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 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 Galphaq, Galphas, and Galphao (), 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.
Roush
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ED
Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Warabi
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K
Kwatra
M M
MM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D018040:Receptors, Neurokinin-1; D013373:Substance P; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate