Mutagenesis of human Mel1a melatonin receptor expressed in yeast reveals domains important for receptor function.

A yeast functional colorimetric assay was employed to test the effects of site-directed point mutations on the function of the human Mel1a melatonin receptor. Seven mutants were created in transmembrane domains III, V, and VII of the receptor to test the rhodopsin-based model of melatonin recognition. Two mutants in transmembrane domains III and VI were created to investigate the mechanisms of G protein activation in the melatonin receptor. Mutations in transmembrane domain V either potentiated agonist efficiencies (H195A) or totally abolished all responses to tested compounds (V192T+H195A). Mutation N124A in the conserved NRY motif in the end of transmembrane domain III seriously impaired receptor activation. Several mutants were found to have decreased ability to activate functional responses, reflecting the importance of these residues for receptor function. These data also suggest that activation of the receptor involves interaction of the 5-methoxy group of melatonin with the conserved histidine H195 in transmembrane domain V.