Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2008., The melanocortin-2 receptor or adrenocorticotropic hormone (ACTH) receptor is the main regulator of glucocorticoid synthesis. Loss of function of MC2 receptors causes a severe glucocorticoid deficiency that, if not treated, results in death. Interestingly, the MC2 receptor requires the MC2 receptor accessory protein (MRAP) for proper trafficking to the plasma membrane. These studies were performed to better understand the structure of the single transmembrane spanning protein MRAP and how it regulates melanocortin receptor trafficking and function. We show that MRAP displays a previously uncharacterized topology. Epitopes on both the N- and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N- and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure. We also showed that an epitope-tagged chaperone for odorant and taste receptors, Receptor Transporting Protein-1S (RTP-1S), also displayed dual topology, suggesting that this unusual structure may be common to several GPCR accessory proteins. We used bimolecular fluorescence complementation to ask where MRAP achieves dual topo