Agonist binding affinity determines palmitoylation of the glucagon-like peptide-1 receptor and its functional interaction with plasma membrane nanodomains in pancreatic beta cells

Abstract

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 ...

Glucagon-like peptide-1 receptor controls exocytosis in chromaffin cells by increasing full-fusion events

“…We cannot offer an easy explanation to these differences; however, differences in the spatial and temporal properties of cAMP increases induced by different stimulus conditions may impact on downstream effectors, as observed in b cells (Buenaventura et al, 2019;Lester et al, 1997).…”

LUXendins reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

“…The presence of nanodomains under non-stimulated conditions might reflect differences in palmitoylation, which has recently been shown to influence GLP1R membrane distribution in response to agonists. 48 Notably, a subpopulation of β-cells appeared to possess highly-concentrated GLP1R clusters. It will be important in the future to investigate whether this is a cell autonomous heterogenous trait, or instead reflects biased orientation of membranes toward specific β-cells.…”

“…INS1 832/3 CRISPR-deleted for the endogenous GLP1R locus (a kind gift from Dr. Jacqui Naylor, MedImmune) 51 were transfected with human SNAP_GLP1R, before FACS of the SNAP-Surface488-positive population and selection using G418. 48 The resulting SNAP_GLP1R_INS1 GLP1R−/− cells were maintained in RPMI-1640 supplemented with 10% FBS, 10 mM HEPES, 2 mM L -glutamine, 1 mM pyruvate, 72 µM β-mercaptoethanol, 1% penicillin/streptomycin and 500 μg/mL G418.…”

New Insights into Beta-Cell GLP-1 Receptor and cAMP Signaling

“…The depolymerization of F-actin was previously shown to disrupt AKAP79-cadherin interaction also resulting in the removal of AKAP from synapses [159]. Ca 2+ -sensitive isoforms of AC, including AC5 and AC8, have been shown to localize to lipid rafts [160] where agonist-bound GLP-1R [107] and the protein components of store-operated Ca 2+ entry (SOCE) also reside [161]. SOCE is reported to activate AC8 and stimulate cAMP production in MIN6 insulinoma cells [156] and may also inhibit AC5 in control liver cells [162].…”

“…It has however not been unambiguously established whether this process occurs endogenously in pancreatic beta cells. Recent work has revealed characteristic differences in how incretin receptors segregate into plasma membrane nanodomains in beta cells [107]. This receptor segregation could influence the formation of Table 1.…”