Functions of DPLIY motif and helix 8 of human melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a member of the family A G protein-coupled receptors (GPCRs). The MC3R remains the most enigmatic of the melanocortin receptors with regard to its physiological functions, especially its role in energy homeostasis. The N/DPxxY motif and the eighth helix (helix 8) in the carboxyl terminus of GPCRs have been identified to be important for receptor expression, ligand binding, signal transduction and internalization. To gain a better understanding of the structure-function relationship of MC3R, we performed a systematic study of all 20 residues in this domain using alanine-scanning mutagenesis. We showed that although all mutants were expressed normally on the cell surface, eleven residues were important for ligand binding and one was indispensable for downstream cAMP generation. F347A showed constitutive activity in cAMP signaling while all the other mutants had normal basal activities. We studied the signaling capacity of nine mutants in the ERK1/2 signaling pathway. All of these mutants showed normal basal ERK1/2 phosphorylation levels. The pERK1/2 levels of six binding- or signaling-defective mutants were enhanced upon agonist stimulation. The unbalanced cAMP and pERK1/2 signaling pathways suggested the existence of biased signaling in MC3R mutants. In summary, we showed that the DPLIY motif and helix 8 was important for MC3R activation and signal transduction. Our data led to a better understanding of the structure-function relationship of MC3R.