Membrane-bound human substance p receptor undergoes agonist-dependent phosphorylation by g protein receptor kinase 2 (grk2) to a high stoichiometry and activates grk2 in the absence of gβγ

We expressed the human substance P receptor (hSPR) in Sf9 cells at high levels (>50 pmol/mg membrane protein) and examined its ability to serve as GRK2 substrate in intact membranes. The hSPR in Sf9 membranes was phosphorylated by purified GRK2 in agonist-dependent manner with a stoichiometry of 19 ±1 mol of phosphate / mol receptor (SEM; n =3). Kinetic analysis indicated that GRK2-catalyzed phosphorylation of agonist-occupied hSPR occurs with a Km of 6.3 ±0.4 nM (SEM; n =3 ) and a Vm" of 1.8 ±0.1 nmol/min/ug. These kinetic parameters were marginally affected by Gpr This was surprising because GpY potently activates GRK2-catalyzed phosphorylation of agonistoccupied rhodopsin and β,-adrenergic receptor (β2AR). To determine whether GRK2 was being activated by GPT endogenously present in Sf9 membranes, the phosphorylation experiments were performed in the presence of guanine nucleotides that suppress (GDPβS) or enhance (GTPyS) the release of Gfr These experiments showed that the effects of GDPβS and GTPyS on GRK2-catalyzed phosphorylation of agonistoccupied hSPR were marginal. In conclusion, our data provide two significant findings: 1) agonist-occupied hSPR in Sf9 membranes is an excellent substrate of GRK2; and 2) hSPR, unlike rhodopsin and β,AR, can almost fully activate GRK2 without G βγ.

Duke Scholars

Author Madan Mohan Kwatra Anesthesiology