Characterization of differences between rapid agonist-dependent phosphorylation and phorbol ester-mediated phosphorylation of human substance P receptor in intact cells

Substance P receptor (SPR), which plays a key role in pain transmission, is known to undergo rapid agonist-dependent desensitization and internalizati on. The present study shows that human SPR undergoes agonist-dependent phos phorylation in intact cells. Immunoprecipitation of SPR from P-32(i)-labele d Chinese hamster ovary cells stably expressing human SPR (CHO-hSPR) indica tes that substance P (SP) causes a rapid (T-1/2 < 1 min), dose-dependent (E C50 = 2 nM), and pronounced (5-fold over basal) phosphorylation of SPR. Bec ause SPR in CHO-hSPR couples to G alpha(q), G alpha(s), and G alpha(o), (Ro ush and Kwatra, 1998), we examined the involvement of various second messen ger-activated protein kinases in SPR phosphorylation. Although increases in intracellular cyclic AMP or treatment with the calcium ionophore A23187 do not cause SPR phosphorylation, treatment with the protein kinase C (PKC) a ctivator phorbol 12-myristate 13-acetate (PMA) causes a 2.5-fold increase i n SPR phosphorylation with a T-1/2 of <1 min. However, PKC inhibitor GF1092 03X has no effect on SP-dependent SPR phosphorylation. Furthermore, althoug h SP treatment phosphorylates SPR on both serine and threonine residues equ ally, PMA treatment phosphorylates the receptor predominantly on serine res idues. Two-dimensional phosphopeptide mapping data indicate that SP-depende nt and PMA-dependent phosphorylations of SPR have some unique differences, Taken together, these data suggest that although activation of PKC by PMA c an lead to SPR phosphorylation, PKC does not mediate SP-dependent phosphory lation of SPR. In conclusion, the present study represents the first demons tration and characterization of agonist-dependent and PMA-mediated phosphor ylation of SPR in intact cells.