Effect of pp120 on Receptor-mediated Insulin Endocytosis Is Regulated by the Juxtamembrane Domain of the Insulin Receptor

Abstract

pp120, a substrate of the insulin receptor tyrosine kinase, does not undergo ligand-stimulated phosphorylation by the insulin-like growth factor-1 (IGF-1) receptor. However, replacement of the C-terminal domain of the IGF-1 receptor ␤-subunit with the corresponding segment of the insulin receptor restored pp120 phosphorylation by the chimeric receptor. Since pp120 stimulates receptor-mediated insulin endocytosis when it is phosphorylated, we examined whether pp120 regulates IGF-1 receptor endocytosis in transfected NIH 3T3 cells. pp120 failed to alter IGF-1 receptor endocytosis via either wild-type or chimeric IGF-1 receptors. Thus, the effect of pp120 on hormone endocytosis is specific to insulin, and the C-terminal domain of the ␤-subunit of the insulin receptor does not regulate the effect of pp120 on insulin endocytosis. Mutation of Tyr 960 in the juxtamembrane domain of the insulin receptor abolished the effect of pp120 to stimulate receptor endocytosis, without affecting pp120 phosphorylation by the insulin receptor. These findings suggest that pp120 interacts with two separate domains of the insulin receptor as follows: a C-terminal domain required for pp120 phosphorylation and a juxtamembrane domain required for internalization. We propose that the interaction of pp120 with the juxtamembrane domain is indirect and requires one or more substrates that bind to Tyr 960 in the insulin receptor.The insulin receptor is essential to mediate the multiple effects of insulin on target cells (1, 2). Insulin binding to the ␣-subunit of its receptor activates the tyrosine kinase of the receptor in the cytoplasmic tail of the ␤-subunit to phosphorylate the receptor (3) and other endogenous substrates, including pp120 1 (4, 5), insulin receptor substrate proteins (IRS-1, -2, -3, and -4) (6 -10), Shc (11,12), and others (reviewed in Ref. 10).Phosphorylated substrates engage in turn the formation of signaling complexes via phosphotyrosine-containing binding motifs with src homology-2 (SH2) domains (13) in order to propagate the signals of insulin in the cell. pp120 is a plasma membrane glycoprotein expressed in the liver as two spliced variants that differ by the inclusion (fulllength) or exclusion (truncated) of 61 out of 71 amino acids of the cytoplasmic domain (14). In contrast to the truncated isoform, full-length pp120 undergoes insulin-stimulated phosphorylation (5). Site-directed mutagenesis revealed that phosphorylation on Ser 503 by cAMP-dependent serine kinase occurs in the absence of insulin and that phosphorylation at this site is required for insulin-stimulated tyrosine phosphorylation on Tyr 488 , the major pp120 phosphorylation site by the insulin receptor kinase (5).In marked contrast to insulin receptors, insulin-like growth factor-1 (IGF-1) receptors failed to mediate pp120 phosphorylation in response to . This is consistent with the predominant expression of pp120 and insulin receptors in the liver, an organ with low levels of IGF-1 receptors (16). Moreover, pp120 phosphorylation by the IGF-1 receptors...

The IR1152 mutant insulin receptor selectively impairs insulin action in skeletal muscle but not in liver.

“…More recently, evidence became available that the proximal events in the insulin signaling machinery may also feature tissue-specific properties (12)(13)(14)(15). For instance, p120, an IRS involved in the internalization of the insulin receptor, is selectively expressed in liver and kidney (14,15,33), and IRSs appear to be differently distributed and regulated in the insulin target tissues (16,17). However, the extent to which differences in these early signaling mechanisms generate diversity in insulin action in the different tissues, as well as the relevance of this diversity to conditions of impaired insulin action, is far from understood.…”

The Differential Effects of pp120 (Ceacam 1) on the Mitogenic Action of Insulin and Insulin-Like Growth Factor 1 Are Regulated by the Nonconserved Tyrosine 1316 in the Insulin Receptor

“…Furthermore, mutating Tyr 960 in the juxtamembrane domain of the ␤-subunit of the insulin receptor did not alter either the effect of pp120 on insulin mitogenesis (Fig. 6) or its phosphorylation by the insulin receptor (42). Taken together, these data suggest that pp120 phosphorylation by the insulin receptor is required and sufficient to regulate its differential downregulatory effect on the mitogenic effects of insulin vis-à-vis IGF-1.…”

“…Unless otherwise indicated, cell lysates were directly subjected to immunoprecipitation with either a monoclonal antibody against pp120/HA4 or ␣-pTyr prior to analysis by 7.5% SDS-PAGE and immunoblotting with horseradish peroxidase (HRP)-coupled ␣-pTyr antibody to detect phosphorylated proteins by the Amersham Enhanced Chemiluminescence (ECL) detection system (41). In some experiments, cell lysates were partially purified by wheat germ agglutinin affinity chromatography (44) prior to being subjected to immunoprecipitation with ␣-pTyr monoclonal antibody to immunoprecipitate phosphorylated pp120 and insulin receptors (42).…”

Interaction between Altered Insulin and Lipid Metabolism in CEACAM1-inactive Transgenic Mice

“…CEACAM1, an insulin receptor substrate in liver, but not in muscle or adipose tissue, regulates insulin action by promoting its receptor-mediated uptake and degradation in a phosphorylation-dependent manner (2)(3)(4).…”