Abstract
Protein kinase C (PKC)-mediated desensitization of the corticotropin releasing factor type 1 (CRF1) receptor was investigated in human retinoblastoma Y79 and transfected COS-7 cells. Because stimulation of Y79 cells with CRF resulted in large (∼30-fold) increases in intracellular cAMP accumulation without changing inositol phosphate levels, the CRF1 receptor expressed in retinoblastoma cells couples to Gs, but not to Gq, and predominantly signals via the protein kinase A cascade. Direct activation of PKC by treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol (DOG) desensitized CRF1 receptors in Y79 cells, reducing the maximum for CRF- (but not forskolin)-stimulated cAMP accumulation by 56.3 ± 1.2% and 40.4 ± 2.1%, respectively (p < 0.001). Pretreating Y79 cells with the PKC inhibitor bisindolylmaleimide I (BIM) markedly inhibited PMA's desensitizing action on CRF-stimulated cAMP accumulation, but did not affect homologous CRF1 receptor desensitization. Retinoblastoma cells were found to express PKCα, βI, βII, δ, λ, and RACK1. When α and β isoforms of PKC were down-regulated 80 to 90% by a 48-h PMA exposure, PMA-induced CRF1 receptor desensitization was abolished. In transfected COS-7 cells the magnitude of CRF1 receptor phosphorylation after a 5-min exposure to PMA was 2.32 ± 0.21-fold greater compared with the basal level. Pretreating COS-7 cells with BIM abolished PMA-induced CRF1 receptor phosphorylation. These studies demonstrate that protein kinase C (possibly α and β isoforms) has an important role in the phosphorylation and heterologous desensitization of the CRF1 receptor.
Footnotes
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Dr. Hauger was supported by a Veterans Affairs (VA) Merit Review grant, the VA Mental Illness Research, Education and Clinical Center (MIRECC) of VISN22, and National Institutes of Health/National Institute on Drug Abuse Grant R01-DA13769-01A1. Dr. Catt was funded by the intramural program of the National Institutes of Health, where he is Chief of the Endocrinology and Reproductive Research Branch (ERRB) of the National Institute of Child Health and Human Development (NICHD). Dr. Olivares-Reyes was supported by a Postdoctoral Visiting Fellow award from the National Institutes of Health.
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DOI: 10.1124/jpet.103.050088.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, GPCR kinase; CRF, corticotropin releasing factor; CRF1, CRF receptor type 1; PKA, protein kinase A; Gs, stimulatory GTP binding protein; PKC, protein kinase C; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; DOG, 1,2-dioctanoyl-sn-glycerol; BIM, bisindolylmaleimide I; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; ANOVA, analysis of variance; RACK, receptors for activated C-kinase; HA, hemagglutinin; PAC1, pituitary adenylate cyclase-activating polypeptide type 1 receptor.
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↵1 Visiting Postdoctoral Fellow, ERRB, NICHD, National Institutes of Health.
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↵2 Current address: Departamento de Bioquimica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, A. P. 14-740, Mexico 07000 D. F., Mexico.
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↵3 Chief, ERRB, NICHD, National Institutes of Health.
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↵4 Current address: CNS Discovery, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium.
- Received February 5, 2003.
- Accepted May 5, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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