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Human Melanocortin Receptor 2 Expression and Functionality: Effects of Protein Kinase A and Protein Kinase C on Desensitization and Internalization

Service d’Endocrinologie, Département de Médecine (Z.K., N.G.-P.), and Département de Physiologie et Biophysique (N.B., P.K., M.D.P.), Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service d’Endocrinologie, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12^e avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet{at}usherbrooke.ca.

The aim of this study was to investigate the short-term regulation of the ACTH receptor human (h) melanocortin receptor 2 (MC2R) by transfection of a c-Myc-tagged hMC2R in the M3 cell line and assess its membrane expression by indirect immunofluorescence. Stimulation with ACTH induced production of cAMP with EC₅₀ values ranging from 7.6–11.9 nM in transient and stable transfectants, respectively. Pretreatment with ACTH induced a dose-dependent loss of cAMP production, from 1 pM up to 10 nM. Desensitization was also time dependent, with 70% loss of maximal responsiveness occurring after 15-min pretreatment with 10 nM ACTH, followed by a plateau up to 60 min. The decrease in hMC2R responsiveness was abrogated by individual treatment with protein kinase A (PKA) or protein kinase C inhibitors, H-89 and GF109203X. However, when added simultaneously, receptor responsiveness was raised over the maximal hMC2R activity observed in control cells. ACTH-induced loss of cAMP production was accompanied by receptor sequestration into intracellular vesicles (maximum after 30-min exposure). Cotransfection of M3 cells with the c-Myc-tagged hMC2R and ß-arrestin-2-green fluorescence protein along with sucrose treatment revealed that ß-arrestin-2-green fluorescence protein and c-Myc-hMC2R were redistributed in similar intracellular vesicles through a clathrin-dependent, but caveolae-independent, process. Sucrose pretreatment blocked receptor desensitization, indicating that hMC2R desensitization and internalization are interrelated. Moreover, preincubation with H-89 abrogated hMC2R internalization, whereas GF109203X had no effect. In conclusion, the present results indicate that PKA and protein kinase C act synergistically to induce hMC2R desensitization, but only PKA is essential for receptor internalization, highlighting the complex nature of the short-term regulatory pattern of this receptor.

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