Identification of amino acids that are selectively involved in Gi/o activation by rat melanin-concentrating hormone receptor 1.

Many G-protein-coupled receptors (GPCRs) are known to functionally couple to multiple G-protein subfamily members. Although promiscuous G-protein coupling enables GPCRs to mediate diverse signals, only a few GPCRs have been identified with differential determinants for coupling to distinct Gα proteins. Mammalian melanin-concentrating hormone receptor 1 (MCHR1) couples to dual G-protein subfamilies. However, the selectivity mechanisms between MCHR1 and different subtypes of Gα proteins are unclear. Our previous studies demonstrated that mammalian MCHR1 couples to both Gi/o and Gq, whereas goldfish MCHR1 exclusively couples to Gq. In this study, we analyzed multiple sequence alignments between rat and goldfish MCHR1s, and designed three multisubstituted mutants of rat MCHR1 by replacing corresponding residues with those in goldfish MCHR1, focusing on regions around the cytosolic intracellular loops. By measurement of intracellular Ca(2+) mobilization, we found that two MCHR1 mutants, i2_6sub and i3_6sub, which contained six simultaneously substituted residues in the second intracellular loop or a combination of substituted residues in the third intracellular loop and fifth transmembrane domain, respectively, significantly reduced Gi/o-sensitive pertussis toxin responsiveness without altering Gq-mediated activity. Analyses of 10 other substitutions revealed that the multiple substitutions in i2_6sub and i3_6sub were necessary for Gi/o-selective responses. As judged by Gi/o-dependent GTPγS binding and cyclic AMP assays, i2_6sub and i3_6sub elicited phenotypes for impaired Gi/o-mediated signaling. We also monitored the dynamic mass redistribution (DMR) in living cells, which reveals receptor activity as an optical trace containing activation of all GPCR coupling classes. Cells transfected with i2_6sub or i3_6sub exhibited reduced Gi/o-mediated DMR responses compared with those transfected with MCHR1. These data suggest that two different regions independently affect the Gi/o-protein preference, and that multiple residues comprise a conformation favoring Gi/o-protein coupling and subsequently result in Gi/o-selective signaling.