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Submitted on August 3, 2005
Accepted on February 3, 2006
Service d'Endocrinologie, Département de Médecine, and Département de Physiologie et Biophysique, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
* To whom correspondence should be addressed. E-mail: Nicole.Gallo-Payet{at}USherbrooke.ca.
The aim of this study was to investigate the short-term regulation of the ACTH receptor hMC2R 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 nM to 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 PKA or PKC 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). Co-transfection of M3 cells with the c-myc-tagged hMC2R and 
-arrestin2-GFP, along with sucrose treatment revealed that -arrestin2-GFP 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 while GF109203 x had no effect. In conclusion, the present results indicate that PKA and PKC act synergistically to induce hMC2R desensitization, but only PKA is essential for receptor internalization, further highlighting the complex nature of the short-term regulatory pattern of this receptor.
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