TY - JOUR T1 - Characterization of Differences Between Rapid Agonist-Dependent Phosphorylation and Phorbol Ester-Mediated Phosphorylation of Human Substance P Receptor in Intact Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 855 LP - 862 VL - 55 IS - 5 AU - Eric D. Roush AU - Kengo Warabi AU - Madan M. Kwatra Y1 - 1999/05/01 UR - http://molpharm.aspetjournals.org/content/55/5/855.abstract N2 - Substance P receptor (SPR), which plays a key role in pain transmission, is known to undergo rapid agonist-dependent desensitization and internalization. The present study shows that human SPR undergoes agonist-dependent phosphorylation in intact cells. Immunoprecipitation of SPR from 32Pi-labeled Chinese hamster ovary cells stably expressing human SPR (CHO-hSPR) indicates that substance P (SP) causes a rapid (T1/2 < 1 min), dose-dependent (EC50 = 2 nM), and pronounced (5-fold over basal) phosphorylation of SPR. Because SPR in CHO-hSPR couples to Gαq, Gαs, and Gαo (Roush and Kwatra, 1998), we examined the involvement of various second messenger-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) activator phorbol 12-myristate 13-acetate (PMA) causes a 2.5-fold increase in SPR phosphorylation with a T1/2 of <1 min. However, PKC inhibitor GF109203X has no effect on SP-dependent SPR phosphorylation. Furthermore, although SP treatment phosphorylates SPR on both serine and threonine residues equally, PMA treatment phosphorylates the receptor predominantly on serine residues. Two-dimensional phosphopeptide mapping data indicate that SP-dependent and PMA-dependent phosphorylations of SPR have some unique differences. Taken together, these data suggest that although activation of PKC by PMA can lead to SPR phosphorylation, PKC does not mediate SP-dependent phosphorylation of SPR. In conclusion, the present study represents the first demonstration and characterization of agonist-dependent and PMA-mediated phosphorylation of SPR in intact cells. The American Society for Pharmacology and Experimental Therapeutics ER -