RESUMO
AIM/HYPOTHESIS: Insulin receptor substrate (IRS) proteins play important roles in insulin action and pancreatic beta-cell function. At least four mammalian IRS molecules have been identified. Although genes and cDNAs encoding human IRS-1, IRS-2, and IRS-4 have been cloned, IRS-3 has been identified only in rodents. Thus, we have attempted to clone the human IRS-3 gene. METHODS: Insulin-stimulated rat or human adipocytes were subjected to Western blot analysis to assess IRS-3 tyrosine phosphorylation. Human liver and adipose cDNA libraries were screened in an effort to clone IRS-3 cDNA. A PCR-based approach was designed to amplify IRS-3 cDNA. Reverse transcription PCR was carried out using mRNA from adipose tissue, liver, and skeletal muscle as templates in combination with an in silico screen using mouse IRS-1, IRS-2 and IRS-3 in a tblastn search of the draft public human genome. RESULTS: In human adipocytes we did not detect a M(r) 60 000 phosphoprotein corresponding to IRS-3, whereas in rat adipocytes IRS-3 protein and insulin-stimulated tyrosine phosphorylation was readily observed. None of the molecular approaches provided evidence for a functional IRS-3gene in human tissue. Two deletions in human IRS-3 gene were identified using bioinformatics. The human IRS-3 gene product is predicted to lack a phosphotyrosine binding domain and also the sequence corresponding amino acid 353-407 of murine IRS-3. The contiguous sequence of genomic DNA between these two homologous regions does not have the coding information for human IRS-3. CONCLUSION/INTERPRETATION: In silico screening of the human IRS-3 genome region, combined with further biological and molecular validation, provides evidence against a functional IRS-3 in humans.
Assuntos
Fosfoproteínas/deficiência , Fosfoproteínas/genética , Adipócitos/metabolismo , Adulto , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , Biologia Computacional , Feminino , Deleção de Genes , Biblioteca Gênica , Genoma Humano , Humanos , Proteínas Substratos do Receptor de Insulina , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfoproteínas/fisiologia , Fosforilação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/metabolismoRESUMO
There is evidence that the atypical protein kinases C (PKC(lambda), PKC(zeta)) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKC(lambda/zeta) from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKC(lambda) is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of alpha- and gamma-adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKC(lambda). Association of the adducins with PKC(lambda) was further indicated by the finding that the adducins coimmunoprecipitated proportionally with PKC(lambda) in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the adducins in a whole cell lysate or on the small portion of the adducins that coimmunoprecipitated with PKC(lambda). PKC(lambda) and the adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKC(lambda) to adducin may function to localize PKC(lambda) in 3T3-L1 adipocytes.
Assuntos
Adipócitos/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteína Quinase C/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/química , Eletroforese em Gel de Poliacrilamida , Insulina/farmacologia , Isoenzimas , Camundongos , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Proteína Quinase C/química , Proteínas/análise , Ratos , Frações Subcelulares/metabolismoRESUMO
In the course of searching for proteins that interact with protein kinase B in 3T3-L1 adipocytes, we isolated a 105 kDa protein from 3T3-L1 adipocytes. Peptides sequenced from the protein were found to be present in several expressed sequence tags. A cDNA containing one of these expressed sequence tags was sequenced and appears to contain the entire coding region. Computer analysis revealed a potential protein kinase domain at the N-terminus; however, the first subdomain and several invariant residues characteristic of protein kinases are absent. An antibody was raised against a peptide from the 105 kDa protein. By immunoblotting, it was found that the protein was widely expressed in mouse tissues, and concentrated in the cytosol and low density microsome fractions of 3T3-L1 adipocytes.
Assuntos
Adipócitos/metabolismo , Fragmentos de Peptídeos/genética , Proteínas Serina-Treonina Quinases , Proteínas/genética , Proteínas Proto-Oncogênicas/química , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Citosol/enzimologia , DNA Complementar/química , Etiquetas de Sequências Expressas , Camundongos , Microssomos/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
The insulin receptor substrates (IRSs) function in insulin signaling. Four members of the family, IRS-1 through IRS-4, are known. Previously, mice with targeted disruption of the genes for IRS-1, -2, and -3 have been characterized. To examine the physiological role of IRS-4, we have generated and characterized mice lacking IRS-4. Male IRS-4-null mice were approximately 10% smaller in size than wild-type male mice at 9 wk of age and beyond, whereas the female null mice were of normal size. Breeding pairs of IRS-4-null mice reproduced less well than wild-type mice. IRS-4-null mice exhibited slightly lower blood glucose concentration than the wild-type mice in both the fasted and fed states, but the plasma insulin concentrations of the IRS-4-null mice in the fasted and fed states were normal. IRS-4-null mice also showed a slightly impaired response in the oral glucose tolerance test. Thus the absence of IRS-4 caused mild defects in growth, reproduction, and glucose homeostasis.
