G-Proteins and Vessel's Response to Injury

Daynene Vykoukal, PhD and Mark G. Davies, MD, PhD, MBA

Intimal hyperplasia is the universal response of a vessel to injury and involves the coordinated stimulation of smooth muscle cells by mechanical, cellular and humoral factors to induce a program of cellular activation that leads to proliferation, migration and extracellular matrix deposition. Signal transduction is the mechanism by which a cell responds to these extracellular signals and converts them to intracellular instructions to coordinate a cell's responses. There are two basic ligand-operated receptor mechanisms responsible for signal transduction; firstly, ligand-modulated release of a secondary intracellular mediator (G-protein coupled receptors) and secondly, ligand-regulated enzyme activity (tyrosine kinase coupled receptors). Targeting signal transduction pathways is a broad and novel avenue for molecular therapeutics.

There are over 1000 G-protein coupled receptors (GPCR) and 400 of these are considered non-sensory GPCRs. They can be grouped into three families based on sequence similarity (Family A, B and C). Family A contains the adrenergic receptors and olfactory receptors, Family B contains the GI peptide hormones and Family C contains the glutamate, GABA and calcium receptors. These transmembrane molecules can be activated by a vast diversity of extracellular inputs such as biogenic amines, amino acids, odorants, lipids, ions, proteases, nucleotides, peptides, large polypeptides, and even photons. 30% of the current pharmaceutical drugs are ligands for GPCRs.

G-proteins classification and action: G-proteins are intrinsic membrane-bound proteins which act as transmembrane signal transducers in cells; they consist of three distinct subunits:   and ; form a tightly associated dimer. G-protein hetero-trimers are classified according to differences in their G subunits. Both G and G subunits have different cellular targets and their activation is intimately associated with receptor activation. Depending on the subtype(s) of the G protein  subunit that a given GPCR interacts with, a single effector or a combination of effectors can be activated. In addition, the  complex, which anchors the  subunit to the plasma membrane, has been shown to activate ion channels. Five subtypes of  subunits and eleven subtypes of  subunits have been identified. A combination of different subtypes of , , and  subunits provides a great diversity of intracellular signaling pathways that can be regulated by GPCRs. A significant number of GPCRs (11% of the GPCRs) have been shown to couple to multiple G protein subtypes. Receptor-G-protein activity is tightly regulated by a series of G-protein related kinases, which dephosphorylate the receptor, and a second family of proteins, regulators of G-protein signaling (RGS) which regulate the GTPase activity of the heterotrimeric units. Thereafter, the process of receptor internalization, which involves the key proteins dynamin, -arrestin and caveolin, regulates downstream secondary mediator activation.

G proteins: The G proteins can be divided into four subfamilies: (1) the ‘Gs’ subfamily that stimulates adenylyl cyclase (Gs and Golf); (2) the ‘Gi/o’ subfamily that inhibits adenylyl cyclase and regulates ion channels (Gi1, Gi2, Gi3, Go1, Go2, Go3, Gz, Gt1, Gt2, and Ggust); (3) the ‘Gq/11’ subfamily that activates phospholipase C (Gq, G11, G14, and G15/16), and (4) the ‘G12/13’ subfamily that activates the Na+/H+exchanger pathway (G12 and G13). In general, Gi protein subtypes (i1, i2, and i3) act to inhibit adenylate cyclase and to decrease intracellular cAMP levels. Gs protein can mediate the activation of adenylate cyclase with an increase in intracellular cAMP and can also regulate calcium channels. Gq subunits have been shown to activate phospholipase C.

G proteins:  subunits have also been shown to activate phospholipase C isozymes (PLC- ,  and ), to activate small GTPases, and to transactivate the epidermal growth factor receptor. The traditional role of the G subunits, released following activation of the Gi proteins, is to bind to the active s-GTP species, thus reducing their activity and leading to decreased cAMP levels. However, Gsubunits of the pertussis toxin sensitive Gi proteins can enhance the effect of Gs on type II and IV adenylate cyclase but can inhibit Gs-stimulated type I adenylate cyclase. The other three Gs-stimulated adenylate cyclases (type III, V and VI) appear not to be modulated by the G subunits.

