Endocytosis following dopamine D2 receptor activation is critical for neuronal activity and dendritic spine formation via Rabex-5/PDGFRß signaling in striatopallidal medium spiny neurons.

Aberrant dopamine D2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/platelet-derived growth factor receptor-ß (PDGFRß)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFRß are then transported to the Golgi apparatus, where those complexes trigger Gαi3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFRß is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.

Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in the young, is an autosomal dominant disease characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Linkage studies and candidate-gene approaches have demonstrated that about half of the patients have mutations in one of six disease genes: cardiac beta-myosin heavy chain (c beta MHC), cardiac troponin T (cTnT), alpha-tropomyosin (alpha TM), cardiac myosin binding protein C (cMBPC), ventricular myosin essential light chain (vMLC1) and ventricular myosin regulatory light chain (vMLC2) genes. Other disease genes remain unknown. Because all the known disease genes encode major contractile elements in cardiac muscle, we have systematically characterized the cardiac sarcomere genes, including cardiac troponin I (cTnI), cardiac actin (cACT) and cardiac troponin C (cTnC) in 184 unrelated patients with HCM and found mutations in the cTnI gene in several patients. Family studies showed that an Arg145Gly mutation was linked to HCM and a Lys206Gln mutation had occurred de novo, thus strongly suggesting that cTnI is the seventh HCM gene.

Review on the in vitro interaction of insulin glargine with the insulin/insulin-like growth factor system: potential implications for metabolic and mitogenic activities.

Insulin analogues provide clinically important benefits for people with diabetes, including more predictable action profiles and lower risk of hypoglycemia compared with human insulin. However, it has been suggested that certain insulin analogues may lead to greater activation of insulin-like growth factor-1 (IGF-1) signaling, with risk for adverse mitogenic effects. This article aims to critically review studies on the mitogenic effects of the insulin analogue insulin glargine (glargine) and its metabolites. A review of in vitro studies suggests that glargine may stimulate mitogenic activity in some cell lines at supraphysiological concentrations (nanomolar/micromolar concentrations). Mitogenicity appeared to be related to the expression of the IGF-1 receptor, being present in cells expressing high levels of the receptor and absent in cells with limited or no IGF-1 receptor expression. In animal studies, glargine did not promote tumor growth, despite administration at supraphysiological concentrations (nanomolar/micromolar), which are unlikely to be observed in clinical practice because the doses needed to produce these concentrations are liable to lead to hypoglycemia. Furthermore, glargine in vivo is rapidly transformed into its metabolites, the metabolic and mitogenic characteristics of which have been shown to be broadly equal to those of human insulin. Thus, the suggestion of increased relative mitogenic potency of insulin glargine seen in some cell lines does not appear to carry over to the in vivo situation in animals and humans.

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

NMDA receptor-dependent refinement of somatotopic maps.

We have examined the role of NMDA receptor-mediated neural activity in the formation of periphery-related somatosensory patterns, using genetically engineered mice. We demonstrate that ectopic expression of a transgene of an NMDAR1 splice variant rescues neonatally fatal NMDAR1 knockout (KO) mice, although the average life span varies depending on the level of the transgene expression. In NMDAR1 KO mice with "high" levels of the transgene expression, sensory periphery-related patterns were normal along both the trigeminal and dorsal column pathways. In the KO mice with "low" levels of the transgene expression, the patterns were absent in the trigeminal pathway. Our results indicate that NMDA receptor-mediated neural activity plays a critical role in pattern formation along the ascending somatosensory pathways.

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity.

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

Caveolin-3 deficiency decreases the gene expression level of osteopontin in mdx mouse skeletal muscle.

Caveolin-3 is a muscle-specific membrane protein that serves as a scaffold of various molecules. As previously reported, caveolin-3 deficiency causes muscle degeneration in mice. In the present study, gene expression profiles, analyzed in the skeletal muscles of caveolin-3 deficient mice using the DNA microarray technique, showed that the gene of osteopontin, a versatile regulator of inflammation and tissue repair, was significantly down-regulated. This is in contrast to mdx mice showing a markedly up-regulated osteopontin gene in their skeletal muscles. Recently, osteopontin has been reported to be important in the pathogenesis of muscular dystrophy. We examined whether up-regulated osteopontin gene expression in mdx muscles is altered by the deficiency of caveolin-3. To this end, we developed caveolin-3 and dystrophin double-deficient mice and used them for the analysis. Levels of osteopontin mRNA and protein in the double-deficient mice clearly decreased compared with those in mdx mice.

Dopamine D2 long receptor-deficient mice display alterations in striatum-dependent functions.

