chico

Protein Interactions

Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the Drosophila Insulin-like receptor (InR) with the extracellular domain of the human insulin receptor (IR) were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bind insulin equally and exhibit an insulin-activated tyrosine kinase activity. The ability of the Drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase is indistinguishable from that of the human IR. The chimeric Drosophila receptors do not bind more phosphatidylinositol 3-kinase (see Phosphotidylinositol 3 kinase 92E) than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors (Yamaguchi, 1995).

Drosophila contain an insulin receptor homolog, encoded by the InR gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene. Six independent mutations that lead to a loss of expression or function of the receptor protein were identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for InR in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).

The cloning and primary structure of the Drosophila insulin receptor gene (InR) is reported, along with functional expression of the predicted polypeptide, and the isolation of mutations in the InR locus. The structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The InR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage that leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90, and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation for both types of beta subunits; in turn, the phosphorylation allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, renders pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among organ systems, since development of the cuticle, as well as the peripheral and central nervous systems are affected by InR mutations (Fernandez, 1995).

The Drosophila insulin receptor (InR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins that binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric InR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediate tyrosine phosphorylation of Shc and activate mitogen-activated protein kinase. IRS-1 is required by the human insulin receptor to activate PI 3-kinase and p70s6k (see Drosophila RPS6-p70-protein kinase), whereas hDIR associate with PI 3-kinase and activates p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the InR possesses additional signaling capabilities when compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells (Yenush, 1996a).

Like the mammalian insulin receptor, the Drosophila insulin receptor (INR)¹ is a tetramer formed by two alpha subunits and two beta subunits. INR alpha and beta subunits are synthesized together as a proreceptor precursor, proteolytically processed, and linked together by disulfide bonds. The alpha subunits, with a molecular mass of 110-120 kDa, are extracellular and contain the ligand binding domains that are capable of binding mammalian insulin with a K_d of 15 nM. The beta subunits traverse the plasma membrane and have an insulin-stimulated tyrosine kinase in the cytoplasmic portion. DNA sequence analysis and expression of the INR beta subunit in mammalian and Drosophila cells indicate that the INR beta subunit is larger than its mammalian homolog and exhibits an apparent molecular mass of ~180 kDa. The increased mass is due to the presence of a 400-amino acid carboxyl-terminal extension. However, the majority of INR beta subunits are processed to 92/102-kDa forms in Drosophila embyros and some cell lines, the difference being due to proteolytic cleavage of the carboxyl-terminal extension. Both truncated and full-length beta subunits are autophosphorylated on tyrosine residues in response to insulin binding (Marin-Hincapie, 1999 and references therein).

The 400-amino acid carboxyl-terminal extension of the beta INR contains clusters of motifs known to be involved in the interaction with SH2 and PTB domain-containing proteins, suggesting a role for this domain in signaling through interaction with other signaling molecules. Interestingly, four tyrosines are found in 'hybrid' amino acid motifs in which residues amino-terminal to each tyrosine form the motif NP X Y, resembling known PTB domain binding sites, and residues carboxyl-terminal to the same tyrosines form the motifs YXXM, YMXM, or YXLLD -- all known to be involved in binding to SH2 domains. Thus, tyrosines 1993 and 2030 appear in the motif SXNPXYXX M; tyrosine 2009 is part of S X NPXYMXM, and tyrosine 1969 appears in the sequence SDNPXYRLLD. Whether these motifs serve to bind SH2 or PTB domain-containing proteins upon tyrosine phosphorylation and whether one is preferred over the other is not clear. The cytoplasmic domain of the INR expressed in cells lacking IRS-1 has been shown to bind PI3-kinase. However, a similar construct expressed in Chinese hamster ovary cells that contain IRS-1 fails to do so. Since a significant percentage of the INR beta subunit undergoes tissue- or stage-specific proteolytic processing in Drosophila embryos to remove the carboxyl-terminal extension and once it is removed it appears not to be phosphorylated, its role in signal transduction by the INR is not clear. Therefore, the signaling capacity conferred by the beta INR carboxyl-terminal extension has been explored by expressing either full-length or truncated INR beta subunit forms in mammalian cells and determining the effect on protein-protein interactions and cell growth (Marin-Hincapie, 1999 and references therein).

