GPCRs undergo cycles of activation and inactivation. In the inactive state, the heterotrimeric G-protein is composed of tightly associated GDP (guanosine-5’-diphosphate)-bound Gα and Gβγ subunits. Upon ligand binding, GPCRs undergo a conformational change, catalyzing Gα to exchange GDP for GTP (guanosine-5’-triphosphate). Then, GTP-bound Gα and Gβγ dissociate from the GPCR and activate downstream signaling effectors. Hydrolysis of Gα-GTP to Gα-GDP causes the reassociation of the heterotrimeric G-protein with the GPCR.
Heterotrimeric G proteins signal through four classes of Gα proteins: Gs/olf, Gi/o, G12/13, and Gq. Gs/olf stimulates adenylyl cyclase (AC) whereas Gi/o inhibits it. G12/13 activates RhoGEFs (Ras homology guanine nucleotide exchange factors). The Gq subfamily contains four types of α-subunits (Gαq, Gα11, Gα14, and Gα16); they all stimulate phospholipase C β (PLC-β); Gαq/11 has also been shown to activate p63RhoGEF. While early work focused on Gα signaling, Gβγ also activates signal transduction cascades.
In the late 1950s, Sutherland and Rall were the first to characterize how GPCRs activate second messenger cascades. They showed that stimulation of various tissues with epinephrine or glucagon (both GPCR agonists) promoted the production of cyclic adenosine monophosphate (cAMP) by AC. Mammals express at least nine different isoforms of AC that vary in their pattern of tissue ex
The production of cAMP by AC can be inhibited as well as stimulated. Gi-coupled GPCRs inhibit AC. Pertussis toxin catalyzes the ADP-ribosylation of Gi, which prevents GPCR coupling and inhibition of AC.
Heterotrimeric G proteins and monomeric Ras GTPases both share the feature of being inactive in the GDP-bound state and active in the GTP-bound state. Guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP to generate the active Ras GTPase. Ras GTPase signaling is terminated via GTPase activating proteins (GAPs), which accelerate the intrinsic GTPase activity of Ras family members.
The Ras-related small GTPase protein super family contains the Rho GTPase subfamily. The human Rho GTPase subfamily contains 22 members; the most extensively characterized members of this family are RhoA, Rac1, and CDC42 due to their ability to cause striking changes in cell morphology upon activation. Rho GTPases have been implicated in a variety of cellular responses including gene transcription, embryogenesis, and actin cytoskeletal dynamics.
Stimulation of G12/13-coupled GPCRs leads to Gα-GTP binding of the RH domain of RhoGEF. This induces a conformational change in RhoGEF, activating it. Activated RhoGEF can then activate the RhoGTPase by promoting the release of bound GDP, which then allows the subsequent binding of GTP.
Most GPCRs that couple to G12/13 also couple to Gq. Gq-coupled GPCRs stimulate PLC-β, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to IP3 and diacylglycerol (DAG). IP3 binds IP3 receptors on the endoplasmic reticulum (ER) and induces calcium release. PLC-β promotes the termination of Gq-GPCR signaling complexes by stimulating GTP hydrolysis of the GTP-bound Gq.
While PLC-β has been thought of as being the canonical Gq effector, evidence from the last decade has shown that Gq-GPCRs are also potent activators of p63RhoGEF. Northern blot analysis and immunohistochemistry suggest that p63RhoGEF is expressed in human heart and brain tissue. Several groups of investigators have shown that p63RhoGEF activates the small GTPase RhoA, but not Rac1 or CDC42.
Astrocytes express GPCRs that signal through all four classes of Gα proteins. Astrocyte signaling via three of those classes Gs, Gi, and G12/13-coupled GPCRs is poorly understood. Astrocytic Gq-GPCRs have been the most extensively studied because of the early development of calcium indicator dyes that allow the monitoring of cytoplasmic calcium concentrations in real time.