Receptor-mediated Internalization of Insulin

Abstract

pp120/HA4 is a hepatocyte membrane glycoprotein phosphorylated by the insulin receptor tyrosine kinase. In this study, we have investigated the role of pp120/HA4 in insulin action. Transfection of antisense pp120/HA4 cDNA in H35 hepatoma cells resulted in inhibition of pp120/HA4 expression and was associated with a 2-3-fold decrease in the rate of insulin internalization. Furthermore, insulin internalization in NIH 3T3 fibroblasts co-transfected with insulin receptors and pp120/HA4 was increased 2-fold compared with cells expressing insulin receptors alone. In contrast, no effect on internalization was observed in cells overexpressing a naturally occurring splice variant of pp120/HA4 that lacks the phosphorylation sites in the intracellular domain. Insulin internalization was also unaffected in cells expressing three site-directed mutants of pp120/HA4 in which the sites of phosphorylation by the insulin receptor kinase had been removed (Y488F, Y488F/Y513F, and S503A). Our data suggest that pp120/HA4 is part of a complex of proteins required for receptor-mediated internalization of insulin. It is possible that this function is regulated by insulin-induced phosphorylation of the intracellular domain of pp120/HA4.Insulin binding to its receptor triggers the rapid endocytosis of the ligand-receptor complex (1, 2). Internalization of the insulin-insulin receptor complex constitutes the major mechanism of insulin degradation and down-regulation of cell surface receptors (3-5). The molecular events involved in the internalization process, however, are yet to be well defined. Specific sequences in the submembranous portion of the receptor are required for internalization to occur. Furthermore, activation of the receptor kinase is required for ligand-induced receptor internalization (6 -15). Following insulin-induced receptor autophosphorylation, several intracellular substrates are phosphorylated, the best characterized of which is insulin receptor substrate-1 (IRS-1) (16). However, the role of substrate phosphorylation in receptor internalization is not established.In the present study, we have investigated the role of pp120/ HA4, a substrate of the insulin receptor kinase that is predominantly expressed in liver, in the internalization process. pp120/HA4 is a transmembrane glycoprotein that is phosphorylated by the insulin receptor tyrosine kinase in intact cells (17)(18)(19)(20) and in cell-free systems (21). It is composed of a large extracellular domain containing 16 sites of potential N-linked glycosylation and a 71-amino acid cytoplasmic domain (22). Studies of site-directed mutants indicate that the intracellular domain contains a site of constitutive serine phosphorylation (Ser 503 ) as well as one tyrosine residue that is phosphorylated in response to insulin (Tyr 488 ) (20,23). Alternative splicing of pp120/HA4 mRNA generates two isoforms, one of which lacks 61 amino acids at the C terminus of the cytoplasmic domain, including all the potential phosphorylation sites (24). The function of pp120/HA4 is still un...

Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells

“…Although enhancement of Tyr phosphorylation using this inhibitor may not be considered to faithfully mimic physiological situations, it should be noted however, that Bgp was demonstrated to be Tyr phosphorylated in other situations. Tyr 488 of Bgp is phosphorylated in response to activation of the insulin receptor (Margolis et al, 1988;Najjar et al, 1995) and phosphorylation of this residue as well as Ser 503 phosphorylation correlate with e cient bile salt extrusion and insulin receptor endocytosis (Sippel et al, 1994;Formisano et al, 1995). Similarly, Bgp was shown to be Tyr phosphorylated by pp60 c-src in human colonic carcinoma cells (BruÈ mmer et al, 1995) and by p53/56 lyn in human neutrophils (Skubitz et al, 1995).…”

“…Bgp has been implicated in many cellular events such as intercellular adhesion, bile salt transport, insulin receptor internalization and inhibition of tumor cell growth (Ocklind and O È brink, 1982;Rojas et al, 1990;McCuaig et al, 1992;Sippel et al, 1993;Kunath et al, 1995;Formisano et al, 1995;Hsieh et al, 1995). All of the above functions, except adhesion (Rojas et al, 1990;McCuaig et al, 1992) depend on the presence of the longer cytoplasmic domain of Bgp .…”

“…Second, Bgp can also promote extrusion of bile salts from cells (Sippel et al, 1993). Third, Bgp has been implicated in mediating the internalization of the insulin receptor in the rat, a process which may relate to the ability of insulin treatment to induce tyrosine phosphorylation of Bgp (Margolis et al, 1988;Najjar et al, 1995;Formisano et al, 1995). Fourth, we and others showed that Bgp expression is down-regulated in intestinal, liver and prostate tumors when compared to their normal counterparts (Hixson et al, 1985;Rosenberg et al, 1993;Neumaier et al, 1993;Hsieh et al, 1994).…”

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

“…Further deletions of a few residues within the Bgp1 cytoplasmic domain (⌬510 mutant) almost completely abrogated Bgp1 Tyr phosphorylation, and association with the phosphatases was undetectable. In addition, elimination of the protein kinase C phosphorylation site at Ser-503, previously shown to regulate both bile transport and internalization of the insulin receptor (43,44), produced minimal differences in Bgp1 Tyr phosphorylation compared with that detected with wildtype Bgp1. However, conversion of Val-518 to Ala abolished SHP-2 binding and is therefore crucial for these protein interactions.…”

“…The ⌬518 mutant was designed to remove the three carboxyl-terminal Lys residues, well conserved (KK(K/ Q)) among the mouse, rat, and human BGP cytoplasmic domains. Furthermore, truncations removing the residues surrounding Tyr-515 (⌬510 mutant), those encompassing a conserved Ser phosphorylation site located at Ser-503 (⌬495 mutant) (43,44), and finally those positioned close to Tyr-488 (⌬483 mutant) were also created. A Ser-503 point mutant converting this residue to a non-phosphorylatable Ala was also designed (S503A mutant).…”