In order to explore the role of the 400 AA extention in INR function, mammalian expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney (MDCK) cells in the absence of the ligand-binding alpha subunits. beta-Myc and betaDelta proteins are constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitates a phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1, Flipper or Chico), whereas INR betaDelta does not, suggesting that the site of interaction is within the carboxyl-terminal extension. IRS-1 is phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase, SHP-2, Raf-1, and JAK2, are not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases confers an equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal extension. However, INR beta-Myc-expressing cells exhibit enhanced survival, relative to parental and betaDelta cells, suggesting that the carboxyl-terminal extension, through its interaction with IRS-1, plays a role in the regulation of cell death (Marin-Hincapie, 1999).

Thus, overexpression of constitutively active INR beta and betaDelta receptors in 293 and MDCK cells promotes cell proliferation, indicating that the INR can engage the mammalian proliferation pathways. The equivalent proliferative responses induced by INR beta-Myc and betaDelta kinases suggests that the growth-promoting function of the INR in these cells is independent of the carboxyl-terminal extension. In contrast, cells expressing the full-length INR beta subunit exhibit significantly enhanced survival as compared with cells expressing the betaDelta INR. Relative to the parental 293 and MDCK cells, the INR beta-Myc and betaDelta proteins confer somewhat different behavior; beta-Myc clearly promotes survival in 293 cells, whereas betaDelta more dramatically accelerates cell death in MDCK cells. Nonetheless, a clear difference in the behavior of cells expressing the full-length or truncated INR beta subunits is evident in both backgrounds. Despite the presence of a juxtamembrane NPXY motif predicted to interact with IRS-1 in both beta-Myc and betaDelta proteins, IRS-1 is not highly phosphorylated in betaDelta cells. This suggests that the carboxyl-terminal extension of the INR beta subunit is required for sustained association and phosphorylation of IRS-1. This persistent IRS-1 phosphorylation distinguishes beta-Myc from betaDelta cells and may be of primary importance in promoting cell survival. Without this sustained interaction, cell death may actually be accelerated, as observed in MDCK cells transfected with the INR betaDelta kinase (Marin-Hincapie, 1999).

IRS-1 that is bound to the INR beta subunit is phosphorylated on tyrosine; however, no evidence has been found for increased association of PI3-kinase or other candidate signaling molecules with this complex. Therefore, the mechanism whereby this association leads to increased cell survival is unclear at present. Interestingly, a recent report demonstrates that expression of a truncated IRS-1 containing only the pleckstrin homology and phosphotyrosine binding domains, without any tyrosine phosphorylation sites, mediates PI3-kinase and phosphotyrosine-independent signals that contribute to the regulation of cell survival and apoptosis. IRS-1 that is bound to the carboxyl-terminal extension of INR in 293 and MDCK cells may have similarly activated pathways that promote cell survival in the absence of PI3-kinase activation (Marin-Hincapie, 1999 and references therein).

Thus, two isoforms of an activated INR beta subunit have been expressed in mammalian cells, and a functional difference between them has been demonstrated. The data presented here indicate that the stimulation of cell proliferation by INR is mediated by the kinase domain independent of the carboxyl-terminal extension. In contrast, the carboxyl-terminal extension mediates an interaction with IRS-1 and influences cell survival. Since an IRS homolog is present in Drosophila, this may reflect an inherent function of the INR which, in flies, is modulated by tissue- or stage-specific processing of the receptor. These data also suggest that in mammalian cells, persistent localization of IRS-1 to membranes via the interaction of IRS-1 with receptors and/or persistent tyrosine phosphorylation generates signals independent of association with PI3-kinase (Marin-Hincapie, 1999 and references therein).

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (InR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that InR might not require an IRS protein to accomplish its signaling functions. However, a cDNA encoding Drosophila IRS (Chico, but referred to in this study as dIRS) has been obtained and one for Chico in a Drosophila cell line has also been demonstrated. Like mammalian IRS proteins, the N-terminal portion of Chico contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with Chico in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of InR) mediates the insulin-stimulated tyrosine phosphorylation of Chico. Mutating the juxtamembrane NPXY motif markedly reduces the ability of the receptor to phosphorylate Chico. In contrast, the NPXY motifs in the C-terminal extension of InR are required for stable association with Chico. Coimmunoprecipitation experiments demonstrate insulin-dependent binding of Chico to phosphatidylinositol 3-kinase and SHP2. However, interactions with Grb2, SHC, or phospholipase C-gamma were not detected. Taken together with published genetic studies, these biochemical data support the hypothesis that Chico functions directly downstream from the insulin receptor in Drosophila (Poltilove, 2000).

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