No abstract

Pancreatic alpha-cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of alpha-cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K(+) (K(V)) and Ca(2+) (Ca(V)) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic alpha-cells and examined their targeting to specialized cholesterol-rich lipid rafts. In alpha-cells, we detected the expression of K(V)4.1/4.3 (A-type current), K(V)3.2/3.3 (delayed rectifier current), Ca(V)1.2 (L-type current), Ca(V)2.2 (N-type current), and the SNARE (synaptosomal-associated protein of 25 kDa, syntaxin 1A, and vesicle-associated membrane protein 2) and SNARE-associated proteins (Munc-13-1 and Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins (syntaxin 1A, synaptosomal-associated protein of 25 kDa, and vesicle-associated membrane protein 2) target to lipid raft domains on alpha-cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin decreased the association of K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins with lipid rafts. This resulted in inhibition of A-type K(V) currents and enhancement of glucagon secretion from alpha-cells. Consistently, capacitance measurements of exocytosis of single alpha-cells showed enhanced exocytosis after membrane cholesterol depletion. Taken together, our results demonstrate the association of K(V)4, Ca(V)1.2, and SNARE proteins with lipid rafts in pancreatic alpha-cells. Glucagon secretion from alpha-cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.

Glucagon-like peptide 1 receptor (GLP-1R) controls diverse physiological functions in tissues including the pancreatic islets, brain, and heart. To understand the mechanisms that control glucagon-like peptide 1 (GLP-1) signaling better, we sought to identify proteins that interact with the GLP-1R using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay. A screen of a human fetal brain cDNA prey library with an unliganded human GLP-1R as bait in yeast revealed 38 novel interactor protein candidates. These interactions were confirmed in mammalian Chinese hamster ovarian cells by coimmunoprecipitation. Immunofluorescence was used to show subcellular colocalization of the interactors with GLP-1R. Cluster analysis revealed that the interactors were primarily associated with signal transduction, metabolism, and cell development. When coexpressed with the GLP-1R in Chinese hamster ovarian cells, 15 interactors significantly altered GLP-1-induced cAMP accumulation. Surprisingly, all 15 proteins inhibited GLP-1-activated cAMP. Given GLP-1's prominent role as an incretin, we then focused on 3 novel interactors, SLC15A4, APLP1, and AP2M1, because they are highly expressed and localized to the membrane in mouse insulinoma β-cells. Small interfering RNA-mediated knockdown of each candidate gene significantly enhanced GLP-1-induced insulin secretion. In conclusion, we have generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling. We suggest that the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.