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An insulin receptor mutant was constructed utilizing site-directed mutagenesis to delete the Arg-Lys-Arg-Arg basic amino acid cleavage site (positions 720-723) from the cDNA encoding the human insulin proreceptor. This mutant was transfected into Chinese hamster ovary cells. Immunoprecipitation of metabolically labeled cells revealed a 205-kDa proreceptor which bound to wheat germ agglutinin. Processed 130-kDa alpha and 95-kDa beta subunits were also observed and contained approximately 20% as much protein as the proreceptor on a molar basis. Trypsin digestion of intact metabolically labeled cells decreased the proreceptor band by 80%. Pulse-chase studies revealed a half-life of 28 h for the proreceptor. When cells were photolabeled with 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1 (NAPA)-insulin, the proreceptor incorporated 10% as much label as the 130-kDa alpha subunit in spite of a 5-fold molar excess. Incubation of NAPA-labeled cells at 37 degrees C for 20 min resulted in 60% of the labeled subunits, but little labeled proreceptor, becoming resistant to trypsin degradation. Immunoprecipitation of NAPA-insulin-stimulated cells with anti-phosphotyrosine antibodies revealed that 62% of the processed labeled receptors, but very little proreceptor, contained phosphotyrosine. Thus, this mutant receptor is synthesized, glycosylated, and expressed on the cell surface as uncleaved proreceptor, although some processing to alpha and beta subunits still occurs. It exhibits a markedly decreased affinity for insulin, and when insulin is bound to, demonstrates defective internalization, down-regulation, and autophosphorylation. These data suggest that cleavage of the mutant proreceptor into subunits is required not only for the development of high affinity binding sites, but also for normal transduction of the signal which activates the beta subunit tyrosine kinase.

In order to test the contribution of the insulin receptor COOH terminus to insulin action, a truncation of 43 COOH-terminal amino acids was engineered by cDNA-based deletion mutagenesis. This cDNA (HIR delta CT), as well as cDNA encoding the complete receptor (HIRc) was transfected into Rat 1 fibroblasts. Cells expressing 6.4 X 10(3) and 1.25 X 10(6) normal receptors and 2.5 X 10(5) HIR delta CT receptors, as well as control Rat 1 fibroblasts were selected for further analysis. All cell lines exhibited insulin binding of similar affinity. Partial tryptic digestion and immunoprecipitation by region-specific antibodies verified that the HIR delta CT receptors were truncated at the COOH terminus. Purified HIRc and HIR delta CT receptors underwent autophosphorylation with similar insulin and ATP sensitivity, although the HIR delta CT receptors were slightly more active in the absence of insulin. Transfected HIRc and HIR delta CT receptors undergo endocytosis in a normal fashion. Insulin internalization and degradation in both HIRc and HIR delta CT cells is increased in proportion to receptor number. Intracellular insulin processing, degradation, and release were qualitatively comparable among the transfected cell lines. Complete and truncated receptors internalize, recycle, and down-regulate normally. We conclude the following: 1) the COOH-terminal portion of the insulin receptor is not necessary for partial autophosphorylation or endocytosis; 2) following internalization the intracellular itinerary of the receptor and ligand appear normal with the truncated receptor; and 3) truncation of the COOH terminus does not impair recycling of the receptor or retroendocytosis of internalized ligand.

Transfected Chinese hamster ovary cell lines were developed that expressed equivalent numbers of either normal human receptor or receptor that had alanine substituted for Lys-1018 in the ATP-binding domain of the beta subunit. The mutated receptor was processed into subunits and bound insulin but lacked protein tyrosine kinase activity. Five effects of insulin were assayed: deoxyglucose uptake, S6 kinase activity, endogenous protein-tyrosine phosphorylation, glycogen synthesis, and thymidine uptake. In each case, cells bearing normal human receptors were 10-100-fold more sensitive to insulin than the parental cells. Cells with the mutant receptor behaved like the parental cells with respect to S6 kinase activation, endogenous substrate phosphorylation, glycogen synthesis, and thymidine uptake, but their deoxyglucose uptake was significantly depressed and relatively insensitive to insulin. The analyses led to the following conclusions: substitution of alanine for lysine at amino acid 1018 inactivates the kinase activity of the receptor; a kinase-negative receptor can be properly processed and bind insulin; insulin-dependent deoxyglucose uptake, S6 kinase activation, endogenous substrate phosphorylation, glycogen synthesis, and thymidine incorporation into DNA are mediated by the normal but not by the kinase-deficient human receptor.

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.