| Biochemical studies have shown that porcine insulin interacts in vitro with a Drosophila melanogaster intrinsic plasma membrane polypeptide complex that shows an insulin regulated protein tyrosine kinase activity. Ligand binding as well as stimulation of the protein kinase activity are highly specific for insulin. These studies have shown that the biochemical functions of the Drosophila insulin receptor subunits, their stoichiometry in mature receptors, and the mechanism by which they interact, are surprisingly homologous to those of the human insulin receptor. An $alpha$ subunit-like polypeptide (Mr: 120 Kd) is responsible for the high affinity and specific ligand binding activity. This $alpha$ subunit, is disulfide linked to two forms (Mr: 95 Kd and 170 Kd) of $beta$ subunit-like polypeptides, which posses intrinsic, insulin stimulated protein tyrosine kinase activity. All these biochemical studies were carried out primarily with antipeptide antibodies elicited to the deduced amino acid sequence of the human insulin receptor. Exploiting the immuno-crossreactivity of these antibodies with the putative Drosophila insulin receptor, biosynthesis and topology studies were also done on metabolically labelled cells, derived from primary cultures of Drosophila embryos or in established Drosophila cell lines. The fly receptor is synthesized as a preproreceptor of 230 Kd, which is glycosylated to a 280 Kd form, before undergoing proteolysis to generate equimolar amounts of $alpha$ (Mr: 120 Kd) and $beta$ (Mr: 170 Kd) subunits. The other form of the $beta$ subunit (Mr: 95 Kd), is generated by a physiological, slow proteolytic processing of the 75 Kd carboxy-terminus of the larger $beta$ form. Independently, we cloned the Drosophila insulin receptor gene. The deduced amino acid sequence of this gene predicts an open reading frame of 225 Kd, which contains all structural motifs expected to be found in such a protein, based on our biochemical data. Moreover, the Drosophila melanogaster insulin receptor turned out to be not only homologous to its human counterpart in both insulin binding and tyrosine kinase domains; but also shows homology, through the unusual, unique carboxy-terminal tail, to the cytosolic protein insulin receptor substrate-1 (IRS-1). Since it is known that in mammalian systems the insulin receptor and IRS-1 are encoded by different genes, we propose that divergent evolution replaced the need for protein-protein interaction in the transduction of some of insulin's signals by the generation of a chimeric protein encoded by a single gene in Drosophila. |
