TY - JOUR
T1 - Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols
AU - Bean, Björn D M
AU - Mulvihill, Colleen J
AU - Garge, Riddhiman K
AU - Boutz, Daniel R
AU - Rousseau, Olivier
AU - Floyd, Brendan M
AU - Cheney, William
AU - Gardner, Elizabeth C
AU - Ellington, Andrew D
AU - Marcotte, Edward M
AU - Gollihar, Jimmy D
AU - Whiteway, Malcolm
AU - Martin, Vincent J J
N1 - Funding Information:
The authors thank Ian Riddington and the mass spectrometry facility at the University of Texas at Austin for their feedback and assistance with GC-MS sample preparation and analysis, and Josh Lutgens at the University of Texas at Austin for setting up and automating combinatorial DNA assembly using an Echo acoustic liquid handling robot. This study was financially supported by FRQNT Team and NSERC Discovery grants to V.J.J.M. and M.W. B.D.M.B. was supported by a Concordia University Horizon Postdoctoral Fellowship, V.J.J.M. is supported by a Concordia University Senior Research Chair and M.W. is supported by a Canada Research Chair. Financial support was also provided by a Cooperative Agreement between the University of Texas at Austin and DEVCOM Army Research Laboratory to A.D.E., E.M.M., and J.D.G. (W911NF-17-2-0091). A.D.E. was also provided support from the NIH’s National Center for Complementary and Integrative Health (5R21AT010777-01,02). R.K.G was supported by the American Heart Association Predoctoral fellowship (#18PRE34060258). E.M.M. acknowledges additional support from the Welch Foundation (F-1515); Army Research Office (W911NF-12-1-0390); and NIH (R35 GM122480). The authors also acknowledge the Center for Microscopy and Cellular Imaging funded by Concordia University, Montréal, Canada, and the Canada Foundation for Innovation.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/5/24
Y1 - 2022/5/24
N2 - The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.
AB - The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.
KW - Cholesterol/metabolism
KW - Humans
KW - Phytosterols/metabolism
KW - Receptors, Opioid/metabolism
KW - Receptors, Opioid, kappa/agonists
KW - Receptors, Opioid, mu/metabolism
KW - Saccharomyces cerevisiae/genetics
KW - Sterols/metabolism
UR - http://www.scopus.com/inward/record.url?scp=85130705062&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85130705062&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-30570-7
DO - 10.1038/s41467-022-30570-7
M3 - Article
C2 - 35610225
SN - 2041-1723
VL - 13
SP - 2882
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 2882
ER -