Evolutionary Identification of the Requirement of the Second Intracellular Loop for the Constitutive Activity of Melanocortin-4 Receptors

Melanocortin-4 receptor (MC4R) functions as a crucial neuroendocrine G protein-coupled receptor (GPCR) in the central nervous system of mammals, displaying agonist-independent constitutive activity that is mainly determined by its N-terminal domain. We previously reported that zebrafish MC4R exhibited a much higher basal cAMP level in comparison to mammalian MC4Rs. However, the functional evolution of constitutive activities in chordate MC4Rs remains to be elucidated. Here we cloned and compared the constitutive activities of MC4Rs from nine vertebrate species and showed that the additive action of the N-terminus with the extracellular region or transmembrane domain exhibited a combined pharmacological effect on the MC4R constitutive activity. In addition, we demonstrated that four residues of F149, Q156, V163, and K164 of the second intracellular loop played a vital role in determining MC4R constitutive activity. This study provided novel insights into functional evolution and identified a key motif essential for constitutive modulation of MC4R signaling.