Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G proteincoupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent highaffinity binding to GLP-1(7-36)NH 2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.allosteric modulation | biased signaling | G protein-coupled receptors | family B GPCRs G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface proteins and play crucial roles in virtually every physiological process. Their widespread abundance, yet selective distribution, and ability to couple to a variety of signaling and effector systems make them extremely attractive targets for drug development (1). Recently, there has been increasing interest in the stoichiometry of receptors involved in GPCR signaling complexes and how this may impact on receptor function and drug discovery (2-4).With the exception of family C GPCRs, where obligate dimerization can occur (4), the role of oligomerization in GPCR function has remained controversial (2-8), and this has been the subject of a number of recent reviews (9-11). Although there is an increasing body of evidence supporting dimerization of GPCRs as a widespread feature of GPCR biology, including numerous studies on family A GPCRs, whether these are stable, transient, constitutive, or ligand dependent, and how they impact on receptor function and drug discovery are less clear, and general rules for oligomeric behavior are not evident (9-16). Even where effects on signaling are studied, these are generally linked to a single pathway and the role of dimerization in the control of receptor engagement and preference for distinct intracellular signaling intermediates (i.e., signal bias) is virtually unstudied.For family B GPCRs, which encompass many ther...

Dimerization of visual pigments in vivo

“…Two dimer interfaces between rhodopsin protomers have been suggested, with the first being formed by transmembrane helices 4 and 5 (i.e., H4/H5-H4/H5) (8, 9) and the second formed by transmembrane helix 1 and cytoplasmic helix 8 (i.e., H1/H8-H1/H8) (24). To determine whether the two interfaces were involved in the R/S-opsin interaction that we observed in vivo, we used peptides derived from these R-opsin domains to see whether they would disrupt, by competition (26)(27)(28), the R/S-opsin interaction and therefore trafficking. Each peptide was encapsulated in biodegradable polylactic acid-polyethylene oxide (PLA-PEO) nanoparticles (NPs) (29) for sustained delivery and was injected subretinally into S-opsin + Rho +/− Lrat −/− mouse eyes.…”

N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway

“…Galectin-9 binding promotes cross-linking of glycoproteins and may facilitate Gcgr dimerization (52) or association with other glycopro- teins. Dimerization of the GLP-1R promotes coupling with G protein-coupled receptors and sensitivity to ligand (53). All nine mammalian adenylyl cyclases share conserved N-glycosylation sites in extracellular loops 5 and 6 (54).…”

Type 2 Diabetes Mellitus and Glucagon Like Peptide-1 Receptor Signalling

“…Dimerisation of the GLP-1R was important for signal bias and discriminated between peptide and non-peptide activation. Additionally, dimerisation was not required for allosteric modulation by compound 2 demonstrating that this small molecule agonist acted in cis [152].…”

“…There is also interest in the development of allosteric agonists and whether they interact with a single receptor (in cis) or across dimers (in trans). Currently, most drug development is dependent on an in cis conformation and mechanism of action [152,153]. The GLP-1R has been shown to form a homodimer through an interface along TM4 and is required for receptor signalling.…”