We have previously described how signaling responses for the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR that plays key roles in metabolic regulation and is a prime type 2 diabetes (T2D) target, are modulated by intracellular membrane trafficking. We have also recently shown that binding to the therapeutic GLP-1R agonist exendin-4 (Exenatide) triggers increased clustering and segregation of biologically active GLP-1Rs into cholesterol-rich plasma membrane nanodomains that enable compartmentalization of acute receptor signaling and clathrin-mediated endocytosis. Both disruption of plasma membrane microarchitecture via cholesterol depletion and mutation of cysteine 438 GLP-1R single palmitoylation site have a substantial impact on acute GLP-1R signaling and endocytosis. 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. Here we present data on the role of three plasma membrane-localized palmitoyltransferases expressed in pancreatic beta cells in the regulation of GLP-1R responses downstream of exendin-4 stimulation. To examine this, we have engineered murine MIN6B1 beta cell lines using CRISPR/Cas9 to delete the expression of the plasma membrane palmitoyltransferases ZDHHC5, 20 and 21. We observe a reduction in exendin-4-induced cAMP production and incretin-stimulated insulin secretion in ZDHHC20 and 21 knockout compared to control cells, while ZDHHC5 deletion leads to increased cAMP and insulin responses downstream of GLP-1R activation. Experiments are underway to analyze the possible effects of knocking out these three palmitoyltransferases on the palmitoylation of the GLP-1R or related downstream effectors such as beta arrestins or G proteins, as well as on the dynamic behavior of the receptor in the lipid bilayer, in order to explain their identified impact on beta cell function.