Determinants of the unique antiparallel homodimer structure of MRAP

Expression of the melanocortin‐2 (MC2) GPCR on the plasma membrane requires MC2 Receptor Accessory Protein (MRAP), a newly recognized protein with a single transmembrane domain. We have shown that endogenous MRAP is inserted in opposite orientations across the membrane and forms antiparallel homodimers. This extraordinary topology raises two fundamental questions: how is dual orientation achieved, and is this topology is required for MRAP function? We engineered chimeras between RAMP3, which is exclusively Nout, and MRAP. Neither the transmembrane domain nor the C‐terminus of MRAP was necessary for dual topology, but the transmembrane domain was essential for MRAP function. Remarkably, the N‐terminus of MRAP conferred dual orientation to RAMP3. Using MRAP deletion mutants, we identified two regions in the N‐terminus of MRAP that promoted one or the other orientation. In every case, loss of dual topology resulted in loss of function. To ask if MRAP is in a dual orientation from the time it is synthesized in the endoplasmic reticulum (ER), we fused MRAP to “split YFP” molecules. MRAP fused N‐terminally to an incomplete YFP and MRAP fused C‐terminally to a complementary YFP fragment were not fluorescent alone but were fluorescent in the ER and on the plasma membrane when expressed together. We conclude that the dual topology of MRAP is coded within the N‐terminal domain, achieved in the ER, and essential for function.