Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes.
In the present study we have investigated the contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling pathways in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulated IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation was barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha revealed a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain showed functional differences. While a strong association between IRS-1/Grb-2 was found, IRS-2/Grb-2 association was virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increased the amount of Grb-2-associated SHC proteins by the same extent. Our results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation that leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2 leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation.
Valverde
A M
AM
Departamento de Bioquimica y Biologia Molecular II, Centro Mixto Consejo Superior de Investigaciones Cientificas/Universidad Complutense, Facultad de Farmacia, Madrid, Spain.
Lorenzo
M
M
Pons
S
S
White
M F
MF
Benito
M
M
D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000378:metabolism / Q000502:physiology*; D002001:Adipose Tissue, Brown / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005333:Fetus; D051380:GRB2 Adaptor Protein; D007328:Insulin / Q000378:metabolism / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526296:Irs1 protein, rat; C526299:Irs2 protein, rat; D010750:Phosphoproteins; D011506:Proteins; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin