The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

Background G protein‐coupled receptors (GPCRs) play crucial roles in regulating islet function, with Gαs‐ and Gαq‐coupled receptors being linked to the stimulation of insulin secretion. We have quantified the mRNA expression of 384 non‐olfactory GPCRs in islets isolated from lean and obese organ donors to determine alterations in islet GPCR mRNA expression in obesity. Methods RT‐qPCR was used to quantify GPCR mRNAs relative to five reference genes (ACTB, GAPDH, PPIA, TBP, and TFRC) in human islets isolated from lean (BMI = 22.6 ± 0.5) and obese (BMI = 32.0 ± 0.8) donors. Results Overall, 197 and 256 GPCR mRNAs were detected above trace level in islets from lean and obese donors, respectively, with 191 GPCR mRNAs being common to the lean and obese groups. 40.9% (n = 157) and 27.1% (n = 104) of the mRNAs were expressed at trace level whilst 7.8% and 6.3% were absent in islets from lean and obese donors, respectively. Hundred and seventeen GPCR mRNAs were upregulated at least twofold in islets from obese donors, and there was >twofold downregulation of 21 GPCR mRNAs. Of particular interest, several receptors signalling via Gαs or Gαq showed significant mRNA upregulation in islets from obese donors (fold increase: PTH2R: 54.0 ± 14.6; MC2R: 34.3 ± 11.5; RXFP1: 8.5 ± 2.1; HTR2B: 6.0 ± 2.0; GPR110: 3.9 ± 1.2; PROKR2: 3.9 ± 0.7). Conclusions Under conditions of obesity, human islets showed significant alterations in mRNAs encoding numerous GPCRs. The increased expression of Gαs‐ and Gαq‐coupled receptors that have not previously been investigated in β‐cells opens up possibilities of novel therapeutic candidates that may lead to the potentiation of insulin secretion and/or β‐cell mass to regulate glucose homeostasis.

30The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 31 2 diabetes and obesity, is known to undergo palmitoylation by covalent ligation of an 32 acyl chain to cysteine 438 in its carboxyl-terminal tail. Work with other GPCRs 33 indicates that palmitoylation can be dynamically regulated to allow receptors to 34 partition into plasma membrane nanodomains that act as signaling hotspots. Here, 35we demonstrate that the palmitoylated state of the GLP-1R is increased by agonist 36 binding, leading to its segregation and clustering into plasma membrane signaling 37 nanodomains before undergoing internalization in a clathrin-dependent manner. Both 38 GLP-1R signaling and trafficking are modulated by strategies targeting nanodomain 39 segregation and cluster formation, including depletion of cholesterol or expression of 40 a palmitoylation-defective GLP-1R mutant. Differences in receptor binding affinity 41 exhibited by biased GLP-1R agonists, and modulation of binding kinetics with the 42 positive allosteric modulator BETP, influence GLP-1R palmitoylation, clustering, 43 nanodomain signaling, and internalization. Downstream effects on insulin secretion 44 from pancreatic beta cells indicate that these processes are relevant to GLP-1R 45 physiological actions and might be therapeutically targetable. 46 47 48 49 50 51 52 53 54 55 56 Introduction 57 58 G protein-coupled receptors (GPCRs), the largest membrane receptor family in 59 eukaryotes (1), are integral membrane proteins and, as such, both their physical 60 organization and their signaling properties are modulated by the lipid composition of 61 the surrounding membrane (2, 3). The localization of GPCRs to dynamic membrane 62nanodomains has been widely reported (4-6). These nanodomains, or membrane 63 rafts, which cannot be directly observed in living cells with current methods (7), are 64 often described as highly organized detergent-resistant, liquid-ordered, 65 glycosphingolipid-and cholesterol-rich platforms where receptor-signaling complexes 66 become compartmentalized, facilitating efficient coupling with G proteins (5, 8, 9). 67Additionally, most GPCRs are modified post-translationally with one or more palmitic 68 acid chains linked covalently, but reversibly, via a thioester bond to cysteines within 69 the intracellular domain of the receptor, in a process known as palmitoylation (2, 10). 70The insertion of acyl chain(s) is regulated by families of acyltransferases (DHHCs) 71 and palmitoyl protein thioesterases (11, 12), and can be either constitutive or 72 modulated by agonist binding (13, 14). GPCR palmitoylation at or near the end of its 73 carboxyl-terminal (C-terminal) tail creates a new membrane anchor and a further 74 intracellular loop (15) that modifies the GPCR structure and its interactions with 75 specific intracellular partners, favoring receptor partitioning into plasma membrane 76 nanodomains (2, 16, 17). 78We have previously described how signaling responses of the glucagon-like peptide-79 1 receptor (GLP-1R), a ...

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