Melanocortin 3 receptor has a 5ʹ exon that directs translation of apically localized protein from the second in-frame ATG

Melanocortin-3 receptor (MC3R) is a canonical MSH receptor that plays an essential role in energy homeostasis. Variants in MC3R have been implicated in obesity in humans and mice. However, interpretation of the functional consequences of these variants is challenging because the translational start site of MC3R is unclear. Using 5ʹ rapid amplification of cDNA ends, we discovered a novel upstream exon that extends the length of the 5ʹ untranslated region (UTR) in MC3R without changing the open-reading frame. The full-length 5ʹ UTR directs utilization of an evolutionarily conserved second in-frame ATG as the primary translation start site. MC3R synthesized from the second ATG is localized to apical membranes of polarized Madin-Darby canine kidney cells, consistent with its function as a cell surface mediator of melanocortin signaling. Expression of MC3R causes relocalization of melanocortin receptor accessory protein 2, an accessory factor for melanocortin-2 receptor, to the apical membrane, coincident with the location of MC3R. In contrast, protein synthesized from MC3R cDNAs lacking the 5ʹ UTR displayed diffuse cytosolic distribution and has no effect on the distribution of melanocortin receptor accessory protein 2. Our findings demonstrate that a previously unannotated 5ʹ exon directs translation of MC3R protein that localizes to apical membranes of polarized cells. Together, our work provides insight on the structure of human MC3R and reveals a new pathway for regulation of energy metabolism.