Human melanocortin receptor 2 expression and functionality: effects of protein kinase A and protein kinase C on desensitization and internalization.

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 EC(50) 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 beta-arrestin-2-green fluorescence protein along with sucrose treatment revealed that beta-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.