Insulin receptor substrate-1 pleckstrin homology and phosphotyrosine-binding domains are both involved in plasma membrane targeting.

The localization of insulin receptor substrate (IRS) molecules may be responsible for the differential biological activities of insulin and other peptides such as platelet-derived growth factor. The subcellular localization of IRS-1 is controversial, with some reports suggesting association with the cytoskeleton and other studies reporting membrane localization. In this study, we used immunofluorescence microscopy to define the localization of IRS-1. In the basal state, recombinant IRS-1 was localized predominantly in the cytoplasm. In response to insulin, recombinant IRS-1 translocated to the plasma membrane. We have also studied the localization of green fluorescent protein (GFP) fusion proteins. Unlike native IRS-1, a fusion protein containing GFP plus full-length IRS-1 appeared to localize in inclusion bodies. In contrast, when GFP was fused to the N terminus of IRS-1 (i.e. the pleckstrin homology and phosphotyrosine-binding domains), this fusion protein was targeted to the plasma membrane. Mutations of phosphoinositide-binding sites in both the pleckstrin homology and phosphotyrosine-binding domains significantly reduced the ability of Myc-tagged IRS-1 to translocate to the plasma membrane following insulin stimulation. However, these mutations did not cause a statistically significant impairment of tyrosine phosphorylation in response to insulin. This raises the possibility that IRS-1 tyrosine phosphorylation may occur prior to plasma membrane translocation.

Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases.

Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

Activation of serum- and glucocorticoid-induced protein kinase (Sgk) by cyclic AMP and insulin.

Sgk (serum- and glucocorticoid-induced protein kinase) is a serine/threonine-specific protein kinase that is transcriptionally regulated by serum, glucorticoids, and mineralocorticoids. Sgk regulates the amiloride-sensitive sodium channel in kidney principal cells. Insulin and insulin-like growth factor-1 stimulate activity of Sgk by a mechanism mediated by phosphoinositide-dependent kinases (PDK)-1 and -2. In this study, we demonstrate that incubation of transfected cells with 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 0.2 mm) led to a 2-fold activation of recombinant Sgk expressed in COS7 cells. Furthermore, the combination of insulin plus 8CPT-cAMP elicited a larger response than either agent alone. The effect of insulin was inhibited by wortmannin (100 nm), but not by the cyclic AMP-dependent protein kinase (PKA) inhibitor, H89 (10 microm). As expected, the effect of 8CPT-cAMP was completely blocked by H89. Surprisingly, the effect of 8CPT-cAMP was also inhibited by wortmannin, suggesting that phosphorylation of Sgk by PDK-1 and/or -2 is required for activation by 8CPT-cAMP. Mutational analysis led to similar conclusions. The Thr(369) --> Ala mutant, lacking the PKA phosphorylation site, was activated by insulin but not 8CPT-cAMP. In contrast, the Ser(422) --> Ala mutant, lacking a PDK-2 phosphorylation site, was inactive and resistant to activation by either insulin or 8CPT-cAMP. In summary, Sgk is subject to complex regulatory mechanisms. In addition to regulation at the level of gene expression, the enzymatic activity of Sgk is regulated by multiple protein kinases, including PKA, PDK-1, and PDK-2. Cross-talk among these signaling pathways may play an important role in the pathogenesis of the hypertension associated with hyperinsulinemia, obesity, and insulin resistance.

Identification and characterization of SNX15, a novel sorting nexin involved in protein trafficking.

Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.

Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes.

Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.

Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory beta-cell hyperplasia.

To investigate the role of insulin receptor substrate (IRS)-2 in vivo, we generated IRS-2-deficient mice by gene targeting. Although homozygous IRS-2-deficient mice (IRS-2-/- mice) had a body weight similar to wild-type mice, they progressively developed type 2 diabetes at 10 weeks. IRS-2-/- mice showed insulin resistance and a defect in the insulin-stimulated signaling pathway in liver but not in skeletal muscle. Despite insulin resistance, the amount of beta-cells was reduced to 83% of that in wild-type mice, which was in marked contrast to the 85% increase in the amount of beta-cells in IRS-1-deficient mice (IRS-1-/- mice) to compensate for insulin resistance. Thus, IRS-2 plays a crucial role in the regulation of beta-cell mass. On the other hand, insulin secretion by the same number of cells in response to glucose measured ex vivo was significantly increased in IRS-2-/- mice compared with wild-type mice but was decreased in IRS-1-/- mice. These results suggest that IRS-1 and IRS-2 may play different roles in the regulation of beta-cell mass and the function of individual beta-cells.

Lipoatrophy revisited.

The lipoatrophy syndromes are a heterogeneous group of syndromes characterized by a paucity of adipose tissue. Severe lipoatrophy is associated with insulin-resistant diabetes mellitus (DM). The loss of adipose tissue can have a genetic, immune, or infectious/drug-associated etiology. Causative mutations have been identified in patients for one form of partial lipoatrophy--Dunnigan-type familial partial lipodystrophy. Experiments using lipoatrophic mice demonstrate that the diabetes results from the lack of fat and that leptin deficiency is a contributing factor. Thiazolidinedione therapy improves metabolic control in lipoatrophic patients; the efficacy of leptin treatment is currently being investigated.

Efficacy and safety of troglitazone in the treatment of lipodystrophy syndromes.

