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.

Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM.

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 ...