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Molecular Pharmacology

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Research ArticleArticle

Characterization of Differences Between Rapid Agonist-Dependent Phosphorylation and Phorbol Ester-Mediated Phosphorylation of Human Substance P Receptor in Intact Cells

Eric D. Roush, Kengo Warabi and Madan M. Kwatra
Molecular Pharmacology May 1999, 55 (5) 855-862;
Eric D. Roush
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Kengo Warabi
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Madan M. Kwatra
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Abstract

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.

Footnotes

  • Send reprint requests to: Madan M. Kwatra, Ph.D., Box 3094, Duke University Medical Center, Durham, NC 27710. E-mail:kwatr001{at}mc.duke.edu

  • This work was supported by National Institutes of Health Grant NS33405 (M.M.K.). Dr. Kwatra is a senior fellow of the Center of Study of Aging and Human Development, Duke University Medical Center.

  • Abbreviations:
    AC
    adenylyl cyclase
    CHO-hSPR
    Chinese hamster ovary cells stably expressing human SPR
    CHO-K1
    Chinese hamster ovary cells, strain K1
    DAG
    diacylglycerol
    GPCR
    G protein-coupled receptor
    GRK
    G protein-coupled receptor kinase
    hSPR
    human substance P receptor
    hSPR-Ab
    human substance P receptor antibody
    PLC
    phospholipase C
    PKA
    protein kinase A
    PKC
    protein kinase C
    PMA
    phorbol 12-myristate 13-acetate
    PVDF
    polyvinylidene difluoride
    PAGE
    polyacrylamide gel electrophoresis
    SP
    substance P
    SPR
    substance P receptor
    TLC
    thin layer chromatography
    TTBS
    20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.02% Tween-20
    • Received February 1, 1999.
    • Accepted February 11, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 55 (5)
Molecular Pharmacology
Vol. 55, Issue 5
1 May 1999
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Research ArticleArticle

Characterization of Differences Between Rapid Agonist-Dependent Phosphorylation and Phorbol Ester-Mediated Phosphorylation of Human Substance P Receptor in Intact Cells

Eric D. Roush, Kengo Warabi and Madan M. Kwatra
Molecular Pharmacology May 1, 1999, 55 (5) 855-862;

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Research ArticleArticle

Characterization of Differences Between Rapid Agonist-Dependent Phosphorylation and Phorbol Ester-Mediated Phosphorylation of Human Substance P Receptor in Intact Cells

Eric D. Roush, Kengo Warabi and Madan M. Kwatra
Molecular Pharmacology May 1, 1999, 55 (5) 855-862;
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