In the present study, we synthesized a novel functional analog of GLP-1 conjugated to tetramethyl rhodamine to monitor the internalization of the receptor. Our data show that after being internalized the receptor is sorted to lysosomes. In endosomes, receptor-ligand complex is found to be colocalized with adenylate cyclase. Pharmacological inhibition of endocytosis attenuates GLP-1R-mediated cAMP generation and consequent downstream protein kinase A substrate phosphorylation and glucose-stimulated insulin secretion. Our study underlines a paradigm shift in GLP-1R signaling and trafficking. The receptor ligand complex triggers cAMP generation both in plasma membrane and in endosomes, which has implications for receptor-mediated regulation of insulin secretion.

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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.

ROLE OF THE PTDINS(4)P/GOLPH3 COMPLEX IN GOLGI FORWARD TRAFFICKING GOLPH3 (also known as GMx33 and GPP34) was first discovered by proteomic studies of purified Golgi fractions (6, 7). Further investigation revealed GOLPH3 to be a peripheral membrane protein, highly localized to the trans-Golgi and to vesicles budding from the trans-Golgi (6-8). The yeast ortholog of GOLPH3, VPS74, was also found to function at the Golgi (9, 10). However, the mechanism of localization to the Golgi and the purpose of GOLPH3 at the Golgi remained uncertain. During a genome-wide, proteomic screen for phosphoinositide binding proteins, we identified GOLPH3 as a protein that binds tightly and specifically to PtdIns(4)P (see Fig. 1) (11). We found that GOLPH3 binding to PtdIns(4)P is responsible for its localization to the Golgi in yeast and mammalian cells (11). GOLPH3 is a highly abundant (10 6 molecules per cell), ubiquitously expressed protein (11, 12). Thus, it is a major effector of PtdIns(4)P at the Golgi. PtdIns(4)P was already well known to be highly enriched at the trans-Golgi (13-15). From yeast to humans, PtdIns(4) P at the Golgi is required for Golgi-to-plasma membrane trafficking (16-19). Certainly, many effectors of PtdIns(4) P besides GOLPH3 have been described. These have a variety of activities, including several that function as nonvesicular lipid transporters, and have been the subject of many previous reviews (20-22). Two phosphatidylinositol-4-kinases (PI-4-kinases), PI4KII and PI4KIII, localize to the Golgi to produce PtdIns(4)P (13, 14, 23). Currently, we lack a detailed understanding of the differential roles for Abstract GOLPH3 is a peripheral membrane protein localized to the Golgi and its vesicles, but its purpose had been unclear. We found that GOLPH3 binds specifically to the phosphoinositide phosphatidylinositol(4)phosphate [PtdIns(4) P], which functions at the Golgi to promote vesicle exit for trafficking to the plasma membrane. PtdIns(4)P is enriched at the trans-Golgi and so recruits GOLPH3. Here, a GOLPH3 complex is formed when it binds to myosin18A (MYO18A), which binds F-actin. This complex generates a pulling force to extract vesicles from the Golgi; interference with this GOLPH3 complex results in dramatically reduced vesicle trafficking. The GOLPH3 complex has been identified as a driver of cancer in humans, likely through multiple mechanisms that activate secretory trafficking. In this review, we summarize the literature that identifies the nature of the GOLPH3 complex and its role in cancer. We also consider the GOLPH3 complex as a hub with the potential to reveal regulation of the Golgi and suggest the possibility of GOLPH3 complex inhibition as a therapeutic approach in cancer.-Kuna, R. S., and S. J. Field. GOLPH3: a Golgi phosphatidylinositol(4)phosphate effector that directs vesicle trafficking and drives cancer.