The dopamine D2 receptor (D2) system has been implicated in several neurological and psychiatric disorders, such as schizophrenia and Parkinson's disease. There are two isoforms of the D2 receptor: the long form (D2L) and the short form (D2S). The two isoforms are generated by alternative splicing of the same gene and differ only by 29 amino acids in their protein structures. Little is known about the distinct functions of either D2 isoform, primarily because selective pharmacological agents are not available. We generated D2L receptor-deficient (D2L-/-) mice by making a subtle mutation in the D2 gene. D2L-/- mice (which still express functional D2S) displayed reduced levels of locomotion and rearing behavior. Interestingly, haloperidol produced significantly less catalepsy and inhibition of locomotor activity in D2L-/- mice. These findings suggest that D2L and D2S may contribute differentially to the regulation of certain motor functions and to the induction of the extrapyramidal side effects associated with the use of typical antipsychotic drugs (e.g., haloperidol). Quinpirole induced a similar initial suppression of locomotor activity in both D2L-/- and wild-type mice. In addition, the D2S receptor in the mutant mice functioned approximately equally well as did D2L as an impulse-modulating autoreceptor. This suggests that the functions of these two isoforms are not dependent on the formation of receptor heterodimers. Our findings may provide novel information for potentially developing improved antipsychotic drugs.

Alteration of insulin-receptor kinase activity by high-fat feeding.

It has been demonstrated in in vivo and in vitro experiments that high-fat (HF) feeding causes insulin resistance. To elucidate the mechanism for this effect, we have measured the kinase activity of the insulin receptor purified from livers of HF-fed rats that showed impaired insulin action in isolated rat adipocytes. In adipocyte experiments, HF feeding led to a 65% decrease in the maximal response stimulated by insulin in a 2-deoxyglucose uptake study. Although insulin binding to adipocytes of HF-fed rats also decreased to 50% of control due to decreased binding affinity, the postbinding defect should be accounted for by decreased insulin action in view of the presence of spare receptor. In contrast to adipocytes, insulin binding to the lectin-purified insulin receptor from livers showed no difference in receptor-binding affinity between HF-fed and control rats. Insulin-stimulated phosphorylation of the beta-subunit of the insulin receptor was decreased to almost 50% throughout the entire dose-response curve. The study of glutamine-tyrosine (4:1) phosphorylation by the insulin-receptor kinase showed results similar to those of the autophosphorylation study. These results suggest that an HF diet causes insulin resistance by affecting insulin-receptor kinase, which plays an important role in transmembrane signaling between insulin binding and insulin action.

Insulin resistance by uncleaved insulin proreceptor. Emergence of binding site by trypsin.

Two sisters presented with severe insulin resistance and markedly decreased insulin binding to erythrocytes, cultured fibroblasts, and transformed lymphocytes. The dose-response curve of insulin-stimulated amino acid uptake in the fibroblasts was shifted to the right. The molecular weight of the insulin receptor on the transformed lymphocytes from the patients was 210,000 and could not be dissociated to alpha- and beta-subunits by dithiothreitol treatment. However, the proreceptor was cleaved by trypsin, and this led to production of a 135,000-Mr alpha-subunit. Insulin binding to the trypsin-treated cells increased to the normal level, and insulin action was normalized. These results suggest that the failure of proreceptor cleavage produces hormone-resistant states and that a proreceptor syndrome may be a unique disease entity for hormone resistance.

Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor.

Receptor binding and biological action of insulin and insulin-like growth factor I (IGF-I) were studied in fibroblasts from a patient with leprechaunism and a patient with type A syndrome of insulin resistance. Insulin binding was reduced to 18.8 and 27.7% of control value, respectively. In contrast, IGF-I binding was normal in both patients. In competitive binding studies, IGF-I had 0.2% of the ability of insulin to compete with 125I-labeled insulin binding, and insulin had 0.1% of the ability of IGF-I to compete with 125I-labeled IGF-I binding in control subjects and patient fibroblasts. The dose-response curves of insulin stimulation assessed by glucose incorporation and alpha-aminoisobutyric acid uptake showed normal responsiveness, and ED50 was significantly shifted to the right in fibroblasts from both patients. However, normal responsiveness and sensitivity were observed in thymidine incorporation studies. For IGF-I, dose-response curves of glucose incorporation, alpha-aminoisobutyric acid uptake, and thymidine incorporation were all normal in both patients. These results indicate that 1) the defect is specific to the insulin-receptor binding in these patients, 2) insulin and IGF-I activate glucose incorporation and alpha-aminoisobutyric acid uptake mainly through their own specific receptors, but 3) the IGF-I receptor appears to have a more important role in stimulating thymidine incorporation than the insulin receptor in physiological condition or, alternatively, an unknown postreceptor process with cascade signal transmission may overcome the decreased insulin-receptor binding to produce a normal dose-response curve.