Assuntos
Glicemia/metabolismo , Camundongos Knockout/crescimento & desenvolvimento , Camundongos Knockout/fisiologia , Fosfoproteínas/fisiologia , Reprodução/fisiologia , Animais , Feminino , Teste de Tolerância a Glucose , Crescimento/fisiologia , Homeostase/fisiologia , Insulina/sangue , Insulina/fisiologia , Proteínas Substratos do Receptor de Insulina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Fosfoproteínas/genéticaRESUMO
Signaling from the activated insulin receptor is initiated by its tyrosine phosphorylation of the insulin receptor substrates (IRSs). The IRSs then act as docking/effector proteins for various signaling proteins containing src homology 2 domains. Four members of the IRS family, designated IRS-1 through IRS-4, have been identified. Although these IRSs show considerable structural homology, the extent to which they overlap in functions has not been explored in detail. The 32D hematopoietic cell line, which contains no detectable amounts of any IRS, provides a system in which to determine whether an IRS supports cell proliferation. Previous studies have shown that introduction of IRS-1 or -2 into 32D cells overexpressing the insulin and IL-4 receptors (32D-R cells) enables the cells to undergo mitogenesis in response to insulin and IL-4. In the present study, we have examined IRS-4, a member of the IRS family that we recently discovered, in this system. Expression of IRS-4 in 32D-R cells permitted the cells to undergo mitogenesis and continuous proliferation in response to insulin and IL-4. Immunoblotting of phosphotyrosine proteins showed that insulin and IL-4 elicited the tyrosine phosphorylation of IRS-4 in these cells. Thus, IRS-4, like IRS-1 and -2, can function in the signal transduction pathways linking insulin and IL-4 receptors to cell proliferation.
Assuntos
Sistema Hematopoético/citologia , Insulina/farmacologia , Interleucina-4/farmacologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular , DNA/biossíntese , Sistema Hematopoético/efeitos dos fármacos , Sistema Hematopoético/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina , Camundongos , Peso Molecular , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
The insulin receptor substrates (IRS) 1 and 2 are required for normal growth and glucose homeostasis in mice. To determine whether IRS-3, a recently cloned member of the IRS family, is also involved in the regulation of these, we have generated mice with a targeted disruption of the IRS-3 gene and characterized them. Compared with wild-type mice, the IRS-3-null mice showed normal body weight throughout development, normal blood glucose levels in the fed and fasted state and following an oral glucose bolus, and normal fed and fasted plasma insulin levels. IRS-3 is most abundant in adipocytes and is tyrosine-phosphorylated in response to insulin in these cells. Therefore, isolated adipocytes were analyzed for changes in insulin effects. Insulin-stimulated glucose transport in the adipocytes from the IRS-3-null mice was the same as in wild-type cells. The extent of tyrosine phosphorylation of IRS-1/2 following insulin stimulation was similar in adipocytes from IRS-3-null and wild-type mice, and the insulin-induced association of tyrosine-phosphorylated IRS-1/2 with phosphatidylinositol 3-kinase and SHP-2 was not detectably increased by IRS-3 deficiency. Thus, IRS-3 was not essential for normal growth, glucose homeostasis, and glucose transport in adipocytes, and in its absence no significant compensatory augmentation of insulin signaling through IRS-1/2 was evident.
Assuntos
Glicemia/metabolismo , Fosfoproteínas/genética , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Transporte Biológico , Peso Corporal , Feminino , Marcação de Genes , Glucose/metabolismo , Homeostase/genética , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismoRESUMO
In the course of characterizing proteins present in a preparation of vesicles from rat adipocytes containing glucose transporters, we examined a protein that migrated at 115 kDa upon SDS gel electrophoresis (designated vp115). Sequences of tryptic peptides were obtained, and from this information the cDNA for rat vp115 was cloned. The cDNA encodes an open reading frame for a protein of 121 kDa. Computer-aided sequence analysis predicted that vp115 has a potential membrane-inserted or membrane-spanning domain toward its amino terminus, followed by five C2 domains. Immunoblotting revealed that vp115 was not actually a component of the glucose transporter-containing vesicles, was most abundant in the plasma membranes and high density microsome fractions of rat adipocytes, and was expressed in all the major rat tissues.
