henhaobio
Sign in

Journal of Biological ChemistryPharmacology & ToxicologyPeer-reviewed journal article

Increased RAMP3 expression enhances GLP-1 mediated Glucose Stimulated Insulin Secretion in a Manner Dependent on Ca2+ Mobilisation

Abigail PearceiD, Poonam KumariiD, Claudia M. SiskiD, Matthew HarrisiD, Ho Yan YeungiD, Sabrina Winfield, Kathleen M. CaroniD, Graham LaddsiD

PDFDOIPreprint

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (13 Aug 2025) · CC BY 4.0

RAMP3 complexes with GLP-1R at the plasma membrane, shifts signalling toward Ca2+ mobilisation, and enhances insulin secretion in INS-1 832/3 cells; Ramp3 loss blunts GLP-1 responses in mice.

GLP-1R signalling varies with cell background and interacting proteins, which complicates drug development. Earlier GLP-1R-RAMP studies relied on lysates or whole-cell energy transfer and had not tested insulin secretion; this work examines plasma-membrane interactions and insulin secretion.

Key results

  1. ShownGLP-1R interacts at the cell surface with RAMP3 but not RAMP1 or RAMP2. The complex binds a fluorescent exendin-4 ligand without changing receptor expression or agonist affinity.

    Cell-surface NanoBRET in Cos7: saturating BRET with SNAP-RAMP3 only (n=4). Ex-4-Red Kd 6.7nM at the LgBiT-GLP-1R/SmBiT-RAMP3 complex, displaced by 1μM GLP-1; not seen with RAMP1 or RAMP2. No pKi change for any peptide (n=3). · GLP-1R specifically interacts with RAMP3; FLAG-RAMP3 has no effect on GLP-1R expression or…; Figure legends

  2. ShownIn HEK293T cells, RAMP3 expression lowered GLP-1R cAMP potency and raised Ca2+ mobilisation, in an agonist-dependent way.

    GLP-1 cAMP pEC50 fell from 11.4±0.3 to 10.4±0.2 at 8 min. GLP-1 Ca2+ pEC50 rose from 7.2±0.2 to 8.1±0.2 (p<0.05). Exendin-4 Ca2+ Emax rose from 43.6±5.30 to 71.61±4.64 percent of ionomycin at 1:1 (p<0.05) and to 97.6±6.8 on overexpression. No significant effect for liraglutide. · Second messenger signalling is influenced by…

  3. ShownTRUPATH biosensor assays point to Gαi/o as the main change, with smaller agonist-specific effects on Gαs and Gαq.

    Gαi1 coupling potency rose with increasing RAMP3 (no change for Gαz). Gαq(R183Q) maximal dissociation by GLP-1 reached 119 ±5.5% of receptor alone. Oxyntomodulin Gαs maximum fell to 58.9% ±5.8. Wild-type Gαq gave no quantifiable response. · RAMP3 expression influences G protein coupling

  4. SuggestedRAMP3 overexpression in rat INS-1 832/3 cells enhanced GLP-1 potentiation of insulin secretion. Inhibitor data suggest Gαq/11 and Gαi/o involvement and no PKA contribution; EPAC inhibition lowered secretion with or without RAMP3.

    10nM GLP-1: 2.36±0.30 fold over 2.8mM glucose (vector) vs 3.94±0.33 (RAMP3). RAMP1 had no effect. PKA inhibitor had no effect. 100nM YM-254890 and PTX reduced secretion only with RAMP3. INS-1 Ca2+ pEC50 6.94±0.11 to 7.41±0.14. · Signalling in an insulin secreting cell line; Insulin secretion is enhanced by RAMP3

  5. SuggestedReducing endogenous Ramp3 weakened GLP-1 responses in mouse systems, and the GLP-1 insulin rise and GLP-1 enhancement of insulin's glucose lowering were lost in Ramp3 knockout mice.

    Min6 B1 RAMP3 siRNA (mRNA to around 50% of control; n=4) modestly attenuated GLP-1 insulin secretion; cAMP unchanged. In male Ramp3-/- mice (n=5), the GLP-1 insulin rise 15 min after glucose was lost, as was GLP-1 enhancement of insulin's blood glucose lowering. · RAMP3 is important for GLP-1R mediated insulin…; Mouse intraperitoneal glucose tolerance test…; Figure legends

Methods, data and reproducibility

Cell-surface NanoBRET/NanoBiT interaction and ligand-binding assays, then signalling assays (cAMP, Ca2+, TRUPATH G proteins, β-arrestin, internalisation) in HEK293T and INS-1 cells. Insulin secretion was tested with inhibitors, siRNA and Ramp3-/- mice.

