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The Melanocortin 2 Receptor Accessory Protein Exists as a Homodimer and Is Essential for the Function of the Melanocortin 2 Receptor in the Mouse Y1 Cell Line

Centre for Endocrinology (S.N.C., I.A.D.V., T.R.W., J.P.C., A.J.L.C.) and Clinical Pharmacology (K.-Y.L.), William Harvey Research Institute, Barts and the London, London EC1M 6BQ, United Kingdom; School of Biological and Chemical Sciences (I.A.D.V., M.E., M.R.E.), Queen Mary University of London, London E1 1BB, United Kingdom; and Division of Molecular and Cellular Neuroscience (M.E.C.), University College of London Institute of Ophthalmology, London EC1V 9EL, United Kingdom

Address all correspondence and requests for reprints to: Professor Adrian J. L. Clark, William Harvey Research Institute, Barts and the London, Queen Mary University of London, West Smithfield, London EC1M 6BQ, United Kingdom. E-mail: a.j.clark{at}qmul.ac.uk.

The ACTH receptor [melanocortin 2 receptor (MC2R)] gene produces a functional receptor only when transfected into cells of adrenocortical origin, implying that it may require an adrenal-specific accessory factor. Recently we showed that the MC2R accessory protein (MRAP) is essential for the cell surface expression of the MC2R in such models. Using RNA interference (RNAi) technology, we have further explored the action of MRAP in the functioning of the MC2R in Y1 mouse adrenocortical cells that endogenously express MRAP and MC2R. We created stable cell lines expressing mouse MRAP short hairpin RNA (shRNAs) by transfecting cells with an expression vector containing the MRAP small interfering RNA sequence. The knockdown of MRAP resulted in a reduction in MC2R signaling. The overexpression of a mouse MRAP-Flag construct did not restore the expression of MRAP due to its degradation by the mouse shRNAs. The introduction of human MRAP that is resistant to silencing by mouse MRAP shRNAs resulted in the rescue of the MC2R signaling. MRAP migrates on SDS-PAGE with markedly lower mobility than predicted for a 14.1-kDa protein. Coimmunoprecipitation and mass spectroscopy suggests that MRAP exists as a homodimer that is resistant to dissociation by sodium dodecyl sulfate and reducing agents.