Assuntos
Adipócitos/metabolismo , Proteínas de Membrana/genética , Adipócitos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Immunoblotting , Proteínas de Membrana/análise , Proteínas de Membrana/química , Dados de Sequência Molecular , Ratos , Homologia de Sequência , Frações Subcelulares/metabolismoRESUMO
In the course of our examination of proteins associated with the GLUT4-containing vesicles of rat adipocytes we have identified a new 22 kDa member of the family of endoplasmic reticulum (ER) proteins known as reticulons. The protein, which we refer to as vp20, was purified from a preparation of GLUT4-containing vesicles of rat adipocytes, and tryptic peptides were micro-sequenced. From this information a cDNA encoding a single open reading frame for a protein of 22 kDa was cloned. This protein is homologous to known members of the reticulon protein family. vp20 has two hydrophobic stretches of about 35 amino acids that could be membrane spanning domains and an ER retention motif at its carboxy-terminus. vp20 was most abundant in the high density microsome fraction of adipocytes, which is the fraction most enriched in ER. Only a small fraction of vp20 was present in the GLUT4 vesicle population, and that fraction appears to be due to ER vesicles that were non-specifically bound to the adsorbent. Analysis of tissue distribution of vp20 in rats revealed that it is concentrated in muscle, fat and the brain.
Assuntos
Adipócitos/metabolismo , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar/química , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Microssomos/metabolismo , Dados de Sequência Molecular , Músculos/metabolismo , Proteínas da Mielina , Proteínas Nogo , RatosRESUMO
The insulin receptor substrates (IRSs) are key proteins in signal transduction from the insulin receptor. Recently, we discovered a fourth member of this family, designated IRS-4, cloned its complementary DNA from the human embryonic kidney 293 cell line, and characterized its signaling properties in this cell line. As part of an investigation of the physiological role of this IRS, we have now cloned the mouse IRS-4 gene and determined its tissue expression and chromosomal location. The coding region of the mouse IRS-4 gene contains no introns, and in this regard is the same as that of the genes for IRS-1 and -2. The predicted amino acid sequence of mouse IRS-4 is highly homologous with that of human IRS-4; the pleckstrin homology domain, the phosphotyrosine-binding domain, and the tyrosine phosphorylation motifs are especially well conserved. The tissue distribution of IRS-4 in the mouse was determined by analysis for the expression of its messenger RNA by RT-PCR and for the protein itself by immunoprecipitation and immunoblotting. The messenger RNA was detected in skeletal muscle, brain, heart, kidney, and liver, but the protein itself was not detected in any tissue. These results indicate that IRS-4 is a very rare protein. The chromosomal locations of the mouse IRS-4 and IRS-3 genes were determined by interspecific back-cross analysis and were found to be on chromosomes X and 5, respectively. As the mouse genes for IRS-1 and -2 are on chromosomes 1 and 8, respectively, each IRS gene resides on a different chromosome.
Assuntos
Mapeamento Cromossômico , Regulação da Expressão Gênica/fisiologia , Fosfoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Desenvolvimento Embrionário e Fetal/fisiologia , Humanos , Proteínas Substratos do Receptor de Insulina , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
We have recently purified and cloned a new member of the insulin receptor substrate family, designated insulin receptor substrate 3 (IRS-3), from rat adipocytes. The amino acid sequence of IRS-3 shows multiple potential sites for tyrosine phosphorylation in motifs which engage specific SH2 domain-containing proteins. In order to determine which SH2 domain proteins complex with IRS-3, we have searched for coimmunoprecipitation from lysates of untreated and insulin-stimulated adipocytes. Phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP-2 complexed with the tyrosine phosphorylated form of IRS-3, whereas the phospholipase Cgamma did not, and the adaptor Grb2 did so to a much lesser extent. These findings complete the survey of SH2 domain proteins associated with each of the four known members of the IRS family and provide the framework for further analysis of the role of IRS-3 in insulin signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Adipócitos/metabolismo , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Ativação Enzimática , Proteína Adaptadora GRB2 , Técnicas In Vitro , Insulina/metabolismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Oligonucleotídeos Antissenso/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Fosfoproteínas/química , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/química , Receptor de Insulina/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Tirosina/química , Domínios de Homologia de srcRESUMO
We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.