Growth hormone induces cellular insulin resistance by uncoupling phosphatidylinositol 3-kinase and its downstream signals in 3T3-L1 adipocytes.

Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1-associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110(CAAX)) or Akt activation stimulated by platelet-derived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1-associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1-associated PI 3-kinase.

Pioglitazone ameliorates tumor necrosis factor-alpha-induced insulin resistance by a mechanism independent of adipogenic activity of peroxisome proliferator--activated receptor-gamma.

Tumor necrosis factor (TNF)-alpha is one of the candidate mediators of insulin resistance associated with obesity, a major risk factor for the development of type 2 diabetes. The insulin resistance induced by TNF-alpha is antagonized by thiazolidinediones (TZDs), a new class of insulin-sensitizing drugs. The aim of the current study was to dissect the mechanism whereby pioglitazone, one of the TZDs, ameliorates TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes. Pioglitazone restored insulin-stimulated 2-deoxyglucose (DOG) uptake, which was reduced by TNF-alpha, with concomitant restorations in tyrosine phosphorylation and protein levels of insulin receptor (IR) and insulin receptor substrate (IRS)-1, as well as association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 and PI 3-kinase activity. Adenovirus-mediated gene transfer of either wild-type human peroxisome proliferator-activated receptor (PPAR)-gamma2 or a mutant carrying a replacement at the consensus mitogen-activated protein kinase phosphorylation site (hPPAR-gamma2-S112A) promoted adipogenesis of 3T3-L1 fibroblasts and restored TNF-alpha-induced decrease of triglyceride in adipocytes as effectively as pioglitazone. Overexpression of the PPAR-gamma proteins in TNF-alpha-treated adipocytes restored protein levels of IR/IRS-1, but did not improve insulin-stimulated tyrosine phosphorylation of IR/IRS-1 or insulin-stimulated 2-DOG uptake. These results indicate that the ability of pioglitazone to restore insulin-stimulated tyrosine phosphorylation of IR/IRS-1, which is necessary for amelioration of TNF-alpha-induced insulin resistance, may be independent of the adipogenic activity of PPAR-gamma that regulates protein levels of IR/IRS-1.

Intracellular signalling pathways of okadaic acid leading to mitogenesis in Rat1 fibroblast overexpressing insulin receptors: okadaic acid regulates Shc phosphorylation by mechanisms independent of insulin.

Okadaic acid is a powerful inhibitor of serine/threonine protein phosphatases 1 and 2A. Although it is known as a potent tumour promoter, the intracellular mechanism by which okadaic acid mediates its mitogenic effect remains to be clarified. We investigated the effect of okadaic acid on the activation of mitogenesis in Rat1 fibroblasts overexpressing insulin receptors. As previously reported, insulin induced Shc phosphorylation, Shc-Grb2 association, MAP kinase activation, and BrdU incorporation. Okadaic acid also stimulated tyrosine phosphorylation of Shc and its subsequent association with Grb2 in a time- and dose-dependent manner without affecting tyrosine phosphorylation of insulin receptor beta-subunit and IRS. However, to a lesser extent, okadaic acid stimulated MAP kinase activity and BrdU incorporation. Interestingly, preincubation of okadaic acid potentiated insulin stimulation of tyrosine phosphorylation of Shc (213% of control), Shc-Grb2 association (150%), MAP kinase activity (152%), and BrdU incorporation (148%). These results further confirmed the important role of Shc, but not IRS, in cell cycle progression in Rat1 fibroblasts. Furthermore, serine/ threonine phosphorylation appears to be involved in the regulation of Shc tyrosine phosphorylation leading to mitogenesis by mechanisms independent of insulin signalling.

Delivery of alpha- and beta-sarcoglycan by recombinant adeno-associated virus: efficient rescue of muscle, but differential toxicity.

