Comparative analyses of site-directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor.

Affiliation

Nakamura Y(1)(2), Asama R(2), Tabata T(2), Morita K(3), Maruyama T(3), Kondo A(1)(2)(3)(4), Ishii J(1)(2).
Author information:
(1)Engineering Biology Research Center, Kobe University, Kobe, Japan.
(2)Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
(3)Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
(4)Center for Sustainable Resource Science, RIKEN, Yokohama, Japan.

Abstract

Melatonin is an indoleamine neurohormone made by the pineal gland. Its receptors, MTNR1A and MTNR1B, are members of the G-protein-coupled receptor (GPCR) family and are involved in sleep, circadian rhythm, and mood disorders, and in the inhibition of cancer growth. These receptors, therefore, represent significant molecular targets for insomnia, circadian sleep disorders, and cancer. The yeast Saccharomyces cerevisiae is an attractive host for assaying agonistic activity for human GPCR. We previously constructed a GPCR-based biosensor employing a high-sensitivity yeast strain that incorporated both a chimeric yeast-human Gα protein and a bright fluorescent reporter gene (ZsGreen). Similar approaches have been used for simple and convenient measurements of various GPCR activities. In the current study, we constructed a fluorescence-based yeast biosensor for monitoring the signaling activation of human melatonin receptors. We used this system to analyze point mutations, including previously unreported mutations of the consensus sequences of MTNR1A and MTNR1B melatonin receptors and compared their effects. Most mutations in the consensus sequences significantly affected the signaling capacities of both receptors, but several mutations showed differences between these subtype receptors. Thus, this yeast biosensor holds promise for revealing the functions of melatonin receptors.

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