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.     

Characterization of an endogenous substrate of the insulin receptor in cultured cells

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.     

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.     

Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.     

A novel fetal insulin-like growth factor (IGF) I receptor. Mechanism for increased IGF I- and insulin-stimulated tyrosine kinase activity in fetal muscle

Insulin and insulin-like growth factor (IGF) I receptors from fetal and adult rat skeletal muscle were compared in order to gain insight into the evolving functions of the hormones during development. Basal, insulin-stimulated, and IGF I-stimulated receptor phosphorylation and tyrosine kinase activity are severalfold higher in partially purified receptor preparations from fetal muscle in comparison with equal numbers of receptors from adult muscle. There are distinct insulin and IGF I receptors with Mr 95,000 beta subunits in adult muscle, as evidenced by hormone dose-response curves, immunoprecipitation with specific antibodies, binding to insulin and IGF I affinity columns, and analysis of tryptic phosphopeptides. In addition to these two receptor species, fetal muscle contains a receptor with a Mr 105,000 beta subunit. The fetal receptor is structurally more closely related to the IGF-I receptor than the insulin receptor on the basis of its precipitation with specific antibodies, binding to an IGF I affinity column, and tryptic phosphopeptide map. The fetal receptor does not appear to bind insulin but, unlike the IGF-I receptor, its phosphorylation is stimulated by low physiological concentrations of both insulin and IGF I. This could be explained by the cross-phosphorylation of fetal receptors by activated insulin receptors. Expression of the fetal receptor is highest in the fetus and decreases markedly during the first 2 weeks of postnatal life. The fetal receptor appears to account for the high tyrosine kinase activity of fetal muscle and may be an important mediator of responses to both insulin and IGF I early in development.     

Madin-Darby canine kidney cells display type I and type II insulin-like growth factor (IGF) receptors

Using affinity cross-linking techniques, we report the presence of type I IGF and type II IGF receptors in Madin-Darby canine kidney cells, a line of cells lacking insulin receptors. The IGF receptors were further characterized by competition binding studies and found to be similar to IGF receptors in other tissue types. In Madin-Darby canine kidney cells, the type I IGF receptor binds IGF-I greater than IGF-II greater than insulin and the type II IGF receptor binds IGF-II and IGF-I with approximately the same affinity, but does not bind insulin.     

Tyrosine phosphorylation of pp185 by insulin receptor kinase in a cell-free system

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the beta-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the beta-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.     

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.     

The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase.

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.     

Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Insulin-like growth factor-mediated phosphorylation and protooncogene induction in Madin-Darby canine kidney cells.

We have characterized the role of tyrosine phosphorylation in protooncogene induction mediated by insulin-like growth factors I and II (IGF-I and IGF-II) in the Madin-Darby canine kidney (MDCK) cell line. These cells possess few, if any, insulin receptors, thus allowing determination of the effects of these growth factors in the absence of any secondary signal mediated through the insulin receptor. We found that IGF-I produced a specific stimulation of tyrosine kinase activity of the 97-kDa beta-subunit of the IGF-I receptor, resulting in autophosphorylation of the receptor and an increase in kinase activity toward a synthetic peptide substrate. This was associated with a gradual decrease in the level of phosphorylation of pp120, the major constitutive phosphotyrosine-containing protein of MDCK cells, and an increase in the ratio of serine to tyrosine phosphorylation. This was followed by a rapid, but transient, induction of c-fos gene expression, with no change in the levels of c-myc mRNA. Cycloheximide treatment resulted in a superinduction of both c-fos and c-myc and prevented any further stimulation by IGF-I. IGF-II did not stimulate tyrosine phosphorylation of its own receptor, but was 25% as active as IGF-I in stimulating phosphorylation of the IGF-I receptor. Despite this, IGF-II did not significantly enhance the expression of either nuclear protooncogene. Insulin also produced a delayed stimulation of IGF-I receptor phosphorylation, but was unable to stimulate biological effects in these cells. Under these conditions neither of the IGFs nor insulin produced any significant stimulation of thymidine incorporation into DNA. These data indicate that the IGF-I receptor can be activated upon binding of IGF-I, and to a lesser extent IGF-II, in intact cells to mediate cellular events. The nature of the signal generated by the IGF-I receptor appears to vary depending on the ligand that occupies it.     

Characterization of insulin-like growth factor I receptors on Madin-Darby canine kidney (MDCK) cell line

Specific insulin-like growth factor I (IGF-I) receptors on the Madin-Darby canine kidney (MDCK) cell line were identified and characterized. [125I]IGF-I specifically bound to the cells, but [125I]insulin bindings to the cells was minimal. Unlabeled IGF-I displaced both the IGF-I and insulin bindings with potencies that were 100 and 10 times as great as insulin. By an affinity labeling technique, IGF type I receptors were present in the MDCK cells. IGF-I stimulated DNA synthesis and cell proliferation at physiological concentrations. On the other hand, insulin had a little effect on DNA synthesis. These data suggest that IGF type I receptors as demonstrated in MDCK cells are involved in DNA synthesis and cell proliferation.     

An altered IGF-I receptor is present in human leukemic cells.

We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.     

