhere 2 , 9.
Therefore, speculation mounted that, in vivo , an additional protein was rapidly terminating signal transduction, returning the system to an agonist-responsive state. Most recently, Scholich et al. To date, more than 30 mammalian RGS proteins have been identified , — , each containing 23 conserved hydrophobic residues at the core of the RGS domain , , , However, in vivo this is not the case.
Our laboratory and others , have demonstrated that in native retinal preparations, RGS9 requires effector activation for the full expression of RGS GAP activity. These studies suggest that in vivo the noncatalytic domains regulate RGS GAP activity through interactions with cellular factors.
Noncatalytic domains of RGS proteins have also been suggested to mediate signal transduction pathway specificity and subcellular targeting of RGS proteins , For example, Ras GAP inserts a catalytic Arg residue into the active site that participates in the hydrolysis step 2. However, this Arg finger is provided by the helical domain in heterotrimeric G proteins and mediates intrinsic GTP hydrolysis as discussed above 2.
The mechanism for effector-mediated GAP activity has not been clearly delineated. By analogy, effector-mediated GAP activity may also occur through a similar stabilizing mechanism. These studies suggest that RGS proteins may be regulated through their participation in a signal transduction complex that may include receptors and effectors and may be localized near the plasma membrane.
A similar suggestion was proposed by Chidiac and Ross Further in-depth discussion of RGS proteins can be found in one of several reviews on this topic — , , N-myristoylation results from cotranslational addition of the saturated carbon fatty acid myristate to a Gly residue at the second position after the removal of the initiating Met by the enzyme methionine amino-peptidase A stable amide bond links the myristate to the protein.
Hence, this myristoylation is essentially an irreversible modification. Palmitoylation of proteins results from the esterification of Cys thiol groups by palmitate. Due to its unstable character, palmitoylation is readily reversible and subject to regulation , As yet, palmitoylation cannot be accurately predicted based on primary sequence.
However, palmitoylation occurs frequently in proximity to other lipid modifications such as myristoylation or prenylation. One clear function of fatty acid acylation is to serve as a hydrophobic membrane anchor. Removal of the palmitoylation site while preserving myristoylation results in a partial shift in localization from the membrane to the cytoplasm — Myristoylation serves as the initial signal bringing the protein to the membrane, and palmitoylation is the second signal that further secures this interaction.
In addition, palmitoylation may specifically target G proteins to the plasma membrane rather than to intracellular organelle membranes , , Because most studies investigating the role of palmitoylation have relied on mutating Cys residues, further studies are needed to determine whether the significance of palmitoylation itself has been overestimated thus far.
Indeed, a paper by Fishburn et al. Lipid modifications also regulate protein-protein interactions. Thus, the palmitoylation state of G proteins can affect their ability to serve as signaling molecules.
As part of a feedback mechanism, palmitate turnover can also be regulated by receptor activity , These studies also provide a framework for conducting structural, functional, and biochemical experiments that can extend our understanding of G proteins along with their various signaling partners. Because only a few G proteins have been crystallized to date see Table 3 , interpretations and conclusions from these structures may not reflect the full complexity of subunit combinations.
Moreover, the static nature of such structures may actually limit our understanding of the dynamic nature of G protein signaling. To more accurately assess G protein interactions with receptors, effectors, and regulators of G protein signaling, it will be necessary to take advantage of new techniques that can provide insights into the complex nature of G protein activation.
A few of these techniques are described below. Fluorescence spectroscopic techniques continue to play an important role in determination of G protein conformational changes. In particular, fluorescence resonance energy transfer provides a real-time measurement of activation, deactivation, and protein-protein interactions under basal and stimulated conditions. Fluorescence resonance energy transfer involves attachment of different fluorescent donor and acceptor probes at known residues.
Changes in tertiary structure as a result of binding or activation, which result in the donor fluorophore coming into close proximity to the acceptor fluorophore, result in a quenching of donor emission and a simultaneous increase in acceptor emission as energy is transferred.
This can be measured as a ratio between donor and acceptor emission in specific timed intervals, resulting in a real-time measurement of dynamic changes in protein conformation that is both sensitive and specific to labeled regions of the proteins. In conjunction with stopped-flow fluorescence measurements, the kinetics of the binding reaction can also be determined. Spin labeling can also be used to examine changes in protein conformation in real time.
