Mathews S T, Chellam N, Srinivas P R, Cintron V J, Leon M A, Goustin A S, Grunberger G
Department of Internal Medicine, Wayne State University, Detroit, MI 48201, USA.
Mol Cell Endocrinol. 2000 Jun;164(1-2):87-98. doi: 10.1016/s0303-7207(00)00237-9.
Human fetuin, [alpha2-Heremans Schmid Glycoprotein (alpha2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that alpha2-HSG inhibits the mitogenic pathway without affecting the metabolic arm of insulin signal transduction. In this study, we demonstrate the time-course and specificity of inhibition, its interaction with IR and probable physiological role. In intact rat1 fibroblasts overexpressing the human insulin receptor (HIRc B), incubation of recombinant human alpha2-HSGbac (1.8 microM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of alpha2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, alpha2-HSGbac at similar concentrations (0.9 or 1.8 microM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-alpha2-HSG immunoprecipitates of alpha2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that alpha2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of alpha2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR alpha-chain and constitutive activation of IR-TKA. We demonstrate that alpha2-HSGbac (0.1 microM) completely inhibited trypsin-activated IR autophosphorylation and TKA in vitro indicating that this effect was not mediated by its interaction with the proximal 576 amino acid residues of the IR alpha-subunit. The physiological relevance of these observations was explored by characterizing the effects of alpha2-HSG injection in rats. Alpha2-HSGbac (2 microM), acutely injected through the portal vein of normal rats, inhibited insulin-stimulated IR autophosphorylation and IRS-1 phosphorylation in liver and hindlimb muscle. Taken together our results suggest that alpha2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.
人胎球蛋白，即α2-赫雷曼斯·施密德糖蛋白（α2-HSG），是胰岛素受体酪氨酸激酶活性（IR-TKA）的天然抑制剂。此前，我们已经证明α2-HSG可抑制有丝分裂途径，而不影响胰岛素信号转导的代谢分支。在本研究中，我们证明了抑制作用的时间进程和特异性、其与IR的相互作用以及可能的生理作用。在过表达人胰岛素受体（HIRc B）的完整大鼠成纤维细胞中，重组人α2-HSGbac（1.8微摩尔）孵育可使胰岛素诱导的IR自磷酸化抑制超过80%。α2-HSGbac对胰岛素诱导的IR自磷酸化的这种抑制作用在60分钟内减弱了一半。有趣的是，相似浓度（0.9或1.8微摩尔）的α2-HSGbac对EGF或IGF-I诱导的同源受体自磷酸化没有影响。α2-HSGbac处理的HIRc B细胞裂解物的抗α2-HSG免疫沉淀物显示存在IR。我们的数据表明α2-HSGbac优先与活化的IR相互作用。为了进一步表征相互作用位点，研究了α2-HSGbac对胰蛋白酶处理的IR自磷酸化的影响。对完整的HIRc B细胞进行胰蛋白酶处理会导致IRα链的蛋白水解和IR-TKA的组成性激活。我们证明α2-HSGbac（0.1微摩尔）在体外完全抑制了胰蛋白酶激活的IR自磷酸化和TKA，表明这种作用不是通过其与IRα亚基近端576个氨基酸残基的相互作用介导的。通过表征α2-HSG注射对大鼠的影响来探索这些观察结果的生理相关性。α2-HSGbac（2微摩尔）通过正常大鼠的门静脉急性注射，抑制了肝脏和后肢肌肉中胰岛素刺激的IR自磷酸化和IRS-1磷酸化。综上所述，我们的结果表明，α2-HSG通过与IR相互作用，特异性抑制胰岛素刺激的IR自磷酸化，并可能在胰岛素信号调节中发挥生理作用。
