pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of fulllength pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were coexpressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with ␣-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrincoated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins.Insulin binding to the extracellular ␣-subunits of its receptor triggers activation of the tyrosine kinase in the intracellular domain of the ␤-subunits. Activation of the tyrosine kinase causes phosphorylation of the receptor and of endogenous substrates, including pp120, a plasma membrane glycoprotein of M r ϳ120,000 that is expressed in the liver as two spliced variants differing by the inclusion (full-length) or exclusion (truncated) of a 61 amino acid (aa) 1 segment in the C terminus of its cytoplasmic domain (1). The truncated isoform lacks all phosphorylation sites. Site-directed mutagenesis studies in NIH 3T3 mouse skin fibroblasts revealed that full-length pp120 is constitutively phosphorylated by cAMP-dependent serine kinase on Ser 503 and that this phosphorylation is required for its phosphorylation on Tyr 488 residue by the insulin receptor tyrosine kinase (2). Tyr 513 , the other tyrosine residue in the cytoplasmic domain of pp120, is not involved in insulinmediated pp120 phosphorylation (2).Binding of insulin to its receptor triggers the entry of the insulin-receptor complex into clathrin-coated pits, which pinch off the surface membrane to form clathrin-coated vesicles (3). The clathrin coat is formed upon receptor activation by the sequestration of the cytoplasmic adaptor proteins 2 (AP2) to the inner surface of the membrane where they bin...

pp 120, a plasma membrane glycoprotein expressed by hepatocytes, is a substrate of the insulin receptor tyrosine kinase. Since insulin-like growth factor-1 (IGF-1) and insulin receptors are structurally homologous, we investigated whether pp120 is also a substrate of the IGF-1 receptor tyrosine kinase. IGF-1 receptor failed to phosphorylate pp120 in response to IGF-1 in stably transfected NIH 3T3 fibroblasts. However, replacement of the C-terminal domain of the beta-subunit of the IGF-1 receptor with the corresponding fragment in the insulin receptor restored ligand-stimulated pp120 phosphorylation, suggesting that this domain plays a regulatory role in pp120 phosphorylation. Since pp120 is the first identified substrate specific for the insulin vis-à-vis the IGF-1 receptor tyrosine kinase, the pp120 signaling pathway may constitute a novel mechanism for the distinct cellular effects of insulin and IGF-1, the former being principally involved in metabolism, and the latter in mitogenesis.

The insulin receptor is expressed as two variably spliced isoforms that differ by the absence (isoform A) or presence (isoform B) of a 12-amino acid sequence encoded by exon 11 at the carboxy terminus of the α-subunit. Coexpression of the A isoform and pp120, a substrate of the insulin receptor tyrosine kinase, in NIH 3T3 fibroblasts increased receptor A-mediated insulin endocytosis and degradation by two- to threefold compared with cells expressing receptors alone. Because B is the predominant isoform in the liver and binds insulin with lower affinity than A, we have examined the effect of pp120 on receptor B-mediated endocytosis. In contrast to isoform A, the effect of pp120 on isoform B-mediated insulin internalization and degradation in stably transfected NIH 3T3 cells was minimal.

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...