Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment

Abstract

ObjectiveUpon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells.MethodsWe studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein–protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes.ResultsOur data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment.ConclusionsThe findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

Hardwiring wire-less networks: spatially encoded GPCR signaling in endocrine systems

“…For other GPCRs, such as the GLP-1 receptor (GLP-1R), the role of GPCR/Gs/arrestin in endosomal signaling and its downstream functions maybe more complex. GLP-1R/-arrestin/Gs interactions have been shown to mediate a sustained cAMP via Rab5 EEs, when activated by the ligand GLP-1, which was required for insulin secretion in a pancreatic beta-cell line [13,14]. In contrast, manipulations of the trafficking machinery that partially inhibit internalization of GLP-1R, or promote receptor recycling, improve insulin secretion, while loss of arrestins had no effect [15].…”

Glucagon-like Peptide-1 (GLP-1)

“…Following subsequent dissociation of the ligand and the arrestin, the re-sensitized receptor either travels back to the plasma membrane or is degraded in the lysosomes [437,453]. Ligand-induced internalization and recycling of GLP-1R has been demonstrated in Chinese hamster lung fibroblasts [453], in rat insulinoma cells [429,453,454], and in rat pancreatic BRIN BD11 cells [452]. In vitro studies using 125I-labeled GLP-1 (7-36amide) in rat insulinoma cells indicate that internalization of the ligand-GLP-1R complex is saturable and time-and temperature-dependent [454].…”

“…In vitro studies using 125I-labeled GLP-1 (7-36amide) in rat insulinoma cells indicate that internalization of the ligand-GLP-1R complex is saturable and time-and temperature-dependent [454]. At the plasma membrane, GLP-1R recruitment of arrestin is observed as early as 1 min after stimulation with exendin-4 [452]. Surface reexpression of GLP-1R after ligand-induced GLP-1R endocytosis appears with a half-time of 15 min with no GLP-1R endocytosis upon treatment of cells with exendin (9e39) [453].…”

“…Continued cAMP generation after GLP-1R sequestration (internalization) is accompanied by joint translocation of the activated Gas subunit along with GLP-1R into the endosome [452]. Interestingly, generation of cAMP by the internalized GLP-1R persists after recruitment of arrestin, which indicates that arrestin recruitment is not essential for GLP-1R desensitization [452].…”

“…Continued cAMP generation after GLP-1R sequestration (internalization) is accompanied by joint translocation of the activated Gas subunit along with GLP-1R into the endosome [452]. Interestingly, generation of cAMP by the internalized GLP-1R persists after recruitment of arrestin, which indicates that arrestin recruitment is not essential for GLP-1R desensitization [452]. In contrast to these findings, siRNA mediated knockdown of b-arrestin in INS-1 cells attenuates GLP-1induced phosphorylation of CREB, ERK1/2, and IRS-2 and diminishes the insulinotropic effect of GLP-1 in conditions of low (2.5 mM) and high glucose (25 mM) [429].…”