Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314

Fabian Bumbak; James B. Bower; Skylar C. Zemmer; Asuka Inoue; Miquel Pons; Juan Carlos Paniagua; Fei Yan; James Ford; Hongwei Wu; Scott A. Robson; Ross A.D. Bathgate; Daniel J. Scott; Paul R. Gooley; Joshua J. Ziarek

doi:10.1038/s41467-023-38894-8

Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314

Fabian Bumbak^*, James B. Bower, Skylar C. Zemmer, Asuka Inoue, Miquel Pons, Juan Carlos Paniagua, Fei Yan, James Ford, Hongwei Wu, Scott A. Robson, Ross A.D. Bathgate, Daniel J. Scott, Paul R. Gooley, Joshua J. Ziarek^*

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

The neurotensin receptor 1 (NTS₁) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS₁ structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used ¹³C^εH₃-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS₁:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

Original language	English (US)
Article number	3328
Journal	Nature communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-38894-8
State	Published - Dec 2023

Funding

We are grateful to Prof. Aashish Manglik (UCSF) for providing the \u03B2Arr1 construct used in this study. We would like to thank Prof. Andrew L. Lee (UNC) for fruitful discussions. The 14.1\u00A0T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. Studies at the Florey Institute were supported by the Victorian Government\u2019s Operational Infrastructure Support Program. The project was funded by: KAKENHI 21H04791 (A.I.), 21H051130 (A.I.), and JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.), and BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST Program JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigaci\u00F3n, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovaci\u00F3n, Mar\u00EDa de Maeztu grant CEX2021-001202-M (J.C.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.).; and National Institutes of Health (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). We are grateful to Prof. Aashish Manglik (UCSF) for providing the \u03B2Arr1 construct used in this study. We would like to thank Prof. Andrew L. Lee (UNC) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. Studies at the Florey Institute were supported by the Victorian Government\u2019s Operational Infrastructure Support Program. The project was funded by: KAKENHI 21H04791 (A.I.), 21H051130 (A.I.), and JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.), and BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST Program JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigaci\u00F3n, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovaci\u00F3n, Mar\u00EDa de Maeztu grant CEX2021-001202-M (J.C.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.).; and National Institutes of Health (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.).

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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10.1038/s41467-023-38894-8

Cite this

Bumbak, F., Bower, J. B., Zemmer, S. C., Inoue, A., Pons, M., Paniagua, J. C., Yan, F., Ford, J., Wu, H., Robson, S. A., Bathgate, R. A. D., Scott, D. J., Gooley, P. R., & Ziarek, J. J. (2023). Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314. Nature communications, 14(1), Article 3328. https://doi.org/10.1038/s41467-023-38894-8

@article{609b77c75e234c478d1576b703a415b7,

title = "Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314",

abstract = "The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor{\textquoteright}s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.",

author = "Fabian Bumbak and Bower, {James B.} and Zemmer, {Skylar C.} and Asuka Inoue and Miquel Pons and Paniagua, {Juan Carlos} and Fei Yan and James Ford and Hongwei Wu and Robson, {Scott A.} and Bathgate, {Ross A.D.} and Scott, {Daniel J.} and Gooley, {Paul R.} and Ziarek, {Joshua J.}",

note = "Funding Information: We are grateful to Prof. Aashish Manglik (UCSF) for providing the βArr1 construct used in this study. We would like to thank Prof. Andrew L. Lee (UNC) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. Studies at the Florey Institute were supported by the Victorian Government{\textquoteright}s Operational Infrastructure Support Program. The project was funded by: KAKENHI 21H04791 (A.I.), 21H051130 (A.I.), and JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.), and BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST Program JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigaci{\'o}n, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovaci{\'o}n, Mar{\'i}a de Maeztu grant CEX2021-001202-M (J.C.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.).; and National Institutes of Health (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). Funding Information: We are grateful to Prof. Aashish Manglik (UCSF) for providing the βArr1 construct used in this study. We would like to thank Prof. Andrew L. Lee (UNC) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. Studies at the Florey Institute were supported by the Victorian Government{\textquoteright}s Operational Infrastructure Support Program. The project was funded by: KAKENHI 21H04791 (A.I.), 21H051130 (A.I.), and JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.), and BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST Program JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigaci{\'o}n, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovaci{\'o}n, Mar{\'i}a de Maeztu grant CEX2021-001202-M (J.C.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.).; and National Institutes of Health (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). Publisher Copyright: {\textcopyright} 2023, The Author(s).",

year = "2023",

month = dec,

doi = "10.1038/s41467-023-38894-8",

language = "English (US)",

volume = "14",

journal = "Nature communications",

issn = "2041-1723",

publisher = "Nature Research",

number = "1",

}

Bumbak, F, Bower, JB, Zemmer, SC, Inoue, A, Pons, M, Paniagua, JC, Yan, F, Ford, J, Wu, H, Robson, SA, Bathgate, RAD, Scott, DJ, Gooley, PR & Ziarek, JJ 2023, 'Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314', Nature communications, vol. 14, no. 1, 3328. https://doi.org/10.1038/s41467-023-38894-8

TY - JOUR

T1 - Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314

AU - Bumbak, Fabian

AU - Bower, James B.

AU - Zemmer, Skylar C.

AU - Inoue, Asuka

AU - Pons, Miquel

AU - Paniagua, Juan Carlos

AU - Yan, Fei

AU - Ford, James

AU - Wu, Hongwei

AU - Robson, Scott A.

AU - Bathgate, Ross A.D.

AU - Scott, Daniel J.

AU - Gooley, Paul R.

AU - Ziarek, Joshua J.

N1 - Funding Information: We are grateful to Prof. Aashish Manglik (UCSF) for providing the βArr1 construct used in this study. We would like to thank Prof. Andrew L. Lee (UNC) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. Studies at the Florey Institute were supported by the Victorian Government’s Operational Infrastructure Support Program. The project was funded by: KAKENHI 21H04791 (A.I.), 21H051130 (A.I.), and JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.), and BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST Program JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigación, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovación, María de Maeztu grant CEX2021-001202-M (J.C.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.).; and National Institutes of Health (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). Funding Information: We are grateful to Prof. Aashish Manglik (UCSF) for providing the βArr1 construct used in this study. We would like to thank Prof. Andrew L. Lee (UNC) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. Studies at the Florey Institute were supported by the Victorian Government’s Operational Infrastructure Support Program. The project was funded by: KAKENHI 21H04791 (A.I.), 21H051130 (A.I.), and JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.), and BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST Program JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigación, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovación, María de Maeztu grant CEX2021-001202-M (J.C.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.).; and National Institutes of Health (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). Publisher Copyright: © 2023, The Author(s).

PY - 2023/12

Y1 - 2023/12

N2 - The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

AB - The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

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DO - 10.1038/s41467-023-38894-8

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VL - 14

JO - Nature communications

JF - Nature communications

IS - 1

M1 - 3328

ER -

Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314

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