α2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor

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Abstract

Human fetuin, (α2-Heremans Schmid Glycoprotein2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that α2-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 α2-HSGbac (1.8 μM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of α2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, α2-HSGbac at similar concentrations (0.9 or 1.8 μM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-α2-HSG immunoprecipitates of α2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that α2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of α2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR α-chain and constitutive activation of IR-TKA. We demonstrate that α2-HSGbac (0.1 μM) 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 α-subunit. The physiological relevance of these observations was explored by characterizing the effects of α2-HSG injection in rats. α2-HSGbac (2 μM), 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 α2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.

Introduction

The insulin receptor (IR) is a disulfide-linked heterotetrameric protein complex consisting of two hormone-binding α-subunits and two signaling α-subunits containing tyrosine kinase activity (TKA). The IR-TKA plays an important role in mediating the myriad of signaling cascades and cellular activities (White and Kahn, 1994). An impairment of the IR signaling capacity has been reported in insulin resistant conditions (Maegawa et al., 1991; Bulangu et al., 1990). However, the molecular mechanism(s) of such regulation is not completely understood. Physiological modulators of IR function are potential candidates in the pathogenesis of insulin resistance. Factors such as hyperinsulinemia, hypoinsulinemia; agonists including catecholamines, adenosine and phorbol esters (Häring and Mehnert, 1993); and TNF-α (Hotamisligil et al., 1996), G proteins (Moxham and Malbon, 1996), PC-1 (Maddux et al., 1995) and protein tyrosine phosphatases (Ahmad et al., 1997) have been shown to regulate insulin receptor function. Additionally, α2-Heremans Schmid Glycoprotein (α2-HSG) has been identified as a candidate for regulation of insulin signal transduction (Srinivas et al., 1993).

α2-HSG, the human homolog of bovine fetuin, is a 60 kD glycoprotein secreted by the liver. Plasma concentration of α2-HSG ranges from 0.4 to 0.6 mg/ml (Putnam, 1984). The circulating form of α2-HSG consists of an N-terminal heavy chain of 321 amino acids, attached by disulfide bonds to a C-terminal light chain of 27 amino acids (Kellermann et al., 1986). α2-HSG and other fetuins are members of the cystatin superfamily of proteins possessing two NH2-terminally located cystatin-like domains and a unique COOH-terminal domain. Native α2-HSG undergoes several post-translational modifications including N-glycosylation, O-glycosylation, proteolytic processing and phosphorylation on serine residues (Jahnen-Dechent et al., 1994). α2-HSG is a negative acute phase protein and its levels decrease significantly following infection, inflammation, malignancy and in Paget’s disease (Ashton and Smith, 1980, Putnam, 1984). Although several biological functions have been proposed for α2-HSG, none has been unequivocally established viz., bone formation, bone resorption, opsonization and cellular immunity (Dziegielewska and Brown, 1995). α2-HSG inhibits apatite formation (Schinke et al., 1996) and is a natural antagonist of transforming growth factor-β, bone morphogenetic protein function (Demetriou et al., 1996) and hepatocyte growth factor binding (Ohnishi et al., 1997). Rat fetuin, whose functional identity was earlier refuted as a cloning artifact (Brown et al., 1992), has been conclusively demonstrated to be an in vitro inhibitor of IR-TKA (Auberger et al., 1989; Rauth et al., 1992). We have earlier reported that plasma α2-HSG acts as a specific inhibitor of IR-TKA (Srinivas et al., 1993). However, some investigators claim plasma α2-HSG to be devoid of this inhibitory activity (Jahnen-Dechent et al., 1994, Kalabay et al., 1998). Recently, we have shown that recombinant α2-HSG (α2-HSGbac) inhibits insulin-stimulated IR autophosphorylation/TKA and signaling through the Ras/Raf/MAPK pathway (Srinivas et al., 1995, Srinivas et al., 1996). This inhibition does not extend to insulin’s metabolic effects, viz., 2-deoxy glucose uptake or amino acid transport. Kalabay et al. (1998) also reported that recombinant α2-HSG inhibits IR-TKA and IR autophosphorylation. In this study, we explored the mechanism of α2-HSG’s IR-TK inhibitory action by characterizing the time-course of inhibition, specificity, interaction with IR and its effect on a truncated IR. Further we tested the effect of α2-HSG to inhibit insulin-induced insulin receptor autophosphorylation in vivo by injecting α2-HSG into rats. Our data indicates that α2-HSGbac is a specific inhibitor of IR autophosphorylation. Acute injection of α2-HSGbac inhibits insulin-stimulated tyrosine phosphorylation of IR α-subunit and IRS-1, both in liver and muscle of normal rats. Further, we present evidence for interaction of α2-HSG with IR in vitro and demonstrate that α2-HSG does not require the proximal 576 amino acids of the IR α-subunit for its IR autophosphorylation/TKA-inhibitory action.