Assuntos
Rim/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Substâncias de Crescimento/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina , Rim/citologia , Rim/embriologia , Microscopia Imunoeletrônica , Fosfoproteínas/genética , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1. 6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.
Assuntos
Aminopeptidases/metabolismo , Proteínas Musculares , Células 3T3 , Adipócitos/metabolismo , Animais , Transporte Biológico , Biotinilação , Diferenciação Celular , Cistinil Aminopeptidase , Fibroblastos/metabolismo , Transportador de Glucose Tipo 4 , Insulina/metabolismo , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismoRESUMO
Vesicles containing the glucose transporter GLUT4 from rat adipocytes contain a major protein of 110 kDa. We have isolated this protein, obtained the sequences of peptides, and cloned a large portion of its cDNA. This revealed that the protein is sortilin, a novel membrane protein that was cloned in another context from a human source while this work was in progress. Subcellular fractionation of rat and 3T3-L1 adipocytes, together with GLUT4 vesicle isolation, showed that sortilin was primarily located in the low density microsomes in vesicles containing GLUT4. Insulin caused a 1.7-fold increase in the amount of sortilin at the plasma membranes of 3T3-L1 adipocytes, as assessed by cell surface biotinylation. The expression of sortilin in 3T3-L1 cells occurred only upon differentiation. Previous characterization of sortilin has led to the suggestion that it functions to sort lumenal proteins from the trans Golgi. The significance of its insulin-stimulated increase at the cell surface and of its expression upon differentiation will require definitive delineation of its function.
Assuntos
Adipócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Proteínas do Tecido Nervoso/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , DNA Complementar , Transportador de Glucose Tipo 4 , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismoRESUMO
In fat and muscle cells insulin causes the marked translocation of the glucose transporter GLUT4 from its intracellular location to the plasma membrane. We and others have discovered an insulin-regulated membrane aminopeptidase (designated IRAP) that colocalizes with intracellular GLUT4 and also translocates markedly in response to insulin. This study describes the trafficking kinetics of IRAP in 3T3-L1 adipocytes. By means of a surface biotinylation method, the half-time for the increase in IRAP at the plasma membrane in response to insulin was found to be 2 min. The increase was completely blocked by the phosphatidylinositol 3-kinase inhibitor, wortmannin. In insulin-treated cells, biotinylated IRAP, initially at the plasma membrane, equilibrated with the intracellular pool with a half-time of 2 min. Thus, IRAP continuously recycles. Finally, vesicles isolated from the intracellular membranes with antibodies against IRAP and GLUT4 showed the same protein composition. In conjunction with results in the literature, these findings indicate that IRAP and GLUT4 traffic through the same intracellular compartments.
Assuntos
Adipócitos/enzimologia , Aminopeptidases/metabolismo , Proteínas Musculares , Células 3T3 , Androstadienos/farmacologia , Animais , Transporte Biológico , Biotinilação , Membrana Celular , Cistinil Aminopeptidase , Inibidores Enzimáticos/farmacologia , Transportador de Glucose Tipo 4 , Insulina/metabolismo , Antagonistas da Insulina/farmacologia , Cinética , Camundongos , Peso Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Propriedades de Superfície , WortmaninaRESUMO
We have previously identified a 160-kDa protein in human embryonic kidney (HEK) 293 cells that undergoes rapid tyrosine phosphorylation in response to insulin (PY160) (Kuhné, M. R., Zhao, Z., and Lienhard, G. E. (1995) Biochem. Biophys. Res. Commun. 211, 190-197). The phosphotyrosine form of PY160 was purified from insulin-treated HEK 293 cells by anti-phosphotyrosine immunoaffinity chromatography, the sequences of peptides determined, and its cDNA cloned. The PY160 cDNA encodes a 1257-amino acid protein that contains, in order from its N terminus, a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and, spread over the C-terminal portion, 12 potential tyrosine phosphorylation sites. Several of these sites are in motifs expected to bind specific SH2 domain-containing proteins: YXXM (7 sites), phosphatidylinositol 3-kinase; YVNM (1 site), Grb-2; and YIEV (1 site), either the protein-tyrosine phosphatase SHP-2 or phospholipase Cgamma. Furthermore, the PH and PTB domains are highly homologous (at least 40% identical) to those found in insulin receptor substrates 1, 2, and 3 (IRS-1, IRS-2, and IRS-3). Thus, PY160 is a new member of the IRS family, which we have designated IRS-4.
