Abstract
Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome hepatic protein fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with insulin receptor (IR) internalization. The IR interactome (IRGEN) connects ATIC to AMPK within the Golgi/endosome protein network (GEN). Forty-five percent of the IR Golgi/endosome protein network have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalized within the plasma membrane (PM) and Golgi/endosome fractions after insulin stimulation and that ATIC later accumulates in the Golgi/endosome fraction. Using an in vitro reconstitution system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPK-Thr172 phosphorylation in IR complexes. We observed that IR internalization was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signaling mechanism that senses adenylate synthesis, ATP levels, and IR activation states and that acts in regulating IR autophosphorylation and endocytosis.
Highlights
Allosteric activator of AMPK, increased insulin receptor (IR) endocytosis in cultured cells and in the liver
We show here the presence of mechanism involving an ATIC protein network and the metabolite AICAR that acts in regulating IR autophosphorylation and endocytosis
We identified the following five primary proteins that interacted with internalized IR complexes 15 min after insulin stimulation: Grb14, ATIC, PTPLAD1, delta-COP, and plasmalemma vesicle-associated protein (PLVAP) (Table I, supplemental Fig. S2 and supplemental Table S2)
Summary
Reagents and Antibodies—Porcine insulin (I5523) was obtained from Sigma-Aldrich (St. Louis, MO). We used a cluster analysis to determine the dense regions of highly interconnected proteins with confident functional relations with the receptor complex (IRGEN) to analyze the environment that was most similar to that of the internalized IR. We used this method with the affinity propagation algorithm implemented in Cytoscape software using clusterMaker plugin [30]. After stopping the reaction at the indicated times, membranes were solubilized, IR was immunoprecipitated (insulin Rb (C19): sc-711, Santa Cruz Biotechnology Inc., Santa Cruz, CA), and a phosphotyrosine content analysis was performed using Western blotting. The surface trypsinization assay was performed as previously reported [32], except that the ␣22 IR tetramer was detected by Western blotting under nondenaturing conditions [33] using 5% resolving gels
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