Section snippets

Source of α2-HSG

Recombinant α2-HSG, expressed in baculovirus (Srinivas et al., 1995) was used throughout this study. Supernatants, from virus-infected high five cells (Pharmingen, CA), containing α2-HSG were purified on anti-human α2-HSG immunoaffinity column. After extensive washing with PBS, α2-HSGbac was eluted with 0.2 M glycine-HCl, pH 2.8 and immediately neutralized. The purity of the eluted α2-HSGbac was confirmed by silver staining and Western blotting. α2-HSGbac protein concentration was determined by

Dose response and time–course of α2-HSGbac inhibition of insulin-induced IR autophosphorylation

In intact HIRc B cells, α2-HSGbac inhibited insulin-induced autophosphorylation of IR β-subunit maximally (80%) at 1.8 μM (Fig. 1). The half-maximal inhibitory dose was 0.5 μM (Fig. 1). To study the time–course of inhibition, serum-starved HIRc B cells were preincubated with 1.8 μM α2-HSGbac (maximal inhibitory concentration) for 15 min followed by incubation with insulin (100 nM) for 1, 5, 30 or 60 min. α2-HSGbac was present in the incubation media during all insulin-stimulation time-points

Discussion

Fetuin homologs have been identified in several species including rat, sheep, pig, mouse and human (α2-HSG) with 60–70% homology at the amino acid level and 80–90% homology at the cDNA level (for a review, see Dziegielewska and Brown, 1995). Several lines of evidence suggest a role for ‘fetuins’ in modulating insulin action (Auberger et al., 1989, Rauth et al., 1992, Srinivas et al., 1993, Mathews et al., 1997). The biological activities of insulin are initiated by binding of insulin to its

References (46)

  • T. Ohnishi et al.

    Purification, characterization and studies on biosynthesis of a 59-kDa bone sialic acid-containing protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human α2-HS glycoprotein and is synthesized by both hepatocytes and osteoblasts

    J. Biol. Chem.

    (1991)
  • W.M. Pardridge et al.

    In vivo quantification of receptor-mediated uptake of asialoglycoproteins by rat liver

    J. Biol. Chem.

    (1983)
  • T. Schinke et al.

    The serum protein α2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells

    J. Biol. Chem.

    (1996)
  • R. Schumacher et al.

    Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors

    J. Biol. Chem.

    (1991)
  • S.E. Shoelson et al.

    Tryptic activation of the insulin receptor: Proteolytic truncation of the α-subunit releases the β-subunit from inhibitory control

    J. Biol. Chem.

    (1988)
  • P.R. Srinivas et al.

    Baculoviral expression of a natural inhibitor of the human insulin receptor tyrosine kinase

    Biochem. Biophys. Res. Commun.

    (1995)
  • P.R. Srinivas et al.

    Recombinant human α2-HS glycoprotein inhibits insulin-stimulated mitogenic pathway without affecting metabolic signalling in Chinese hamster ovary cells overexpressing the human insulin receptor

    Cell Signal

    (1996)
  • J.M. Tavaré et al.

    Mutational analysis of insulin receptor function: consensus and controversy

    Biochim. Biophys. Acta

    (1993)
  • M.F. White et al.

    The insulin signaling system

    J. Biol. Chem.

    (1994)
  • P.A. Wilden et al.

    The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities: analysis with insulin and anti-receptor antibodies

    J. Biol. Chem.

    (1992)
  • F. Ahmad et al.

    Alterations in skeletal muscle protein tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes

    J. Clin. Invest.

    (1997)
  • B.A. Ashton et al.

    Plasma α2HS-glycoprotein concentration in Paget’s disease of bone: its possible significance

    Clin. Sci.

    (1980)
  • L. Bulangu et al.

    Insulin-sensitive tyrosine kinase: relationship with in vivo insulin action in humans

    Am. J. Physiol.

    (1990)
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