SELECTIVE REGULATION OF GALPHA Q/11 BY AN RGS DOMAIN IN THE G PROTEIN-COUPLED RECEPTOR KINASE, GRK2

Abstract G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211–212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF4 −-dependent binding to G protein α subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF4 −. This revealed the specific ability of bovine brain Gαq/11 to bind to both GRK2 and GRK3 in an AlF4 −-dependent manner. In contrast, Gαs, Gαi, and Gα12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Gαq/11 could also bind to an N-terminal construct of GRK2, while no binding of Gαq/11, Gαs, Gαi, or Gα12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Gαq revealed significant binding of both GαqGDP/AlF4 − and Gαq(GTPγS), but not Gαq(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Gαq(R183C) but not wild type Gαq. In vitro analysis revealed that GRK2 possesses weak GAP activity toward Gαq that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Gαq-mediated activation of phospholipase C-β both in vitro and in cells, possibly through sequestration of activated Gαq. These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.