title: Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Wayne State University, Detroit, MI 48201, USA.
authors.forename: S T
authors.initials: ST
authors.lastname: Mathews
authors.forename: N
authors.initials: N
authors.lastname: Chellam
authors.forename: P R
authors.initials: PR
authors.lastname: Srinivas
authors.forename: V J
authors.initials: VJ
authors.lastname: Cintron
authors.forename: M A
authors.initials: MA
authors.lastname: Leon
authors.forename: A S
authors.initials: AS
authors.lastname: Goustin
authors.forename: G
authors.initials: G
authors.lastname: Grunberger
mesh_terms: D000818:Animals; D001798:Blood Proteins; D015891:Cystatins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D013997:Time Factors; D060749:alpha-2-HS-Glycoprotein; D000509:alpha-Fetoproteins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*
chemical_list: C556354:AHSG protein, human; D001798:Blood Proteins; D015891:Cystatins; D060749:alpha-2-HS-Glycoprotein; D000509:alpha-Fetoproteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin