The C-terminal domains of melanocortin-2 receptor (MC2R) accessory proteins (MRAP1) influence their localization and ACTH-induced cAMP production.

Abstract

ACTH binding to the human melanocortin-2 receptor (MC2R) requires the presence of the MC2R accessory protein1 isoforms, MRAPα or MRAPβ. This study evaluated the role of the isoform-specific C-terminal domains of MRAP with regard to their cellular localization, topology, interaction with MRAP2 and cAMP production. When stably expressed in HEK293/FRT cells or in B16-G4F mouse melanoma cells (an MSH receptor-deficient cell clone), MRAPα and MRAPdCT (truncated MRAP1, N-terminal only) localized mainly around the nuclear envelope and within dense intracellular endosomes, while MRAPβ exhibited a strong localization at the plasma membrane, and partially with rapid recycling endosomes. MRAPβ and MRAPdCT both exhibited dual-topology (N(cyto)/C(exo) and N(exo)/C(cyto)) at the plasma membrane whereas MRAPα exhibited only N(cyto)/C(exo) topology at the plasma membrane while adopting dual-topology in intracellular compartments. Both MRAPα and MRAP2 colocalized in intracellular compartments, as opposed to weak colocalization between MRAPβ and MRAP2. MRAP2 and MC2R enhanced the expression of MRAP1 isoforms and vice versa. Moreover, in both HEK293/FRT and B16-G4F cells, ACTH failed to activate MC2R unless MRAP1 was present. MRAP1 expression enhanced MC2R cell-surface expression as well as concentration-dependent cAMP accumulation. In the presence of human or zebrafish MC2R, MRAPβ induced the highest cAMP accumulation while MRAPdCT induced the lowest. Together, the present findings indicate that the C-terminal domains of MRAP dictate their intracellular localization in addition to regulating ACTH-induced cAMP production. These preferential localizations suggest that MRAPα is involved in MC2R targeting to the plasma membrane, while MRAPβ may enhance ACTH-MC2R coupling to cAMP production.

• Authors: Simon Roy, Sébastien J Roy, Sandra Pinard, Maria Josep Agulleiro, José Miguel Cerdá-Reverter, Jean-Luc Parent, Nicole Gallo-Payet
• Journal: General and comparative endocrinology (Gen Comp Endocrinol) Vol. 176 Issue 2 Pg. 265-74 (Apr 01 2012) ISSN: 1095-6840 [Electronic] United States
• PMID: 22366472 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier Inc. All rights reserved.

Chemical References

• MRAP protein, human
• Membrane Proteins
• Protein Isoforms
• Receptor, Melanocortin, Type 2
• Adrenocorticotropic Hormone
• Cyclic AMP

Topics

• Adrenocorticotropic Hormone (pharmacology)
• Animals
• Blotting, Western
• Cell Line
• Cyclic AMP (metabolism)
• Enzyme-Linked Immunosorbent Assay
• Humans
• Immunoprecipitation
• Membrane Proteins (chemistry, metabolism)
• Mice
• Microscopy, Fluorescence
• Protein Binding
• Protein Isoforms
• Receptor, Melanocortin, Type 2