This technique requires introduction of a nitroxide side chain at specific residues and electron paramagnetic resonance signal from the nitroxide spin label can detect and report subtle changes in its local environment. It is possible to determine changes in solvent accessibility, dynamics, and intermolecular distances of side chains in solution in real time, yielding information about the time scale and magnitude of structural changes in the labeled region of the protein.
Changes can be measured on a millisecond time scale. Farrens et al. This suggests some type of crystallization artifact, leaving a question as to the relevance of the N terminus present in this 2. Another powerful technique for measuring protein-protein interactions in real time is surface plasmon resonance.
This technique measures changes in refractive index on the surface of a chemically modified sensor chip as a binding event occurs. The resultant binding curve allows for a quantitative measure of affinity of the binding interaction. Figler et al. Current advances include development of methods to immobilize vesicles to a sensor chip derivatized with lipophilic alkyl chains, thus anchoring intact vesicles and providing a physical and chemical environment similar to that of cell membranes, which can be used to measure protein-protein interactions of membrane-associated proteins Computational approaches such as structure prediction and three-dimensional modeling and mathematical techniques such as monte-carlo simulations all provide valuable insights into G protein signaling.
More importantly, they are valuable tools that serve to direct further biochemical and functional experiments. These approaches, combined with genetics, can be used to define and examine key components of the signaling pathway, which will both broaden our understanding of the complex nature of G protein signaling and lead to new questions for further investigations.
Structural and functional aspects of heterotrimeric G proteins, their binding partners, and the signaling networks in which they participate are the subjects of intense investigation, and dramatic progress has been made in recent years.
The next frontier is to understand how signaling pathways interact with each other to form signaling networks Cells are bombarded with a multiplicity of ligands, and the cellular response is somehow integrated based on all its responses. The experimental approaches to this problem are beginning to be available, but are in their infancy. Certainly, many new approaches to these issues of complexity in cellular signaling will need to be pioneered, and will surely lead to new insights.
We apologize to all whose work could not be recognized because of page restrictions. We are grateful to Ms. Simona Ioffe and Ms. Anjela Papassavas for their assistance in gathering information for this review. Hamm HE The many faces of G protein signaling. J Biol Chem : — Google Scholar.
Sprang SR G protein mechanisms: insights from structural analysis. Annu Rev Biochem 66 : — Nature : — Science : — Cell 83 : — Genomics 62 : — Biochemistry 34 : — J Biol Chem 26 : — Annu Rev Neurosci 9 : 87 — Marin EP , Krishna AG , Sakmar TP Rapid activation of transducin by mutations distant from the nucleotide-binding site: evidence for a mechanistic model of receptor-catalyzed nucleotide exchange by G proteins.
J Cell Biochem 76 : — Biochemistry 38 : — Biochemistry 39 : — Bourne HR How receptors talk to trimeric G proteins. Curr Opin Cell Biol 9 : — Curr Opin Biotechnol 8 : — Nature : 35 — Mol Cell Endocrinol : — Mol Neurobiol 19 : — Wess J G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition.
Cell 73 : — J Biol Chem : 23 — A direct evaluation of selectivity in receptor G protein coupling. Mol Pharmacol 50 : — Neuron 3 : — Mol Pharmacol 47 : — Mazzoni MR , Hamm HE Interaction of transducin with light-activated rhodopsin protects it from proteolytic digestion by trypsin.
Nat Biotechnol 17 : — Cell 91 : — FEBS Lett : 8 — Adv Pharmacol 42 : — Walther W , Stein U Targeted vectors for gene therapy of cancer and retroviral infections. Mol Biotechnol 6 : — J Pharm Sci 87 : — Endogenous 5-HT 2C receptors in choroid plexus epithelial cells. J Cell Biochem 31 : — Taussig R , Zimmermann G Type-specific regulation of mammalian adenylyl cyclases by G protein pathways.
Adv Sec Mess Phosphoprot Res 32 : 81 — Cell 68 : — J Cell Biol : — FEBS Lett : — Activation of the members of the Rho family is via GTP binding.