The sarcoglycanopathies are a group of four autosomal recessive limb girdle muscular dystrophies (LGMD 2D, 2E, 2C, and 2F), caused by mutations of the alpha-, beta-, gamma-, or delta-sarcoglycan genes, respectively. The delta-sarcoglycan-deficient hamster has been the most utilized model for gene delivery to muscle by recombinant adeno-associated virus (AAV) vectors; however, human patients with delta-sarcoglycan deficiency are exceedingly rare, with only two patients described in the United States. Here, we report construction and use of AAV vectors expressing either alpha- or beta-sarcoglycan, the genes responsible for the most common forms of the human sarcoglycanopathies. Both vectors showed successful short-term genetic, biochemical, and histological rescue of both alpha- and beta-sarcoglycan-deficient mouse muscle. However, comparison of persistence of expression in 51 injected mice showed substantial differences between AAV alpha-sarcoglycan (alpha-SG) and beta-sarcoglycan (beta-SG) vectors. AAV-beta-SG showed long-term expression with no decrease in expression for more than 21 months after injection, whereas AAV-alpha-SG showed a dramatic loss of positive fibers between 28 and 41 days post-injection (p = 0.006). Loss of immunopositive myofibers was correlated with significant inflammatory cell infiltrate, primarily macrophages. To determine whether the loss of alpha-sarcoglycan-positive fibers was due to an immune response or cytotoxic effect of alpha-sarcoglycan overexpression, severe combined immunodeficient (SCID) mouse muscle was assayed for cytotoxicity after injection with AAV-alpha-SG, AAV-beta-SG, or phosphate-buffered saline. The results were consistent with overexpression of alpha-sarcoglycan causing significant cytotoxicity. The cytotoxicity of alpha-sarcoglycan, and not beta- or delta-sarcoglycan overexpression, was consistent with biochemical studies of the hierarchical order of assembly of the sarcoglycan complex. Our data suggest that even closely related proteins might require different levels of expression to avoid toxicity and achieve long-term tissue rescue.

Functional characterization of hybrid receptors composed of a truncated insulin receptor and wild type insulin-like growth factor 1 or insulin receptors.

To assess the characteristics of hybrid receptors composed of one kinase-inactive alpha beta-insulin half-receptor and one endogenous alpha beta-insulin-like growth factor 1 (IGF-1) or insulin half-receptor, a cell line expressing an insulin receptor truncated by 365 amino acids (HIR delta 978) was studied, which lacks most of the cytoplasmic beta-subunit. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions revealed four distinct receptor species: endogenous receptors, the more rapidly migrating HIR delta 978 homodimer, and two intermediate species representing HIR delta 978/IGF-1 hybrid receptors and HIR delta 978/IR hybrid receptors. In vivo ligand-binding affinity of the hybrid receptors was studied by receptor-ligand cross-linking, and the delta 978/IGF-1R hybrid receptor was found to have a high affinity for IGF-1, whereas its affinity for insulin was low. Autophosphorylation studies of lectin-purified receptors revealed that neither the HIR delta 978 holoreceptor nor the hybrid receptors underwent autophosphorylation in response to either ligand, despite the presence of intact IGF-1 or insulin half-receptors in the hybrids. Neither hybrid receptor underwent ligand-induced endocytosis, as assessed with the bioactive photoaffinity probes B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin and N-epsilon B28-monoazidobenzoyl-IGF-1. In conclusion, the HIR delta 978/IGF-1R hybrid receptor has a high in vivo affinity for IGF-1 but not for insulin. Neither the delta 978/IGF-1R nor the delta 978/IR hybrids undergo autophosphorylation or ligand-induced endocytosis in response to either ligand, indicating that intramolecular trans-, rather than cis-, signal transduction is important in mediating autophosphorylation and endocytosis.

Insulin and epidermal growth factor influence guanine nucleotide-releasing factor by distinct mechanisms.

Both insulin and epidermal growth factor (EGF) significantly increased the activation of p21Ras in Rat-1 fibroblasts transfected with human insulin receptors. As both growth factors have been shown to stimulate guanine nucleotide exchange on p21Ras in permeabilized cells, we examined their effects on total cellular guanine nucleotide exchange factor (GEF) activity in a direct assay, using the rate of dissociation of [3H]GDP from p21Ras as a measure of GEF activity. Insulin increased GEF activity in a time-dependent manner, whereas EGF had no effect on GEF activity at any time point studied. To assess whether EGF stimulates guanine nucleotide exchange by recruiting Grb-2-Sos complexes to the plasma membrane, we measured GEF activity in both the cytosol and the plasma membrane fractions of the insulin- and EGF-treated cells. Insulin increased GEF activity in both fractions (48 +/- 12% [3H]GDP released vs. 24 +/- 6% in control plasma membranes, and 65 +/- 13% vs. 13 +/- 4% in control cytosolic fractions), whereas EGF enhanced only the plasma membrane-associated activity (43 +/- 12% of [3H]GDP release in the plasma membrane fraction and 10 +/- 2% in the cytosol). Western blotting of the subcellular fractions with Grb-2 and Sos antibodies revealed translocation of these elements to the plasma membrane after stimulation of cells with either insulin or EGF. Thus, whereas insulin stimulates guanine nucleotide exchange on Ras by both translocating Grb-2-Sos complexes to the plasma membrane and increasing the GEF activity of Sos, EGF does so by causing a translocation of Grb-2-Sos without increasing GEF activity.

