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Diabetologia ; 45(12): 1697-702, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12488959

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/metabolismo
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