Comparative analyses of site‐directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor. Issue 2 (6th November 2020)

• Record Type: Journal Article
• Authors: Nakamura, Yasuyuki; Asama, Ririka; Tabata, Takuya; Morita, Kenta; Maruyama, Tatsuo; Kondo, Akihiko; Ishii, Jun
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 2(2021)
• Page Start: 863
• Page End: 876
• Publication Date: 2020-11-06
• Subjects: G‐protein signaling -- G‐protein‐coupled receptor -- melatonin receptor -- Saccharomyces cerevisiae -- site‐directed mutagenesis; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27609
• Languages: English
• ISSNs: 0006-3592

Abstract

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. Abstract : A Saccharomyces cerevisiae fluorescence‐based biosensor to detect signaling by human melatonin receptors (MTNR1A and MTNR1B) in response to melatonin was constructed in this study, which incorporates a bright fluorescent reporter (ZsGreen) and a chimeric yeast‐human Gα protein, and provides a higher dynamic range and high sensitivity compared to other biosensors. Nakamura and coworkers demonstrated the utility of the GPCR‐based biosensor for analyzing site‐directed mutants of human melatonin receptors. The constructed yeast biosensor holds promise for revealing the functions of melatonin receptors.