CHARACTERIZATION OF GRK2-CATALYZED PHOSPHORYLATION OF THE HUMAN SUBSTANCE-P RECEPTOR IN SF9 MEMBRANES

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupi ed G protein-coupled receptors (GPCRs), resulting in GPCR desensitizat ion. GRK2 is one of the better studied of the six known GRKs and phosp horylates several GPCRs. In a previous study, we documented that GRK2 and GRK3 phosphorylate purified and reconstituted rat substance P rece ptor (rSPR) [Kwatra et al. (1993) J. Biol. Chem. 268, 9161-9164]. Here , we characterize in detail GRK2-catalyzed phosphorylation of human SP R (hSPR) in intact membranes. GRK2 phosphorylates hSPR in urea-washed Sf9 membranes in an agonist-dependent manner with a stoichiometry of 1 9 +/- 1 mol of phosphate/mol of receptor, which increases slightly (1. 3-fold increase) in the presence of G beta gamma. Kinetic analyses ind icate that receptor phosphorylation occurs with a K-m of 6.3 +/- 0.4 n M and a V-max of 1.8 +/- 0.1 nmol/min/mg; these kinetic parameters are only slightly affected by G beta gamma [K-m = 3.6 +/- 1.0 nM and V-ma x = 2.2 +/- 0.2 nmol/min/mg]. The lack of a strong stimulatory effect of G beta gamma on GRK2-catalyzed phosphorylation of hSPR is surprisin g since G beta gamma potently stimulates GRK2-catalyzed phosphorylatio n of beta(2)-adrenergic receptor and rhodopsin. Involvement of G beta gamma endogenously present in membranes is ruled out as a source of hi gh levels of hSPR phosphorylation, since receptor phosphorylation was not affected by guanine nucleotides that suppress or enhance the relea se of endogenous G beta gamma. The present study determines, for the f irst time, the kinetics of phosphorylation of a receptor substrate of GRK2 in intact membranes. Further, our results identify hSPR as a uniq ue substrate of GRK2 whose phosphorylation is strong even in the absen ce of G beta gamma.