Comparison of the Signaling Abilities of the Drosophila and Human Insulin Receptors in Mammalian Cells

Abstract

Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the drosophila insulin receptor (IR) with the extracellular domain of the human 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 bound insulin equally and exhibited 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 was found to be indistinguishable from that of the human IR. The chimeric drosophila receptors did not bind more phosphatidylinositol 3-kinase 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.

The Drosophila Insulin Receptor Activates Multiple Signaling Pathways but Requires Insulin Receptor Substrate Proteins for DNA Synthesis

“…The intracellular portion of the DIR ␤ subunit is similar to the ␤ subunit of the human IR, especially around the tyrosine autophosphorylation sites in the regulatory and juxtamembrane regions, and was shown previously to display a similar signaling capacity (37). The broader signaling potential of the DIR revealed by our results is apparently due to the 368-amino-acid extension at the COOH terminus (DIR tail).…”

“…2). Thus, hDIR was activated by human insulin in 32D cells, as previously shown in other cell systems (37).…”

“…Since hDIR stimulates both IRS-1-dependent and IRS-1-independent pathways, it was expected to mediate insulin-stimulated DNA synthesis without IRS-1. However, DNA synthesis in 32D hDIR cells is insensitive to insulin, indicating that the phosphorylation of Shc and the activation of PI 3-kinase, p70 Other studies investigating the signaling capacity of the Drosophila and mammalian IRs have suggested that there are no differences in function (37). However, these studies are confounded by the presence of endogenous IRS proteins in the cell systems used.…”

“…1A). The DIR binds human insulin with slightly decreased affinity, but its signaling capacity is similar to that of the human IR, suggesting that essential functions in the ␤ subunit were conserved from insects to mammals (8,37). The DIR is essential for Drosophila development and appears to be required for the epidermis and the nervous system (8).…”

Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function.

“…Cells were lysed in lysis buffer containing 50 mM Hepes (pH 7.6), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 10 mM NaF, aprotinin (10 ,tg/ml), leupeptin (10 ,ug/ml), 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 20 mM sodium pyrophosphate. Cell lysates or immunoprecipitates were separated by SDS/PAGE in 10% polyacrylamide gels and then examined by immunoblot analysis as described (8). The polyclonal antibody to Grb-IR was generated against the purified SH2 domain containing glutathione S-transferase (GST) fusion protein (GST-Grb-IRc) described below.…”