Insulin and insulin-like growth factor I (IGF I) stimulate phosphorylation of a Mr 175,000 cytoskeleton-associated protein in intact FRTL5 cells

FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells.     

Different effects of two proteinase inhibitors on insulin-induced cellular responses in rat adipocytes

Among various proteinase inhibitors, N-acetyl-L-tyrosine ethyl ester (ATEE), a chymotrypsin substrate analog, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, showed significant inhibitory effects on insulin stimulated glucose transport in rat adipocytes. ATEE did not affect insulin binding, but inhibited insulin internalization. In intact adipocytes, ATEE inhibited tyrosine phosphorylation of the beta-subunit of the insulin receptor, a 170 kDa protein and a 60 kDa protein at almost the same concentration (ID50 = 0.24 +/- 0.05 mM, n = 4, mean +/- S.E.), but in a plasma membrane fraction, ATEE did not appreciably inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor, TLCK did not inhibit insulin binding. At 0.25 mM, TLCK did not inhibit insulin internalization, but inhibited 70% of the insulin-stimulated glucose transport (ID50 = 0.19 +/- 0.02 mM, n = 7). TLCK inhibited insulin internalization at more than 0.25 mM. TLCK did not inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor in intact cells or in the plasma membrane fraction. In intact cells, TLCK inhibited the phosphorylation of the 60 kDa protein and simultaneously it stimulated the phosphorylation of the 170 kDa protein more than 3-fold. These results indicate that there are at least two sites in the insulin-induced signal transduction pathway where proteinase inhibitors act to suppress the insulin signal transduction. A major ATEE site is very close to phosphorylation of the beta-subunit of the insulin receptor. On the other hand, TLCK inhibits a step(s) in the signal transduction pathway after the insulin receptor but before the glucose transporter.     

Characterization of the receptor for insulin-like growth factor on Leishmania promastigotes

Insulin-like growth factor (IGF)-I constitutively present in the skin is one of the first growth factors that Leishmania parasites encounter after transmission to the vertebrate host. We have previously shown that IGF-I is a potent growth-promoting factor for Leishmania parasites. IGF-I binds specifically to a single-site putative receptor at the parasite membrane, triggering a cascade of phosphorylation reactions. In the present article we characterize the receptor for IGF-I on Leishmania (Leishmania) mexicana promastigotes. The receptor is a monomeric glycoprotein with a molecular mass of 65 kDa and is antigenically related to the alpha chain of human type 1 IGF-I receptor. Upon IGF-I stimulation the receptor undergoes autophosphorylation on tyrosine residues with activation of its signaling pathway. Activation of the IGF-I receptor also leads to phosphorylation of an 185-kDa molecule that is homologous to the substrate of the insulin receptor present in human cells, the insulin receptor substrate 1 (IRS-1).     

Insulin-like growth factor (IGF)/somatomedin receptor subtypes: structure, function, and relationships to insulin receptors and IGF carrier proteins

The insulin-like growth factors IGF-I and IGF-II are mitogenic polypeptides with a high degree of chemical homology. Two distinct subtypes of receptors for the IGFs have been identified on the basis of structure and binding specificity. Type I IGF receptors bind IGF-I with equal or greater affinity than IGF-II, and also bind insulin with a low but definite affinity. They are structurally homologous to insulin receptors, containing disulfide-linked a-subunits that bind the peptides and beta-subunits that have intrinsic tyrosine-specific kinase activity. Type II IGF receptors typically bind IGF-II with greater affinity than IGF-I, and do not interact with insulin. They consist of a single polypeptide and lack tyrosine kinase activity. Because of the extensive cross-reactivity of IGF-I and IGF-II with both type I and type II receptors, we believe that potentially either receptor may mediate the biological responses of either peptide. Type I IGF receptors have been shown to mediate the mitogenic effects of the IGFs in some cell types. Whether type II IGF receptors mediate the same or different functions remains to be elucidated.     

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.     

Increased autophosphorylation of insulin-like growth factor-I and insulin receptors in placentas of diabetic women

Autophosphorylation of insulin and insulin-like growth factor (IGF)-I receptors were measured in lectin purified receptor preparations from placentas of normal and diabetic patients. The basal and insulin or IGF-I stimulated phosphorylation of the approximately 94 kD protein, corresponding to beta-subunit of the insulin and IGF-I receptors, were approximately 2 times greater (p less than 0.05) in placentas from diabetic patients with poor glycemic control (as judged by their serum HbA1c level) compared to the normals. The magnitude of IGF-I or insulin stimulation of the phosphorylation of the 94 kD protein was comparable in placentas from both diabetic and normal patients. Immunoprecipitation and immunodepletion of IGF-I receptor by alpha-IR3, a monoclonal antibody to IGF-I receptor, revealed the increased basal phosphorylation of the approximately 94 kD protein in placentas of diabetic patients to be associated with IGF-I and insulin receptors. The magnitude of IGF-I and insulin stimulated phosphorylation of the immunoprecipitated and immunodepleted IGF-I receptor, respectively, was the same in both normal and diabetic patients. These results suggested that the increased basal phosphorylation of the 94 kD protein in placentas from diabetic patients may be intrinsic to IGF-I and insulin receptor, however, the regulatory mechanisms effecting the increase may not be dependent on IGF-I or insulin.