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pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the beta-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR(-/-)) revealed that Tyr(1316), which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr(1316) residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the beta-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR(-/-) hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein in the hepatocyte. It is expressed as two spliced isoforms differing by the presence (full length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Because the two isoforms differ by their ability to regulate receptor-mediated insulin endocytosis and degradation, we aimed to investigate the cellular basis for this functional difference by comparing their intracellular trafficking. During its intracellular assembly, pp120 is transported from the trans-Golgi network to the sinusoidal domain of the plasma membrane before its final transcytosis to the bile canalicular domain. Because both isoforms are expressed in hepatocytes, we examined their intracellular trafficking in NIH 3T3 fibroblasts individually transfected with each isoform. Pulse-chase experiments demonstrated that most of the newly synthesized full-length isoform reached complete maturation at about 60 min of chase. By contrast, only about 40% of the newly synthesized truncated isoform underwent complete maturation, even at more prolonged chase. Moreover, a significant portion of the truncated isoform appeared to be targeted to lysosomes. Abolishing basal phosphorylation on Ser(503) by cAMP-dependent serine kinase by mutating this residue to alanine was correlated with incomplete maturation of full length pp120 in NIH 3T3 cells and hepatocytes. This finding suggests that the intracellular domain of pp120 contains information that regulates its vectorial sorting from the trans-Golgi network to the plasma membrane., (Copyright 1999 Wiley-Liss, Inc.)

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 full-length pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were co-expressed. 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 alpha-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrin-coated 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.

pp120, a plasma membrane glycoprotein, is phosphorylated on Tyr488 by the insulin receptor tyrosine kinase. This requires basal phosphorylation on Ser503 by cAMP-dependent serine kinase. Phe513, the other tyrosine residue in the intracellular domain, does not undergo insulin-stimulated phosphorylation. Phe488 mutation abolished basal and insulin-stimulated phosphorylation, whereas, Phe513 plus Phe488 mutation markedly decreased the effect of insulin on pp120 phosphorylation without altering basal phosphorylation in intact cells. To investigate whether basal phosphorylation of pp120 is regulated by a phosphatase activity that requires Tyr513, in vitro phosphorylation assays using partially purified glycoproteins from stably transfected NIH 3T3 cells were performed in the absence of phosphatase inhibitors. Wild-type pp120 was promptly dephosphorylated, whereas, Y513F pp120 was not. Decreasing pp120 expression by antisense cDNA transfection proportionally decreased phosphatase activity in H4-II-E hepatoma cells measured by the p-nitrophenyl phosphate assay. This suggests that pp120 is associated with phosphatase activity that requires an intact Tyr513 residue., (Copyright 1998 Academic Press.)

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 beta-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 beta-subunit of the insulin receptor does not regulate the effect of pp120 on insulin endocytosis. Mutation of Tyr960 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 Tyr960 in the insulin receptor.

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 alpha-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.

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.

In the nonobese diabetic mouse, insulin-dependent diabetes is an autoimmune disease characterized by T cell-mediated invasion and destruction of pancreatic islet beta cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined, since it has been shown that the IR is a chemotactic receptor capable of directing cell movement in response to insulin. Using polyclonal antisera to the IR, phenotypic analysis of purified splenic T cells from diabetic mice showed that about 15% of T cells expressed high density IR (IRhigh). In addition, IRhigh T cells were already a dominant phenotype in the insulitis of young prediabetic mice. To determine the ability of IRhigh T cells to transfer diabetes, cells were sorted by flow cytometry before adoptive transfer into young (6- to 8-wk-old) nondiabetic irradiated nonobese mice. Transfer of as few as 3 x 10(6) purified IRhigh T cells alone resulted in rapid onset of insulitis and diabetes, and IRhigh-depleted T cells were essentially unable to passage either insulitis or diabetes. The adoptive transfer of disease was not due to the transfer of activated cells, since removal of IL-2R+ or transferrin R+ cells did not alter diabetes transfer. Therefore, IRhigh T cells are aggressively diabetogenic, suggesting that increased IR expression may provide a mechanism for delivering potentially autoreactive T cells to the islet, regardless of their activation state.

Cloning of the 5 -flanking region of the rat pp120 gene has indicated that it is a housekeeping gene: it lacks a functional TATA box and contains several Sp1 binding sites and multiple transcription initiation sites at nucleotides -101, -71, -41, and -27 spread over a GC-rich area. A fragment between nucleotides -21 and -1609 exhibited promoter activity when ligated in a sense orientation into a promoterless luciferase reporter plasmid and transiently transfected into rat H4-II-E hepatoma cells. 5' progressive deletion and block substitution analyses revealed that the three proximal Sp1 boxes (boxes 3, 5, and 6) are required for basal transcription of the pp120 gene. Promoter activity was stimulated 2-3-fold in response to insulin, dexamethasone, insulin plus dexamethasone, and cAMP. Although unaltered by phorbol esters alone, promoter activity was stimulated 4-5-fold in response to phorbol esters plus cAMP. Several motifs resembling response elements for insulin (in the rat phosphoenolpyruvate carboxykinase gene), glucocorticoids, cAMP, and phorbol esters as well as a number of putative binding sites for activating proteins-1 (Jun/Fos) and -2, and liver-specific factors were detected. The role of these sites in tissue-specific expression of pp120 remains to be investigated.

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 the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 20,000. Analysis of the deduced amino acid sequence of rat liver pp120/HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr488 and Tyr513), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser503). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe488 and Ala503) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr488 is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser513 may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg(2+)-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg(2+)-dependent ecto-ATPase activity.

The insulin receptor possesses tyrosine kinase activity which is thought to mediate the biological effects of insulin upon target cells. pp120 is a liver-specific glycoprotein of apparent molecular size of 120 kDa that is phosphorylated on tyrosine residues by the receptors for insulin, insulin-like growth factor-I, and epidermal growth factor. Previously, we demonstrated that pp120 is identical to a liver-specific ecto-ATPase. In the present study, we have cloned the rat gene encoding pp120/ecto-ATPase. The gene is contained within approximately 15 kilobases of genomic DNA, and consists of nine exons interrupted by eight introns. Using the reverse transcriptase/polymerase chain reaction, we isolated cDNA clones complementary to rat liver mRNA encoding pp120/ecto-ATPase. Sequence analysis indicated the presence of two populations of cDNA's that differ by the presence or absence of a 53-base pair (bp) fragment encoding the juxta-membrane region of the cytoplasmic domain. By cloning the corresponding region of the ecto-ATPase gene, we demonstrated that the 53-bp represents exon 7 of the gene. This 53-bp exon undergoes alternative splicing, thereby giving rise to two mRNA variants. Deletion of this 53-bp cassette exon introduces a frameshift, and results in a premature chain termination codon that truncates the cytoplasmic domain. The truncated cytoplasmic domain contains 10 rather than 71 amino acid residues. Because the short isoform of ecto-ATPase lacks the putative sites for tyrosine- and serine-specific phosphorylation, this alternative splicing may have a major effect upon the physiological function of the enzyme.