Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.

This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.     

A case of insulin resistant diabetes with possible antibodies to insulin receptors.

A case of a 19-year-old, non-obese female with insulin resistant diabetes mellitus and polycystic ovary syndrome was reported. The maximal insulin requirement attained 360 units per day, but a satisfactory control of diabetes did not follow. The patient's serum contained not only anti-insulin antibodies, but also possible anti-insulin receptor antibodies which were demonstrated by the 125I-insulin binding test using insulin receptors derived from human placental plasma membrane. The insulin resistance in this case was assumed to be caused primarily by possible blocking antibodies to insulin receptors and partly by anti-insulin antibodies because of the following observations. First, high serum free insulin (165 microunits/ml) without hypoglycemia indicates the presence of insulin resistance due to other factors than antiinsulin antibodies. Second, the titer of 125I-insulin binding capacity of serum was not unusually higher than those seen in chronically insulin-treated diabetics. Third, immunologically heterospecies insulin (fish insulin) was also ineffective. The clinical features such as absence of ketoacidosis and association with polycystic ovary syndrome resemble those of an unique diabetic syndrome reported previously though acanthosis nigricans and endogenous hyperinsulinemia were not found in this case. Her insulin resistance remitted spontaneously and over the next 18 months' observation, her diabetes remained regulated without insulin therapy.     

Monoclonal antibodies to the human insulin receptor mimic a spectrum of biological effects in transfected 3T3/HIR fibroblasts without activating receptor kinase

The effects of four monoclonal antibodies to the alpha subunit of the human insulin receptor were studied in transfected mouse 3T3 fibroblasts expressing human insulin receptors (3T3/HIR). Three antibodies, MA-5, MA-20, and MA-51, mimicked insulin stimulation of the uptake of both 2-deoxy-D-glucose and alpha-aminoisobutyrio acid, and S6 kinase activity. Antibody MA-5 also mimicked insulin stimulation of [3H]thymidine incorporation and cell growth. Although these antibodies mimicked insulin stimulation of biological effects, they failed to significantly activate insulin receptor tyrosine kinase activity. These studies suggest, therefore, that the insulin receptor can signal a variety of cellular functions without stimulation of receptor kinase activity.     

Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.     

Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.     

A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface

We have previously shown that a homozygous mutation encoding a substitution of proline for leucine at position 233 in the insulin receptor is linked with the syndrome of leprechaunism, being a lethal form of insulin resistance in newborn children. Specific binding of insulin and insulin-stimulated autophosphorylation of the insulin receptor are nearly absent in fibroblasts from the leprechaun patient. To examine the molecular basis of the observed insulin receptor abnormalities, CHO cell lines overexpressing mutant insulin receptors were made by transfection. The results show that the mutation inhibits cleavage and transport of the proreceptor from intracellular sites to the cell surface. As the mutant receptor is poorly precipitated by two different monoclonal antibodies recognizing epitopes on undenatured wild-type alpha-subunits, the mutation probably affects overall folding of the alpha-subunit. The mutant proreceptor is unable to bind insulin and exhibits no insulin-stimulated autophosphorylation. These data explain the abnormalities seen in the patient's fibroblasts. Pulse-chase labeling experiments on transfected cells show that the mutant precursor has an extended half-life (approximately 5 h) compared to the precursor of wild-type insulin receptors (approximately 2 h). This mutation is the first example of a naturally occurring mutation in the insulin receptor which completely blocks cleavage of the proreceptor and transport to the cell surface.     

Presence of an insulin-stimulated serine kinase in cell extracts from IM-9 cells

Insulin responsive protein kinase activities of wheat germ purified glycoproteins were examined. Glycoproteins were first incubated without or with insulin, and then exposed to a serum containing antibodies to insulin receptor. Thereafter, both immunoprecipitates and supernatants were studied for their kinase activity toward histone. Incubation with anti receptor antibodies promoted insulin receptor beta subunit and histone phosphorylation. More important insulin receptor depleted extract contained a kinase activity toward histone, that was increased by preincubation with insulin. This stimulation was observed only when insulin was added before the immunoprecipitation of insulin receptors. Alkali treatment and phosphoamino acids analysis revealed that the kinase activity remaining in the supernatant is serine specific. These findings suggest, that a serine kinase activity is associated with the insulin receptor, that it can be separated from the insulin receptor with anti receptor antibodies, that the serine kinase is activated by the hormone-receptor complex.     

Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase

The endocytosis, recycling, and degradation of the insulin receptor were studied in IM-9 cells and U-937 cells by employing two monoclonal antibodies directed at the alpha subunit of the human insulin receptor, antibodies MA-5 and MA-10. Antibody MA-5 is an insulin agonist and MA-10 is an insulin antagonist (Forsayeth, J., Caro, J.F., Sinha, M.K., Maddux, B.A., and Goldfine, I.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3448-3451). Both monoclonal antibodies, like insulin, induced the endocytosis of the insulin receptor within 15 min. Upon removal of extracellular ligand the internalized receptor recycled to the cell surface. At this time there was no degradation of the receptor as measured by a sensitive insulin receptor radioimmunoassay. After 20 h of incubation, insulin and MA-5, but not MA-10, induced significant receptor degradation as measured by both insulin receptor radioimmunoassay and metabolic labeling studies. These studies demonstrated, therefore, that: 1) internalization and recycling of the receptor can be induced by antireceptor monoclonal antibodies that are either insulin agonists or insulin antagonists; 2) enhanced receptor degradation can be induced by monoclonal antibodies that are insulin agonists; and 3) the process of receptor internalization does not necessarily lead to enhanced receptor degradation. Since prior studies have indicated that neither MA-5 nor MA-10 enhance insulin receptor kinase activity, the present studies also suggest that insulin receptor endocytosis and degradation induced by ligands different than insulin can occur without activation of this process.     

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation

Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.     

Insulin-like and insulin-inhibitory effects of monoclonal antibodies for different epitopes on the human insulin receptor.

Monoclonal antibodies previously shown to react with five distinct epitopes on the human insulin receptor were tested for their metabolic effects on isolated human adipocytes. Two antibodies which reacted with receptor alpha-subunit and completely inhibited 125I-insulin binding mimicked the actions of insulin to stimulate lipogenesis from [14C]glucose and to inhibit catecholamine-induced lipolysis. On a molar basis, these antibodies were comparable in potency with insulin itself. Two other antibodies which decreased insulin binding only slightly or not at all also mimicked these metabolic effects of insulin. One of these antibodies reacted with receptor beta-subunit. In contrast, a further antibody which reacted with alpha-subunit and inhibited insulin binding did not affect basal lipogenesis or catecholamine-induced lipolysis, but was able to antagonize the effects of insulin on these processes. The same antibody antagonized the insulin-like effect of another antibody with which it competed in binding to insulin receptor, but not the effect of an antibody which bound independently to the receptor. It is concluded that binding of ligand at or close to the insulin-binding site is neither necessary nor sufficient to trigger insulin-like metabolic effects, which may rather depend on some general property of antibodies, such as their ability to cross-link and aggregate receptor molecules.     

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.     

A solid-phase competitive radioimmunoassay for the insulin receptor

A radioimmunoassay for the insulin receptor has been developed. In this assay, unlabeled receptor competes with 125I-labeled receptor for binding to monoclonal anti-receptor antibodies immobilized on microtiter wells coated with affinity-purified anti-mouse immunoglobulin G. This assay was highly reproducible and could detect 7 ng (14 fmol) of insulin receptor. By utilizing monoclonal antibodies to various antigenic regions of the receptor, different parts of the receptor molecule could be examined. By utilizing antibodies to the cytoplasmic domain of the receptor, an assay was developed which was not influenced by the presence of insulin and could equally detect the insulin receptor from different species (rat and human) and different tissues (placenta and brain). By utilizing antibodies to an autophosphorylation site of the receptor, the assay was shown capable of detecting the extent of phosphorylation of the receptor. Finally, this assay was utilized to monitor the decrease in insulin receptors in lysates of insulin-treated human lymphocytes. This radioimmunoassay should be useful for monitoring both the number and status of the insulin receptor under a variety of physiological conditions.     

Preparation of 125I-labelled monoclonal antibodies of the insulin receptor

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.     

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 and partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor alpha subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[gamma-32P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor beta subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.     

Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.     

Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background

The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.

Methodology/Principal Findings

We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t_1/2<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.

Conclusion

It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules.

The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.