title: Mechanisms of melanocortin-2 receptor (MC2R) internalization and recycling in human embryonic kidney (hek) cells: identification of Key Ser/Thr (S/T) amino acids.
abstract: ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.
authors.affiliation: Service d'Endocrinologie, Département de Médecine, Université de Sherbrooke, Québec, Canada.
authors.forename: Simon
authors.initials: S
authors.lastname: Roy
authors.forename: Sébastien Jean
authors.initials: SJ
authors.lastname: Roy
authors.forename: Sandra
authors.initials: S
authors.lastname: Pinard
authors.forename: Louis-Daniel
authors.initials: LD
authors.lastname: Taillefer
authors.forename: Mohamed
authors.initials: M
authors.lastname: Rached
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Parent
authors.forename: Nicole
authors.initials: N
authors.lastname: Gallo-Payet
mesh_terms: D019390:Arrestins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D044103:Receptor, Melanocortin, Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D013912:Threonine / Q000737:chemistry* / Q000235:genetics
chemical_list: D019390:Arrestins; D044103:Receptor, Melanocortin, Type 2; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP; D034281:Dynamins