replying to J. P. Kukkonen Nature Communications 10.1038/s41467-023-38764-3 (2023)
Kukkonen challenges our interpretation of data in Figure 5d of our paper1. In this experiment, we co-transfected HEK293 cells with CMV-driven overexpression plasmids for OX2R and Gαi1 along with a pGloSensor cAMP reporter construct, and measured diminished cAMP levels with OX2R agonists after forskolin stimulation. Results from this experiment showed a weak reduction in cAMP, without a characteristic low-nanomolar EC50 for either TAK-925 or Orexin B (in contrast to the parallel inositol phosphate accumulation assay with overexpressed OX2R and Gαq). We acknowledge the validity of Kukkonen’s arguments that the cAMP data could also be explained by a combination of signaling through different G protein subtypes and/or regulation by PDEs. We also appreciate his arguments that the G protein selectivity or promiscuity exhibited by OX2R will depend on the cell line and experimental setup, and has not been definitively established in CNS neurons.
Acknowledgements
Portions of the original research were supported by the National Institutes of Health (R01NS097594 to J.K.D.B. and D.M.R.; R35GM116387 to D.M.R.), the Welch Foundation (I-1422 to J.K.D.B.; I-1770 to D.M.R.; I-1944 to X.B.), the Edward Mallinckrodt, Jr. Foundation (Scholar Award to D.M.R.), and the Julie and Louis Beecherl, Jr., Chair in Medical Science at The University of Texas Southwestern (to J.K.D.B.). The original OX2R-mGsqiN cryo-EM data were collected at The University of Texas Southwestern Medical Center Cryo-EM Facility, which is funded by the CPRIT Core Facility Support Award RP170644.
Author contributions
J.Y., Y.K., M.F., J.K.D.B., and D.M.R. designed the original research. J.Y., Y.K., K.C., Y.S., R.R., and P.L. performed experiments. J.Y., Y.K., A.P.M., K.C., E.K., A.O., T.K., Y.M., Y.S., R.R., P.L., X.B., J.K.D.B., and D.M.R. analyzed data. J.Y., Y.K., A.P.M., M.S., J.K.D.B., T.K., M.F., and D.M.R. wrote the original paper. Authors from The University of Texas Southwestern Medical Center (J.Y., K.M., R.R., P.L., X.B., J.K.D.B., D.M.R.) obtained structural data for OX2R-mGsqiN bound to TAK-925. Authors from Takeda Pharmaceutical Company Limited (Y.K., A.P.M., M.S., E.K., A.O., T.K., Y.M., Y.S., M.F.) obtained structural data for OX2R-Gi1 bound to TAK-925. All authors contributed to this reply.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work.
Competing interests
J.Y., K.C., R.R., P.L., X.B., J.K.D.B., and D.M.R. declare no competing interests. Y.K., A.P.M., M.S., E.K., A.O., T.K., Y.M., Y.S., and M.F. are current or former employees of Takeda Phamaceutical Company Limited.
Footnotes
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These authors contributed equally: Jie Yin, Yanyong Kang, Aaron P. McGrath.
Contributor Information
Mack Flinspach, Email: mflinspach@gmail.com.
Jef K. De Brabander, Email: jef.debrabander@utsouthwestern.edu
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Reference
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