BACKGROUND: Troglitazone promotes adipocyte differentiation in vitro and increases insulin sensitivity in vivo. Therefore, troglitazone may have therapeutic benefit in lipoatrophic diabetes. OBJECTIVE: To determine whether troglitazone ameliorates hyperglycemia and hypertriglyceridemia or increases fat mass in lipoatrophic patients. DESIGN: Open-labeled prospective study. SETTING: United States and Canada. PATIENTS: 20 patients with various syndromes associated with lipoatrophy or lipodystrophy. INTERVENTION: 6 months of therapy with troglitazone, 200 to 600 mg/d. MEASUREMENTS: Levels of hemoglobin A1c triglycerides, free fatty acids, and insulin; respiratory quotient; percentage of body fat; liver volume; and regional fat mass. RESULTS: In the 13 patients with diabetes who completed 6 months of troglitazone therapy, hemoglobin A1c levels decreased by a mean of 2.8% (95% CI, 1.9% to 3.7%; P < 0.001). In all 19 study patients, fasting triglyceride levels decreased by 2.6 mmol/L (230 mg/dL) (CI, 0.7 to 4.5 mmol/L [62 to 398 mg/dL]; P = 0.019) and free fatty acid levels decreased by 325 micromol/L (CI, 135 to 515 micromol/L; P = 0.035). The respiratory quotient decreased by a mean of 0.12 (CI, 0.08 to 0.16; P < 0.001), suggesting that troglitazone promoted oxidation of fat. Body fat increased by a mean of 2.4 percentage points (CI, 1.3 to 4.5 percentage points; P = 0.044). Magnetic resonance imaging showed an increase in subcutaneous adipose tissue but not in visceral fat. In one patient, the serum alanine aminotransferase level increased eightfold during the 10th months of troglitazone treatment but normalized 3 months after discontinuation of treatment Liver biopsy revealed an eosinophilic infiltrate, suggesting hypersensitivity reaction as a cause of hepatotoxicity. CONCLUSION: Troglitazone therapy improved metabolic control and increased body fat in patients with lipoatrophic diabetes. The substantial benefits of troglitazone must be balanced against the risk for hepatotoxicity, which can occur relatively late in the treatment course.

Characterization of Drosophila insulin receptor substrate.

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitation experiments demonstrated insulin-dependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.

Mutational and haplotype analyses of families with familial partial lipodystrophy (Dunnigan variety) reveal recurrent missense mutations in the globular C-terminal domain of lamin A/C.

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.

Final results of phase III trial in regionally advanced unresectable non-small cell lung cancer: Radiation Therapy Oncology Group, Eastern Cooperative Oncology Group, and Southwest Oncology Group.

STUDY OBJECTIVES: The purpose of this phase III clinical trial was to test whether chemotherapy followed by radiation therapy resulted in superior survival to either hyperfractionated radiation or standard radiation in surgically unresectable non-small cell lung cancer. DESIGN: Patients were prospectively randomized to 2 months of cisplatin, vinblastine chemotherapy followed by 60 Gy of radiation at 2.0 Gy per fraction or 1.2 Gy per fraction radiation delivered twice daily to a total dose of 69.6 Gy, or 2.0 Gy per fraction of radiation once daily to 60 Gy. Patients were enrolled from January 1989 through January 1992, and followed for a potential minimum period of 5 years. SETTING: This trial was an intergroup National Cancer Institute-funded trial within the Radiation Therapy Oncology Group, the Eastern Cooperative Oncology Group, and the Southwest Oncology Group. PATIENTS: Patients with surgically unresectable non-small cell lung cancer, clinical stage II, IIIA, and IIIB, were required to have a Karnofsky Performance Status of > or = 70 and a weight loss of < 5% for 3 months before study entry. Four hundred ninety patients were registered on trial, of which 458 patients were eligible. CONCLUSION: Overall survival was statistically superior for the patients receiving chemotherapy and radiation vs the other two arms of the study. The twice-daily radiation therapy arm, although better, was not statistically superior in survival for those patients receiving standard radiation. Median survival for standard radiation was 11.4 months; for chemotherapy and irradiation, 13.2 months; and for hyperfractionated irradiation, 12 months. The respective 5-year survivals were 5% for standard radiation therapy, 8% for chemotherapy followed by radiation therapy, and 6% for hyperfractionated irradiation.

Overexpression of a novel sorting nexin, SNX15, affects endosome morphology and protein trafficking.

Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins. Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking. Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments. In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures. Later, myc-SNX15 was found in larger rings. Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures. These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network. However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15. In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed. In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited. Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway. This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.

A gene for congenital generalized lipodystrophy maps to human chromosome 9q34.

Congenital generalized lipodystrophy (CGL, Berardinelli-Seip Syndrome, OMIM # 269700) is a rare autosomal recessive disorder characterized by near complete absence of adipose tissue from birth. Affected individuals have marked insulin resistance, hypertriglyceridemia and acanthosis nigricans, and develop diabetes mellitus during teenage years. The genetic defect for CGL is unknown. A semi-automated genome-wide scan with a set of highly polymorphic short tandem repeats (STR) was carried out in 17 well-characterized pedigrees and identified a locus for CGL to chromosome 9q34. The maximum two-point lod score obtained was 3.6 at D9S1818 (theta(max) = 0.05). There was evidence for genetic heterogeneity (alpha = 0.73) and 2 of the pedigrees were unlinked. Multipoint linkage analysis excluding the 2 unlinked families yielded a peak lod score of 5.4 between loci D9S1818 and D9S1826. The CGL1 critical region harbors a plausible candidate gene encoding the retinoid X receptor alpha (RXRA) that plays a central role in adipocyte differentiation. Identification of the CGL gene(s) will contribute to our understanding of the adipocyte differentiation and elucidation of the mechanisms of insulin resistance in disorders of adipose tissue.

Subcellular localization and internalization of the four human leptin receptor isoforms.

There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.

Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins.

Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway.