The class B1 subfamily of G-protein-coupled receptors (GPCRs) comprises 15 members that respond to endogenous peptides to regulate an array of important physiological processes. Activation by peptide ligands is considered to follow a 2-step binding mechanism that involves an initial interaction with the receptor ectodomain (ECD) followed by engagement with transmembrane regions that collectively result in effector coupling. This model suggests that the ECD acts as an affinity trap that generally does not regulate downstream signaling events. Here, we show that members of the glucagon receptor subfamily of class B1 GPCRs contain a conserved 6-amino acid motif within their ECDs and that individual residues regulate cAMP and/or βarr pathways by either allosterically promoting effector coupling or as determinants required for proper receptor expression and function via modulation of posttranslational receptor glycosylation. We also highlight that this conserved motif surprisingly alters observed signaling phenomena in both a ligand- and receptor-dependent manner. Our study unveils an unexpected role of the ECD in regulating class B1 GPCR signaling properties independently of ligand binding affinity and suggests that the well-established 2-step binding paradigm requires further investigation to understand the nuances of activation mechanisms for this receptor class. SIGNIFICANCE STATEMENT: Class B1 G-protein-coupled receptors respond to endogenous peptide ligands to fine-tune biological processes. These receptors contain the hallmark structural feature of a large ectodomain at their N-termini that has been shown to be critical for ligand binding affinity, but whether this region regulates downstream signaling remains unclear. We identify a conserved site within the glucagon-like peptide-1 receptor, glucagon receptor, and glucose-dependent insulinotropic polypeptide receptor localized to the ectodomain that modulates both effector coupling as well as proper receptor expression and functionality in ligand- and receptor-dependent manners. Given the benefit of targeting these receptors in the treatment of type 2 diabetes and obesity, our findings may provide insights toward a better understanding of their activation mechanisms and facilitate rational drug discovery approaches.
Copyright © 2026 The Author(s). Published by Elsevier Inc. All rights reserved.

Parkinson's disease (PD) is a progressive neurological disorder that affects movement, causing symptoms such as tremors, stiffness, slowness, and balance problems due to the degeneration of dopamine-producing neurons in the brain. Nowadays there is no cure for PD. Alpha synuclein (α-syn) aggregates, which are a hallmark of PD, are known to induce microglial activation, specifically the detrimental M1 microglial phenotype, which contributes to neuroinflammation and disease progression. Cannabinoid receptor 2 (CB2R) activation has been shown to counteract neuroinflammation. CB2R is able to interact with N-methyl-D-aspartate (NMDA) receptors (NMDAR), which has also attracted attention in PD research due to its role in excitotoxicity. Here we aimed to study the interaction between CB2R and NMDAR in a PD context in rat tissue. We observed that α-syn fibrils alter CB2R activation and CB2R-NMDAR heteromerization in a heterologous expression system. Furthermore, activation of CB2R counteracted NMDAR signaling. In microglia, α-syn fibrils decreased CB2R-NMDAR heteromer expression while increasing CB2R signaling. Importantly, CB2R activation counteracted the α-syn fibrils-induced increase in M1-activated microglia, while it favored the polarization of microglia to the beneficial M2 phenotype. These results reinforce the idea of using cannabinoids for treating PD, as they provide not only the anti-inflammatory effects of cannabinoids but also counteract the detrimental increase in NMDAR signaling present in this disease.

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.
© 2024, Oqua et al.

Parkinson’s disease (PD) is a progressive neurological disorder that affects movement, causing symptoms such as tremors, stiffness, slowness, and balance problems due to the degeneration of dopamine-producing neurons in the brain. Nowadays there is no cure for PD. Alpha synuclein (α-syn) aggregates, which are a hallmark of PD, are known to induce microglial activation, specifically the detrimental M1 microglial phenotype, which contributes to neuroinflammation and disease progression. Cannabinoid receptor 2 (CB 2 R) activation has been shown to counteract neuroinflammation. CB2R is able to interact with NMDA receptors (NMDAR), which has also attracted attention in PD research due to its role in excitotoxicity. Here we aimed to study the interaction between CB 2 R and NMDAR in a PD context. We observed that α-syn fibrils alter CB2R activation and CB 2 R-NMDAR heteromerization in a heterologous expression system. Furthermore, activation of CB2R counteracted NMDAR signaling. In neurons α-syn fibrils decreased CB2R-NMDAR heteromer expression, while increasing CB 2 R signaling. Importantly, CB 2 R activation counteracted the α-syn fibrils-induced increase in M1 activated microglia, while it favored the polarization of microglia to the beneficial M2 phenotype. These results reinforce the idea of using cannabinoids for treating PD, as they provide not only the anti-inflammatory effects of cannabinoids but also counteract the detrimental increase in NMDAR signaling present in this disease.

The class B1 G protein-coupled receptor (GPCR) subfamily is a class of receptors known for their regulatory roles in metabolism and neuronal activity important as drug targets. Lipids play key functional roles in modulation of GPCR signalling, yet our understanding of the molecular level detail of specific lipid interactions with class B1 GPCRs remains limited. Here we present coarse-grained molecular dynamics (MD) simulations of the active and inactive states of 15 human class B1 family members and use aiida-gromacs to capture full provenance for the set-up of simulations in complex plasma membranes. Receptors exhibit state-dependent lipid interactions with the regulatory sterol cholesterol and phospholipid phosphatidylinositiol-4,5-bisphosphate [PI(4,5)P 2 ] at defined locations on the receptor surface. Global analysis of trends across the subfamily reveals conserved patterns of lipid interaction dynamics. The glycosphingolipid GM3 exerts a modulatory influence on the dynamics of class B1 extracellular domains in both simulations and in vitro time-resolved FRET assays. Teaser Characterisation of regulatory lipid interactions with all class B1 GPCRs.