Assuntos
Insulina/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Humanos , Proteínas Substratos do Receptor de Insulina , Dados de Sequência Molecular , Peso Molecular , Fosfotirosina/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A 97-kDa protein present in the glucose transporter (GLUT4 isotype)-containing vesicles from rat adipocytes has been isolated, the sequences of two tryptic peptides were obtained, and on the basis of these its cDNA partially cloned. The 97-kDa protein is almost certainly identical to a major integral glycoprotein of this size in the rat adipocyte plasma membrane, since its predicted N-terminal sequence is the same as that recently determined for this glycoprotein by amino acid sequencing. Moreover, the predicted partial sequence (322 amino acids) of the 97-kDa protein is highly homologous to the corresponding region of a human placental amine oxidase, which was cloned simultaneously and proposed to be a secreted protein. The amino acid sequence of the 97-kDa rat/human amine oxidase indicates that the protein consists of a very short N-terminal cytoplasmic domain followed by a single transmembrane segment and a large extracellular domain containing the catalytic site. Thus this study establishes the 97-kDa rat/human amine oxidase as the first integral membrane amine oxidase to be cloned. The membrane amine oxidase was more abundant in the plasma membranes than the low density microsomes of the adipocyte, and in contrast to some other proteins found in GLUT4 vesicles, it did not redistribute to the plasma membrane in response to treatment of the cells with insulin.
Assuntos
Adipócitos/enzimologia , Amina Oxidase (contendo Cobre) , Proteínas Sanguíneas/genética , Metaloproteínas/genética , Proteínas Musculares , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Adipócitos/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Celular/enzimologia , Clonagem Molecular , Transportador de Glucose Tipo 4 , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteínas de Transporte de Monossacarídeos/análise , Ratos , Distribuição TecidualRESUMO
We previously discovered that insulin stimulates the marked translocation of a novel membrane aminopeptidase, designated vp165 for vesicle protein of 165 kDa, to the cell surface in adipocytes. To examine the hypothesis that this enzyme acts on peptide hormones, we assessed the relative affinity of the enzyme for 22 peptide hormones by measuring the inhibitory effect of each on the hydrolysis of a fluorogenic substrate, and we directly assayed the cleavage of four of these. Angiotensin III, angiotensin IV, and Lys-bradykinin bound to the enzyme with half-saturation constants between 20 and 600 nM and were cleaved by vp165. Vasopressin bound with lower affinity but at saturation was cleaved more rapidly. Subsequently, the effect of insulin on the rates of cleavage of 125I-labeled vasopressin by intact 3T3-L1 and rat adipocytes was determined. With both cell types, vasopressin cleavage was stimulated approximately threefold. These findings indicate that a physiological role for vp165 may be the processing of peptide hormones and that insulin could enhance the cleavage of extracellular substrates by eliciting the translocation of vp165 to the cell surface.
Assuntos
Adipócitos/metabolismo , Aminopeptidases/metabolismo , Insulina/farmacologia , Vasopressinas/metabolismo , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Adipócitos/efeitos dos fármacos , Aminopeptidases/antagonistas & inibidores , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Animais , Membrana Celular/metabolismo , Cistinil Aminopeptidase , Hidrólise , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Especificidade por SubstratoRESUMO
A 60-kDa protein that undergoes rapid tyrosine phosphorylation in response to insulin and then binds phosphatidylinositol 3-kinase has been previously described in adipocytes and hepatoma cells. We have isolated this protein, referred to as pp60, from rat adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA. The predicted amino acid sequence of pp60 reveals that it contains an N-terminal pleckstrin homology domain, followed by a phosphotyrosine binding domain, followed by a group of likely tyrosine phosphorylation sites, four of which are in the YXXM motif that binds to the SH2 domains of phosphatidylinositol 3-kinase. The overall architecture of pp60 is thus the same as that of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and furthermore both the pleckstrin homology and phosphotyrosine binding domains are highly homologous (about 50% identical amino acids) to these domains in both IRS-1 and IRS-2. Thus, pp60 is a new member of the IRS family, which we have designated IRS-3.
Assuntos
Adipócitos/química , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fosfoproteínas/isolamento & purificação , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosfoproteínas/classificação , Fosfoproteínas/genética , Ratos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.