Characterization of the Regulator of G Protein Signaling Domain of G Protein‐Coupled Receptor Kinase 4

G protein-coupled receptor kinase 4 (GRK4) is a member of the GRK family, and is the least studied of the GRKs. Recently, two independent groups demonstrated that polymorphisms of human GRK4 are associated with essential hypertension. Two of the polymorphisms (GRK4 R65L and GRK4 A142V) lie within the regulator of G protein signaling (RGS) domain. RGS domains are found in many proteins and generally bind to GTP-bound (active), but not GDP-bound (inactive) Gα subunits of the heterotrimeric G proteins. This binding increases the intrinsic GTPase activity of a Gα subunit. There are RGS domains, such as the RGS domain within axin, that have not been found to interact with any Gα subunits. In the case of GRK4, no studies have been conducted on the RGS domain, but GRK4 is known to interact with Gαs-coupled receptors. The hypothesis being tested is that the RGS domain of GRK4 binds to active Gαs, consequently increasing the intrinsic hydrolytic rate of Gαs. Our co-immunoprecipitation data demonstrates that His-GRK4γ interacts with Gαs, and the interaction is increased in the presence of AlF4-. The high basal binding suggests that Gαs binds to GRK4 in the GDP-bound state, yet the increase in binding in the presence of AlF4− indicates that the RGS domain preferentially binds to the GTP-bound state. This data supports the previous findings that GRK4 inhibits D1 dopamine receptor-mediated signaling, and provides a potential molecular mechanism for the previously reported high basal activity of GRK4. The effect of the RGS domain on the hydrolytic rate of Gαs is currently being examined.