Prolonged signaling of backbone-modified glucagon-like peptide- 1 analogues with diverse receptor trafficking.

Brian P Cary; Marlies V Hager; Zamara Mariam; Rylie K Morris; Matthew J Belousoff; Giuseppe Deganutti; Patrick M Sexton; Denise Wootten; Samuel H Gellman

doi:10.1073/pnas.2407574122

Prolonged signaling of backbone-modified glucagon-like peptide- 1 analogues with diverse receptor trafficking.

Brian P Cary, Marlies V Hager, Zamara Mariam, Rylie K Morris, Matthew J Belousoff, Giuseppe Deganutti, Patrick M Sexton, Denise Wootten, Samuel H Gellman

Centre for Health and Life Sciences

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Abstract

Signal duration and subcellular location are emerging as important facets of G protein-coupled receptor (GPCR) function. The glucagon-like peptide-1 receptor (GLP-1R), a clinically relevant class B1 GPCR, stimulates production of the second messenger cyclic adenosine monophosphate (cAMP) upon activation by the native hormone, GLP-1. cAMP production continues after the hormone-receptor complex has been internalized via endocytosis. Here, we report GLP-1 analogues that induce prolonged signaling relative to GLP-1. A single β-amino acid substitution at position 18, with the residue derived from ( , )- -2-aminocyclopentanecarboxylic acid (ACPC), enhances signaling duration with retention of receptor endocytosis. Pairing ACPC at position 18 with a second substitution, α-aminoisobutyric acid (Aib) at position 16, abrogates endocytosis, but prolonged signaling is maintained. Prolonged signaling is sensitive to the structure of the β residue at position 18. Cryoelectron microscopy structures of two GLP-1 analogues bound to the GLP-1R:Gs complex suggest substantial alterations to bound peptide structure and dynamics compared to the GLP-1:GLP-1R:Gs complex. These structural findings strengthen an emerging view that agonist dynamics in the receptor-bound state influence signaling profiles. Our results advance understanding of the structural underpinnings of receptor activation and introduce tools for exploring the impact of spatiotemporal signaling profiles following GLP-1R activation.

Original language	English
Article number	e2407574122
Pages (from-to)	e2407574122
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	122
Issue number	14
Early online date	1 Apr 2025
DOIs	https://doi.org/10.1073/pnas.2407574122
Publication status	Published - 1 Apr 2025

Bibliographical note

Publisher Copyright:
Copyright © 2025 the Author(s).

Funding

This work was supported in part by the NIH (R01 GM056414 and its successor R35 GM151985, to S.H.G.). Additional support was provided by the Vilas Trust. P.M.S. (#1154434, #2025694) and D.W. (#1155302, #2026300) are recipients of fellowships from the National Health and Medical Research Council (NHMRC). P.M.S. is the Director and D.W. the Monash University Node leader of the Australian Research Council Industrial Transformation Training Centre for Cryo-electron Microscopy of Membrane Proteins (#IC200100052). This work was funded in part by an NHMRC Program Grant (#1150083) to P.M.S. and NHMRC Ideas Grant (#1184726) to D.W. B.P.C. and R.K.M. were supported in part by graduate fellowships from the US NSF (DGE-1747503). B.P.C. was supported in part by a Biotechnology Training Grant from the National Institute of General Medical Sciences (NIGMS) (T32 GM008349). M.V.H. was supported in part by a Chemical Biology Interface Training Grant from NIGMS (T32 GM008505). We thank Elle Grevstad for helpful discussions and assistance with optical microscopy. Confocal microscopy was performed at the University of Wisconsin-Madison Biochemistry Optical Core, which was established with support from the University of Wisconsin\u2013Madison Department of Biochemistry Endowment. Cryo-EM samples were imaged at the Bio21 Advanced Microscopy Facility (The University of Melbourne). High-performance computing was supported by Monash MASSIVE.

Funders	Funder number
University of Wisconsin- Madison
University of Melbourne
Monash University
National Health and Medical Research Council
William F Vilas Trust Estate	1154434, 1155302, 2025694, 2026300
Australian Research Council	1150083, 1184726
National Institute of General Medical Sciences	T32 GM008349, T32 GM008505
National Institutes of Health	R35 GM151985, R01 GM056414
National Science Foundation	DGE-1747503

Keywords

Glucagon-Like Peptide-1 Receptor/metabolism
Glucagon-Like Peptide 1/metabolism
Humans
Endocytosis
Signal Transduction
Protein Transport
HEK293 Cells
Cyclic AMP/metabolism
Animals
Aminoisobutyric Acids/metabolism
Cryoelectron Microscopy
peptide
GLP-1
dynamics
cryo-EM
trafficking

ASJC Scopus subject areas

General

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10.1073/pnas.2407574122Licence: CC BY-NC-ND

morris-et-al-prolonged-signaling-of-backbone-modified-glucagon-like-peptide-1-analogues-with-diverse-receptorFinal published version, 1.68 MBLicence: CC BY-NC-ND

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Cary, B. P., Hager, M. V., Mariam, Z., Morris, R. K., Belousoff, M. J., Deganutti, G., Sexton, P. M., Wootten, D., & Gellman, S. H. (2025). Prolonged signaling of backbone-modified glucagon-like peptide- 1 analogues with diverse receptor trafficking. Proceedings of the National Academy of Sciences of the United States of America, 122(14), e2407574122. Article e2407574122. https://doi.org/10.1073/pnas.2407574122

Prolonged signaling of backbone-modified glucagon-like peptide- 1 analogues with diverse receptor trafficking. / Cary, Brian P; Hager, Marlies V; Mariam, Zamara et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 122, No. 14, e2407574122, 01.04.2025, p. e2407574122.

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

Cary, BP, Hager, MV, Mariam, Z, Morris, RK, Belousoff, MJ, Deganutti, G, Sexton, PM, Wootten, D & Gellman, SH 2025, 'Prolonged signaling of backbone-modified glucagon-like peptide- 1 analogues with diverse receptor trafficking.', Proceedings of the National Academy of Sciences of the United States of America, vol. 122, no. 14, e2407574122, pp. e2407574122. https://doi.org/10.1073/pnas.2407574122

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Prolonged signaling of backbone-modified glucagon-like peptide- 1 analogues with diverse receptor trafficking.

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

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