  • Cos7 cells for cell-surface Nluc-GPCR/SNAP-RAMP NanoBRET interaction assays · n = n=4 (Figure 1A)
  • HEK293T cells for binding, cAMP, Ca2+, TRUPATH G protein, β-arrestin and internalisation assays · n = n>3 to n>4 depending on assay
  • INS-1 832/3 rat insulinoma cells (and GLP-1R KO line) for signalling and glucose-stimulated insulin secretion · rat · n = n>7 for secretion with agonists
  • Min6 B1 mouse islet cell line with RAMP3 siRNA knockdown · mouse · n = n=4
  • Wild-type SvEv129/SJ and isogenic Ramp3-/- male mice, IPGTT and ITT with GLP-1 · mouse · n = n=5
Methods & tools
NanoBRET, NanoBiT (LgBiT/SmBiT), Tag-Lite GLP-1R red ligand (Ex-4-Red), TRUPATH G protein biosensors, LANCE ultra cAMP kit, Fluo4-AM Ca2+ assay, YM-254890, Pertussis toxin, ESI-09, RT-PCR with Fiji (ImageJ), GraphPad Prism 10, BD Accuri C6 flow cytometer
Reagents & environment
GraphPad Prism 10 used for all analyses; plasmids include TRUPATH kit (Addgene kit #1000000163).
Reproducibility gaps
Data availability statement says all data are within the manuscript; no deposited datasets, accessions or analysis code are given.
Limitations (6)
  • Partial RAMP3 knockdown in Min6 B1 cells (low endogenous expression) gave only a small attenuation of GLP-1 insulin secretion. · stated by the authors
  • HEK293T effects on cAMP differ from rodent cells (no cAMP change on RAMP3 manipulation in INS-1 or Min6 B1); the authors suggest receptor overexpression or species bias may explain this. · stated by the authors
  • Without structural interrogation, the origin of the agonist-dependent RAMP3 effects cannot be determined. · stated by the authors
  • The PKA inhibitor Rp-8-Br-cAMPs may have reduced affinity for rat kinases, so the absence of a PKA effect in INS-1 cells could reflect the compound rather than the pathway. · stated by the authors
  • In vivo evidence comes from a global Ramp3 knockout in male mice with n=5, so tissue-specific effects, sex effects and human relevance are untested. · noted in this summary
  • The pathway conclusion rests on pharmacological inhibitors in transfected INS-1 cells; no direct Ca2+ or G protein rescue experiments link Gαi/o coupling to the secretion gain. · noted in this summary

Read this if you work on GLP-1R pharmacology, GPCR-RAMP interactions or biased agonism, and want data linking RAMP3 to Ca2+ signalling and insulin secretion.

Citations
8
FWCI
2.41
Field percentile
top 10%

Abstract

The targeting of the Glucagon-like peptide-1 receptor (GLP-1R) for diabetes and obesity is not a novel strategy, with recent therapeutics showing efficacy in weight loss and glycaemic control. However, they are also associated with side effects, including gastrointestinal disruptions and pancreatitis. Developing agonists with different signalling profiles, or that exert some tissue selectivity can circumvent these on-target, unwanted effects. Receptor activity-modifying proteins (RAMPs) offer the potential to do both, through modulation of agonist binding and signalling, as well as surface expression. The GLP-1R was found to interact with RAMP3, with the heterodimer able to bind agonist at the cell surface. RAMP3 expression biased the receptor towards Ca2+ mobilisation, away from the canonical cAMP-driven signalling. When examining G protein coupling, the interaction with RAMP3 reduced activation of the cognate Gs but increased secondary couplings to Gq and Gi. These increased couplings led to an elevation in glucose-stimulated insulin secretion when cells overexpressing RAMP3 were stimulated with GLP-1. A reciprocal effect was observed when looking at reduced expression of endogenous RAMP3, with a loss of sensitivity to GLP-1 in both glucose and insulin tolerance tests in a Ramp3 knockout mouse. The effects of this interaction can then inform selection of models and peptide design when targeting this receptor for therapeutic intervention. Significance StatementG Protein-Coupled Receptors, such as the Glucagon-Like Peptide-1 (GLP-1) Receptor are common drug targets, although not without side effects due to on-target, unwanted signalling outputs. This study identifies an interacting protein which alters its signalling to promote insulin secretion. This may improve the therapeutic potential of GLP-1 mimetics, enhancing effectual signalling over that associated with unwanted effects by directly targeting this receptor complex.

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISHarris '21Lucey '19Manchanda '25Guida '20Jones '20Lucey '21
78% semantic matchPharmacology & Toxicology
M. Harris, D. I. Mackie, J. B. Pawlak+18 more·bioRxiv·Apr 8, 2021