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 AMPK␣2 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. Molecular & Cellular

Rat liver parenchyma Golgi/endosomes fractions harbor a tyrosine-phosphorylated 34-kDa protein. Screening of Golgi, endosomes (ENs), plasmalemma (PM), and cytosolic (Cyt) fractions revealed the presence of the mitotic kinase Cdk2 in ENs, PM, and Cyt. The fluid phase endocytic marker horseradish peroxidase gained access to the endosomal Cdk2, confirming its localization. Cdk2 was shown to be associated to cyclin E and was active in ENs and PM fractions. The administration of a single dose of insulin (1.5 g/100 g, body weight) induced a time-dependent activation of the insulin receptor kinase in these structures. Insulin receptor-kinase activation was followed by the inhibition of immunoprecipitated Cdk2-cyclin E kinase activity in PM and the progressive disappearance of cyclin E. In marked contrast, no such effect was observed in ENs. The injection of a phosphotyrosyl phosphatase inhibitor (bpV-(phen)) increased the levels of cyclin E in ENs and PM. A massive recruitment of p27 kip1 was observed in the Cdk2-cyclin E complexes isolated from PM and Cyt but not from ENs. In vitro, Cdk2-cyclin E complexes have the capacity to inhibit the formation of hybrid structures containing horseradish peroxidase and radioiodinated epidermal growth factor. Therefore, in the PM and ENs of adult rat liver, an active and regulated pool of the mitotic kinase Cdk2-cyclin E and some yet to be defined effectors are present. Cdk2 may contribute to the modulation of transport events and/or maintenance of the topology of endocytic elements.

Despite the identification of many susceptibility genes our knowledge of the underlying mechanisms responsible for complex disease remains limited. Here, we identified a type 2 diabetes disease module in endosomes, and validate it for functional relevance on selected nodes. Using hepatic Golgi/endosomes fractions, we established a proteome of insulin receptor-containing endosomes that allowed the study of physical protein interaction networks on a type 2 diabetes background. The resulting collated network is formed by 313 nodes and 1147 edges with a topology organized around a few major hubs with Cdk2 displaying the highest collective influence. Overall, 88% of the nodes are associated with the type 2 diabetes genetic risk, including 101 new candidates. The Type 2 diabetes module is enriched with cytoskeleton and luminal acidification–dependent processes that are shared with secretion-related mechanisms. We identified new signaling pathways driven by Cdk2 and PTPLAD1 whose expression affects the association of the insulin receptor with TUBA, TUBB, the actin component ACTB and the endosomal sorting markers Rab5c and Rab11a. Therefore, the interactome of internalized insulin receptors reveals the presence of a type 2 diabetes disease module enriched in new layers of feedback loops required for insulin signaling, clearance and islet biology.

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine‐phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine‐phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c‐Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [γ‐32P]ATP. The c‐Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c‐Src is protected in G/E fractions. Following injection of the protein‐tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine‐phosphorylated in an insulin‐dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c‐Src‐containing signalling platforms.

We have described a thyroid hormone receptor in synaptosomes of the chick embryo brain. To understand how the hormones exert their actions at this level, we performed a series of studies to demonstrate that this receptor could be linked to G proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)(100 muM) lowered the binding capacity of the receptor high affinity site from 8.9 +/- 1.3 to 3.4 +/- 1.3 ng T3/mg protein, a finding consistent with the coupling of receptor to G proteins. Furthermore, ADP ribosylation with pertussis toxin showed that thyroid hormones induced a dose-dependent increase in the inactive alpha 0-subunit of the G0 protein. This effect was detected at 10 pM, with a maximal increase (mean +/- SEM, 50 +/- 3.6%) at 100 nM, and T4 was as effective as T3. Both hormones also decreased the intrinsic guanine triphosphatase activity of G proteins by lowering the binding of GTP to the alpha-subunit and their rate of hydrolysis. This inhibition was greater with T4 (25 +/- 5%) than with T3 (14 +/- 2%), suggesting that the former could be the more active hormone at the synaptosomal level. The effect on guanine triphosphatase activity confirms that the synaptosomal thyroid hormone receptor is coupled to a G(zero) protein. These results demonstrate that thyroid hormones increase or favor the ADP ribosylation of G alpha(zero) by pertussis toxin. Thus, they enhance the alpha(zero)-GDP form of the G(zero) protein, namely its inactive conformation. By decreasing the activity of this protein, these hormones may modulate the formation of second messengers in synaptosomes and intervene in the regulation of neuronal proliferation and differentiation induced by several factors. Therefore, thyroid hormones may exert their action on brain maturation at least in part by modulating G alpha(zero) through their synaptosomal receptor.

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In this study we have examined the effect of neonatal hypothyroidism and triiodothyronine (T₃) replacement therapy on the maximal binding capacity (MBC) and the affinity of T₃ receptors prepared from liver, brain and lungs. Rats were radiothyroidectomized at birth and administered T₃ or its placebo starting at 8 days of age. The thyroid state in normal, hypothyroid and hypothyroid T₃-treated rats was assessed by serum determination of thyroxine, T₃ and by the measurement of the hepatic α-glycerophosphate dehydrogenase. The animals were sacrificed at 8, 16 or 24 days of age and the T₃ binding was estimated in isolated nuclei and in salt nuclear extracts. The MBC of the T₃ receptors was higher in the hypothyroid rats at all ages when it was determined in isolated nuclei, but not when was measured in nuclear extracts. At 8 days, the MBC had risen twofold or more in the receptors from brain (1.0 ± 0.37 vs. 0.4 ± 0.1 ng T₃/mg DNA in controls) and from lungs (0.6 ± 0.25 vs. 0.3 ± 0.15 ng T₃/mg DNA in controls), but was only slightly elevated in the hepatic receptor (0.62 ± 0.08 vs. 0.48 ± 0.15 ng T₃/mg DNA in controls). At 16–24 days, the highest value of MBC was observed in the hepatic receptor (0.83 ± 0.04 vs. 0.41 ± 0.1 ng T₃/mg DNA in controls) followed by the brain receptor (0.65 ± 0.03 vs. 0.35 ± 0.02 ng T₃/mg DNA in controls), and that of lung (0.39 ± 0.07 vs. 0.20 ± 0.03 ng T₃/mg DNA in controls). T₃ replacement therapy caused a marked diminution of the MBC of the hepatic receptor (0.54 ± 0.07 ng T₃/mg DNA), but had little effect on brain and lungs. No changes in affinity were observed throughout the experiments. It has been concluded that the increase of MBC in hypothyroidism probably represents a protective mechanism which provides an adequate amount of T₃ to the cellular structure responsible for initiating the hormone action. T₃ replacement therapy restores the MBC to normal in receptors from tissues (liver) that bind especially T₃ from plasma, but not in tissues which need locally generated hormone to occupy their receptors (brain and lungs).