Characterization of the GRK2 Binding Site of Gαq

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The G q subfamily of G␣ subunits couples GPCR activation to the enzymatic activity of phospholipase C-␤ (PLC-␤). Regulators of G protein signaling (RGS) proteins bind to activated G␣ subunits, including G␣ q , and regulate G␣ signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for G␣ subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to G␣ q attenuates PLC-␤ activity. The RH domain of GRK2 interacts with G␣ q/11 through a novel G␣ binding surface termed the "C" site. Here, molecular modeling of the G␣ q ⅐GRK2 complex and site-directed mutagenesis of G␣ q were used to identify residues in G␣ q that interact with GRK2. The model identifies Pro 185 in Switch I of G␣ q as being at the crux of the interface, and mutation of this residue to lysine disrupts G␣ q binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations G␣ q -V240A, G␣ q -D243A, both residues within Switch III, and G␣ q -Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of G␣ q -Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to G␣ q -R183C. Interestingly, the model also predicts that residues in the helical domain of G␣ q interact with GRK2. In fact, the mutants G␣ q -K77A, G␣ q -L78D, G␣ q -Q81A, and G␣ q -R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant G␣ q -T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for G␣ q interaction rely on contacts with distinct regions and different Switch I residues in G␣ q .G protein-coupled receptors (GPCRs) 1 are heptahelical integral membrane proteins responsible for the transmission of extracellular signals, such as light, neurotransmitters, and hormones, to intracellular signaling pathways. Agonist-bound GPCRs directly interact with heterotrimeric (␣␤␥) G proteins and catalyze nucleotide exchange on G␣ subunits (1). Several mechanisms are in place to ensure the appropriate level of response to an agonist. Receptors become desensitized to agonist stimulation upon phosphorylation by GPCR kinases (GRKs) and subsequent binding of arrestins (2-5). The G␣ subunit has intrinsic GTPase activity that returns the G protein to the inactive GDP-bound state, promoting re-association with G␤␥ (6).A third mechanism, which accounts for the rapid desensitization observed in cellular signaling systems such as phototransduction in the eye (7), is attributed to GTPase-activating proteins (GAPs), which bind G␣ subunits and accelerate the GTPase rea...

WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex

“…Our results also indicated that WDR36 can positively contribute to Gaq-mediated activation of PLCb by competing with GRK2 for binding to activated Gaq. Indeed, GRK2 was previously reported to preferentially bind to the transition and active states of Gaq and to sequester it from other effectors (Carman et al, 1999;Day et al, 2004;Sherrill and Miller, 2006;Sterne-Marr et al, 2004).…”

Pleiotropic Phenotype of a Genomic Knock-In of an RGS-Insensitive G184S Gnai2 Allele

“…The analogous mutation (Gly 184 to Ser) in mammalian G␣ o and G␣ i1 prevented the GAP activity of RGS4 and RGS7 and blocked RGS4 binding to aluminum fluoride-activated G␣ subunits (41). These mutations do not affect other functions of the G␣ subunit, such as the intrinsic GTPase activity of the G protein or its coupling to ␤␥ subunits, receptors, GRK, or effectors (adenylyl cyclase [AC]) (18,25). Thus, the only known effect of the G184S mutation in G␣ o and G␣ i is to prevent RGS action on G␣.…”

The fifth class of Gα proteins

“…The helical domain is a divergent region of G␣ proteins in general, but seems to be conserved within a class or a family. Although the functions of this domain have not been fully understood so far, several studies have shown its effect on GTPase activity and involvement in the interaction with GPCRs, regulator and effector proteins, and possibly ␤/␥ heterodimers (13)(14)(15)(16)(17)(18).…”

“…S1). They are located mainly in the helical domain, but also in SWs and may thus be involved in interaction with GPCRs, ␤/␥ heterodimers, regulators, and effectors (4,(13)(14)(15)(16)(17)(18), suggesting that the G␣ v proteins have their specific interaction partners and consequently regulate distinct pathways. Furthermore, both N-and C-terminal regions, which are assumed to interact with GPCRs and ␤/␥ heterodimers (4), contain several Gv-specific amino acids as well ( Fig.…”