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
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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
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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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