β-arrestin: β-arrestins (non-visual arrestins) are ubiquitously expressed proteins that were first described for their role in desensitizing G-protein-coupled receptors (GPCRs). There are two β-arrestin families, namely, β-arrestin1 and βarrestin-2 and β-arrestin-3, respectively. Upon complexing with receptors, β-arrestins can serve as inhibitors of signal transduction by preventing further receptor coupling to G protein signaling cascades.

G-protein-Receptor Kinases: G-protein receptor kinases (GRKs) are necessary to terminate signaling of G-protein coupled receptors through receptor desensitization and downregulation. GRKs comprise a family of seven mammalian serine/threonine protein kinases that phosphorylate and regulate agonist-occupied or constitutively active G-protein coupled receptors. There are three sub-groups within the GRK family. GRK1 and GRK7 form one distinct sub-group, which are only found in retinal cells. The non-visual GRKs divide into two sub-groups: the GRK2 subfamily, consisting of GRK2 (b-ARK1) and GRK3 (b-ARK2), and the GRK4 subfamily (GRK4, GRK5 and GRK6). The different GRKs are highly specific in their receptor preference. In addition, GRKs have been shown to interact with MAPKs, PI-3Ks and GTPase-activating proteins, which are involved in regulating receptor trafficking and signaling. Furthermore, GRK2 and 3 are well-known to bind the G subunit complex, a process that induces activation of these GRKs.

Regulators of G-protein Signaling: Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G subunits. There are 20 RGS, and more than 20 RGS-like, proteins. Receptor-induced GDP/GTP exchange activates G proteins by dissociating G-protein -subunits from the -dimers. Both -subunits and -dimers are involved in effector regulation. The deactivation of these active forms is controlled by the hydrolysis of GTP bound to  -subunits, allowing the inactive heterotrimer to reform. Termination of G-protein-mediated signaling in vivo is 10- to 100-fold faster than the in vitro rate of GTP hydrolysis by  -subunits, suggesting that in analogy to the GTPases of the Ras-superfamily, GTPase-activating proteins (GAPs) are required to achieve timely deactivation. Recently, members of a novel protein superfamily, known as "regulators of G-protein signalling" (RGS), were identified as potent GAPs. RGS proteins utilize both direct and indirect mechanisms to form stable functional pairs with preferred GPCRs to selectively modulate the signaling functions of those receptors and linked G proteins.

Receptor Transactivation: Transactivation of the epidermal growth factor receptor has been shown to play a crucial role in the signaling by G-protein-coupled receptors. At present, the triple membrane passing signaling mechanism of GPCR induced EGFR activation is a widely accepted model of receptor linked tyrosine kinase transactivation. In this model, there is a sequence of three transmembrane signaling events: GPCR activation which is followed by intracellular activation of an extracellular metalloproteinase which induces the release of a tethered ligand. The tethered ligand binds to extracellular domain of epidermal growth factor receptor.

G-proteins and proliferation: The role of G-protein signal transduction in cellular proliferation is being defined. In mesenchymal cells, Gi G-proteins enhance ERK activity, DNA synthesis and cell proliferation. Furthermore, a Gi- regulated role in mitosis has been identified in fibroblast cell lines. In BALB/c3T3 cells, Gi2 and G are responsible for ERK activation and DNA synthesis, while Gi3 is responsible for cell transformation. G subunit activation results in proliferation through the activation of receptor tyrosine kinase pathways, involving G-mediated activation of Src-like kinases to activate Shc-Grb2-Sos and ras pathways. Ras activates raf directly or acts on PI3Kinase to induce raf, protein kinase B (akt) and rac phosphorylation. These various pathways lead to ERK / p38 /JNK activation and subsequent transcription. G-mediated ERK activation has recently been shown to involve transactivation of the epidermal growth factor receptor in smooth muscle cells. β-arrestin1 promotes smooth muscle cell proliferation while β-arrestin2 inhibits smooth muscle cell proliferation.