A truncated human insulin receptor missing the COOH-terminal 365 amino acid residues does not undergo insulin-mediated receptor migration or aggregation.

A previous study of tyrosine kinase-defective insulin receptors demonstrated that receptor autophosphorylation or tyrosine kinase activity was required for concentrating insulin receptors in coated pits, but not for their migration or aggregation on the cell surface. Furthermore, receptor migration and aggregation on the cell surface were not sufficient to cause internalization of the occupied receptors in coated pits. In the present study, biochemical and ultrastructural techniques were used to compare insulin receptor mobility and internalization in Rat 1 fibroblasts expressing wild-type human insulin receptors (HIRc) with those in cells expressing receptors truncated at residues 978 (HIR delta 978) or 1301 of the carboxyl-terminus (HIR delta CT). There were no significant differences in the mobility or internalization of insulin receptors on HIR delta CT cells compared to those of insulin receptors on HIRc cells. Ultrastructural analysis revealed that truncated insulin receptors on HIR delta 978 cells failed to migrate from their initial location on the microvilli, move to the plasma membrane, and aggregate in coated pits. Receptor-mediated insulin internalization in HIR delta 978 cells was markedly decreased due entirely to a decrease in ATP-dependent, coated pit-mediated internalization. ATP-independent endocytosis in non-coated pinocytotic invaginations was not affected by receptor truncations. These results provide evidence of the roles that regions of the beta-subunit play in the processing of occupied insulin receptors. 1) The carboxyl-terminus of the insulin receptor is not involved in the events leading to receptor internalization, i.e. migration, aggregation, and concentration in coated pits. 2) Internalization of insulin receptors by the ATP-independent noncoated invagination pathway is not regulated by residues in the insulin receptor beta-subunit distal to 978. 3) Sequences in the beta-subunit between 978-1300, but not the autophosphorylation and kinase domains, are involved in insulin-induced receptor migration and aggregation.

Evidence for functional roles of Crk-II in insulin and epidermal growth factor signaling in Rat-1 fibroblasts overexpressing insulin receptors.

We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.

Role of the Src homology 2 (SH2) domain and C-terminus tyrosine phosphorylation sites of SH2-containing inositol phosphatase (SHIP) in the regulation of insulin-induced mitogenesis.

To examine the role of SHIP in insulin-induced mitogenic signaling, we used a truncated SHIP lacking the SH2 domain (deltaSH2-SHIP) and a Y917/1020F-SHIP (2F-SHIP) in which two tyrosines contributing to Shc binding were mutated to phenylalanine. Wild-type (WT)-, deltaSH2-, and 2F-SHIP were transiently transfected into Rat1 fibroblasts overexpressing insulin receptors (HIRc). Insulin-stimulated tyrosine phosphorylation of WT-SHIP and deltaSH2-SHIP, whereas tyrosine phosphorylation of 2F-SHIP was not detectable, indicating that 917/1020-Tyr are key phosphorylation sites on SHIP. Although SHIP can bind via its 917/1020-Tyr residues and SH2 domain to Shc PTB domain and 317-Tyr residue, respectively, insulin-induced SHIP association with Shc was more greatly decreased in 2F-SHIP cells than that in deltaSH2-SHIP cells. Insulin stimulation of Shc association with Grb2, which is important for p21ras-MAP kinase activation, was decreased by overexpression of WT- and 2F-SHIP. Importantly, insulin-induced Shc x Grb2 association was not detectably reduced in deltaSH2-SHIP cells. In accordance with the extent of Shc association with Grb2, insulin-induced MAP kinase activation was relatively decreased in both WT-SHIP and 2F-SHIP cells, but not in deltaSH2-SHIP cells. To examine the functional role of SHIP in insulin's biological action, insulin-induced mitogenesis was compared among these transfected cells. Insulin stimulation of thymidine incorporation and bromodeoxyuridine incorporation was decreased in WT-SHIP cells compared with that of control HIRc cells. Expression of 2F-SHIP also significantly reduced insulin-induced mitogenesis, whereas it was only slightly affected by overexpression of deltaSH2-SHIP. Furthermore, the reduction of insulin-induced mitogenesis in WT-SHIP cells was partly compensated by coexpression of Shc. These results indicate that SHIP plays a negative regulatory role in insulin-induced mitogenesis and that the SH2 domain of SHIP is important for its negative regulatory function.