Activation of Rho-mediated signaling pathways can be indirectly mediated by GPCRs, integrins, or receptor tyrosine kinases. Rho kinase and ROCK are stimulated due to the cleavage of activated caspase 3 during apoptosis. These two proteins have an identical number of amino acids and are functionally almost identical.
However, the tissue distribution of the two isoforms is distinct This domain is conserved between all members of the G protein superfamily 6. The GTPase domain contains three switch regions, which are flexible loops that change conformation when bound with GTP.
Thus, EGFR transactivation may occur through both pertussis toxin-sensitive and -insensitive pathways. The independent signaling pathways have made it difficult to envisage a single potential therapeutic target for the inhibition of all GPCR transactivation signaling G proteins in cells can be effectively knocked down utilizing a molecular approach and this has allowed for detailed studies of the function of various G proteins and their interactions. Classic experimental approaches assume that the intervention is specific and does not alter other parameters that would impact on the experimental result of the index intervention.
This is not always the situation and is certainly not the reliable paradigm in the case of the regulation of G proteins. Results of knock down interventions are also not always reciprocal — the knock down of one G protein may lead to a compensatory increase in another G protein family member but the reverse or reciprocal phenomenon may not occur A consequence of the role of post-translational regulation on stability and the cellular levels of G proteins is that the relationship between mRNA and protein levels may be perturbed.
Higher mRNA levels may lead to increased expression of the G protein, but if it is orphaned and free the protein may be degraded providing for high level of mRNA and in the presence of low levels of protein. Molecular approaches to the up- and down-regulation of target proteins are a major component of modern mechanistic studies of cell biology.
However, as exemplified above, alteration of target protein levels may result in compensatory changes in other components of a system and the perturbation might not provide the expected result. Pharmacological approaches nullify the activity or function of a target protein without in most cases altering the level of the target protein. Such studies are currently underway in our laboratory. The compound known as YM, a cyclic depsipeptide isolated from the Chromobacterium sp.
When bound to GDP, the non-polar side chains of YM form hydrogen bonds with the Switch I region; however, this is a conformation that cannot be maintained when bound with GTP Aside from antiplatelet activity, by electrically inducing carotid artery thrombosis in rodents, YM was also shown to have antithrombotic and thrombolytic effects YM was discovered and developed by Yamaguichi Pharmaceuticals, Japan; Yamaguichi subsequently became the property of Astellas Pharmaceuticals, Japan.
YM was made available to researchers 10 years ago and a small number of interesting studies were published. As discussed above, molecular approaches in this area, for example, G protein knock down can lead to rebound increases in other G proteins with unexpected results.
The blood pressure lowering effect was attributed to the ability of UBO-QIC to partially mediate nitric oxide release from endothelial cells and inhibit calcium migration caused by voltage-dependent and receptor-operated channels In , Tanski et al. These compounds have been widely used 47 — 51 although they are not considered to be especially useful and specific agents. Figure 2. The former leads to initiate the release of 1,4,5-inositol tris phosphate IP 3 initiating calcium release, activating protein tyrosine kinase 2 PYK2 , which leads to proto-oncogene tyrosine protein kinase Src activating Ras guanine nucleotide exchange factor Ras GEF , which leads to the activation of MAPK signaling.
Table 1. U and its analog U were used to show the effect of human platelet calcium signaling and protein tyrosine phosphorylation in the presence of thrombin, collagen, and thapsigargin U did not show any calcium inhibitory effect via the activation of PLC but rather showed the calcium inhibitory effect via the upstream activation of cPLA2 in the presence of thapsigargin and collagen This provides a clear indication that U has minimal activity as a PLC inhibitor.
In the presence of U, the phosphorylation of tyrosine kinase was abolished As mentioned above, another agent, which can further be investigated, is neomycin, a PLC inhibitor. Our laboratory has previously reported that neomycin strongly inhibits the formation of IP 3 in rat aortic smooth muscle cells in the presence of endothelin, an agonist that influences contraction in smooth muscle ETR is coupled to PLC via G proteins 55 and its activation acts on the cardiac muscle where it binds to ryanodine located on the SR, which releases calcium mobilization within the cardiac muscle cell As known, the signaling pathways of GPCRs through G proteins contribute to various functions in different cell types such as the contraction of blood vessels and are involved in many diseases such as cancer and cardiovascular disease 16 , In unpublished data, we have found that neomycin has a dose-dependent inhibition of thrombin-mediated release of intracellular calcium in human VSMCs.