G-proteins and migration: The role of G-protein signal transduction in the cellular migration is less well defined. Migration of inflammatory cells is driven in part by families of chemokines, whose receptors are G-protein coupled and pertussis toxin sensitive, suggesting the association of Gi. Further data has demonstrated a role for ras, rac, rho and ERK1/2 in chemokine linked Gi induced cascades. It appears that receptors coupled to Gi can mediate chemotaxis, even when they lack the distinguishing features of chemokine receptors and when their agonist ligands are not classical chemokines. Activation of Gi is required but probably not sufficient for chemotaxis. Although Gi coupled receptors undergo internalization during many cell-induced responses such as mitogenesis, similar Gi receptors can mediate chemotaxis without undergoing agonist-induced internalization. Irrespective of the Gi, G is an essential mediator of chemotaxis. Integrin signaling in cells is mediated through Focal Adhesion Kinase (FAK) but is also associated with an integrin-associated protein, which is G-protein coupled and pertussis toxin sensitive. Thrombin induces migration that is pertussis toxin sensitive. Thrombin also involves transactivation of the epidermal growth factor receptor by G PDGF and IGF both have pertussis toxin sensitive elements when they are used as chemoattractants in migration assays. PDGF activation induces formation of sphingose-1-phosphate which in turn binds to its receptors and is responsible for the migration to PDGF.

G-protein expression and response to injury: In vein grafts, there is enhanced expression of Gi2 and G subunits and de novo expression of Gi3. These dramatic changes in G-protein expression were associated with a change in the functional coupling of serotonergic receptors to G-proteins; the vein grafts developed pertussis toxin (PTx) sensitive responses, while the control veins had a pertussis toxin insensitive response. The changes in the expression of G-proteins in the vein grafts, particularly the Gi3 and G subunits, paralleled the formation of intimal hyperplasia. A similar pattern of changes occurs during the development of intimal hyperplasia after arterial injury. These results suggest that the smooth muscle cell phenotype involved in the intimal hyperplasia response undergoes changes in membrane transducing systems with the expression of new components and changes in the coupling of these components to surface receptors.

Given the changes in G-proteins, modulating G-protein expression or function should affect the intimal hyperplastic response. G signaling is common to all G-proteins and was targeted by a gene encoding a specific Ginhibitor, the carboxyl terminus of the -adrenergic receptor kinase ARKCT). Intimal hyperplasia both in vein grafts and following arterial injury was significantly reduced by a single application of this inhibitor. The altered G protein-coupling after vein grafting (i.e. development of pertussis toxin sensitivity) was blocked by ARKCT treatment. It was recently demonstrated that one time exposure to pertussis toxin, a Gi inhibitor, will also reduce intimal hyperplasia in vivo. Thus, Gi /G-mediated pathways appear to play a major role in intimal hyperplasia development. Mice lacking β-arrestin1 display enhanced neointimal hyperplasia and smooth muscle cell proliferation. -arrestin2 knockout mice display less severe neointimal hyperplasia. These data support a bidirectional role for β-arrestin1 versus β-arrestin2 in injury-provoked neointimal hyperplasia suggesting that inhibition of β-arrestin2 while stimulation of βarrestin1 mediated pathways might be therapeutically beneficial. Expression of RGS-5 is suppressed in the developing neointima.

Conclusion: G-proteins represent a significant, little explored signal transduction system in the development of vascular disease. Targeting G-protein coupled responses, instead of individual receptors, appears to offer a novel therapeutic strategy.

Figure Legend: Life Cycle of the GPCR. Binding of an agonist to the G-protein coupled receptor (GPCR) leads to dissociation of the G and G subunits. Gdimers recruit GRKs, which phosphorylate the agonist-associated receptor. This, in turn, leads to recruitment of arrestin to the receptor and induces dynamin mediated Clatherin Pit formation. The receptor is internalized and undergoes either phosphorylation or is recycled to the cell surface.

Updated January 2010

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