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Submitted on January 22, 2007
Accepted on April 18, 2007
and 
isogenic HEK293 cells
Service d'Endocrinologie, Département de Médecine, 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 ACTH receptor (melanocortin-2 receptor, MC2R) is the smallest known G protein-coupled receptor (GPCR). Herein, human MC2R accessory protein (MRAP) isoforms 
and 
, cloned from a human fetal adrenal gland, were expressed with c-Myc-tagged MC2R (Myc-MC2R) in 293/FRT cells by homologous recombination. Although insertion of Myc-MC2R at the plasma membrane occurred without MRAP assistance, ACTH stimulation of cAMP production was only detected in cells co-expressing MC2R with either MRAP isoform. On the other hand, a MC2R-GFP fusion transfected with either MRAP or MRAP was impaired both in cell membrane localization and signaling. MRAP isoforms were also tagged with either Flag or 6xHis epitopes. In cell populations co-expressing transiently and/or stably Myc-MC2R with MRAP or MRAP, stimulation with ACTH induced production of cAMP with EC50 values lower in MRAP- than in MRAP-expressing cells. ACTH only bound Myc-MC2R in the presence of MRAP. Higher Myc-MC2R cell surface density was observed in the presence of MRAP comparatively to MRAP, possibly contributing to higher ACTH binding capacity and higher maximal cAMP responses observed in MRAP-expressing cells. Immunofluorescence studies indicated that MRAP isoforms were localized near the plasma membrane and in the vicinity, but not colocalized, with Myc-MC2R. In summary, through the generation of a new all-human experimental model devoid of endogenous MCRs, we present evidence that human MRAP isoforms, while not essential for MC2R localization at the plasma membrane, are essential for ACTH binding and ACTH-induced cAMP production and that they differentially regulate, although modestly, cell membrane density and functional properties of MC2R.
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