Ionomycin is a calcium ionophore, which elevates intracellular calcium Radioactive sulfate incorporation into proteoglycans was unaffected by ionomycin, providing support that calcium regulation is not involved in proteoglycan synthesis Similarly, BAPTA-AM, a chelator of calcium ions, which prevents an elevation of intracellular calcium by acting as a calcium buffer 52 had no effect on proteoglycan synthesis The interpretation therefore concluded that there were no effects on calcium ion stimulation hence intracellular calcium does not play a role in VSMC proteoglycan synthesis The ROCK family of kinases is involved in Rho-induced formation of actin-stress fibers and focal adhesion as well as the down-regulation of myosin light chain MLC phosphatases.
Deng et al. Subsequent experiments showed that blockade of Rho kinase signaling is not essential for CCL2 protein production but is important in the release of CCL2 from the cell, as thrombin-mediated CCL2 levels are inhibited by Y Both protein subunit families contributed significantly to RhoA activation by thrombin A luciferase reporter system with a chimera that contains promoter elements that drive Gs, Gq, and G12 signals and another chimera with promoters to drive Gi signals revealed neuromedin U receptor 1 activating Gq, neuromedin U receptor 2 activating Gi, and luteinizing hormone receptor activating Gq and Gs proteins Furthermore, the protein contains a switch mechanism and this can be targeted as it is the target of the YM class of inhibitors Such situations are common in therapeutics but in most cases can only be established experimentally.
G protein coupled receptor signaling is a major area of cell biology and therapeutics. The functioning of the seven transmembrane GPCR has been one of the most intensively studied areas of protein function.
For multiple reasons, mostly the limited availability of pharmacological agents, which inhibit G protein function, the role of G proteins in GPCR signaling has been severely under-studied relative to the intense activity around the GPCRs. Given the broad involvement of GPCRs in cellular functioning, this is a major deficit in cellular signaling studies and potentially more importantly in the search for new drug targets. HK and MB provided chemical insight about cyclic depsipeptide.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Physiological regulation of G protein-linked signaling. Physiol Rev 79 — G-protein signaling: back to the future. Cell Mol Life Sci 62 — Impact of GPCRs in clinical medicine: monogenic diseases, genetic variants and drug targets.
Biochim Biophys Acta — Wess J. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition.
Google Scholar. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.
Mol Pharmacol 63 — Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol 9 — Roles of G protein subunits in transmembrane signalling. Nature — Hein P, Bunemann M. Coupling mode of receptors and G proteins. Naunyn Schmiedebergs Arch Pharmacol — Lipid modifications of trimeric G proteins. J Biol Chem —6.
G protein pathways. Science —9. Strathmann M, Simon MI. G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines.
Cell Mol Life Sci 72 4 — Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type I receptor.
J Biol Chem — J Biol Chem 10 —9. The paradigm of g protein receptor transactivation: a mechanistic definition and novel example. ScientificWorldJournal 11 — J Pharm Pharmacol 65 — Little PJ. GPCR responses in vascular smooth muscle can occur predominantly through dual transactivation of kinase receptors and not classical Galphaq protein signalling pathways.
Life Sci 92 —6. Liebmann C. Mol Cell Endocrinol — Endothelin-1 stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by endothelin receptor transactivation of the transforming growth factor-[beta] type I receptor.
J Cardiovasc Pharmacol 56 —8. Mammalian RGS proteins: barbarians at the gate. Endothelin-1 and endothelin-3 stimulate calcium mobilization by different mechanisms in vascular smooth muscle. Biochem Biophys Res Commun — Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration.
Circ Res 84 — Biochem Pharmacol 77 — Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins. Cell Signal 18 — Defective platelet activation in G alpha q -deficient mice. Nature —6. J Biol Chem —5. Role of Galphaq in smooth muscle cell proliferation.
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