High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I receptor, was employed over 60% of 125I-insulin binding was inhibited. The B29-MAB-125I-insulin photoprobe was then cross-linked to MCF-7 membranes. Cross-linking was inhibited by both unlabeled insulin and IGF-I. Further, the B29-MAB-125I-insulin photoprobe cross-linked to MCF-7 membranes was strongly immunoprecipitated by alpha-IR3. Employing sequential affinity chromatography with insulin-Affi-gel followed by insulin receptor monoclonal antibody agarose, atypical insulin binding activity was separated from insulin receptor binding activity. This atypical receptor had intrinsic tyrosine kinase activity. Both insulin and IGF-I stimulated the phosphorylation of the receptor's beta subunit. In MCF-7 cells both IGF-I and insulin stimulated [3H]thymidine incorporation; alpha-IR3 blocked all of the IGF-I effect but only 50-60% of the insulin effect. This study demonstrates in MCF-7 cells that, in addition to typical insulin and IGF-I receptors, there is another receptor that binds both insulin and IGF-I with high affinity.

Muscle cell differentiation is associated with increased insulin receptor biosynthesis and messenger RNA levels.

Muscle is a major tissue for insulin action. To study the effect of muscle differentiation on insulin receptors, we employed cultured mouse muscle BC3H-1 and C2 cells. In both cell lines differentiation from myoblasts to myocytes was associated with a 5-10-fold increase in specific 125I-insulin binding to intact cells. When 125I-insulin binding was carried out on solubilized myocytes and myoblasts, 125I-insulin binding to myoblasts was low. After differentiation the number of insulin receptors increased 5-10-fold. In contrast to insulin binding, insulin growth factor I receptor binding was elevated in myoblasts and was decreased by 50% in myocytes. Specific radioimmunoassay of the insulin receptor indicated that the increase in insulin binding to myocytes was due to an increase in insulin receptor content. Studies employing [35S]methionine indicated that this increase in insulin-binding sites reflected an increase in insulin receptor biosynthesis. To study insulin receptor gene expression, myoblast and myocyte mRNA was isolated and analyzed on Northern and slot blots. Differentiation from myoblasts to myocytes was accompanied by a 5-10-fold increase in insulin receptor mRNA. These studies demonstrate, therefore that differentiation in muscle cells is accompanied by increased insulin receptor biosynthesis and gene expression.

Membrane glycoprotein PC-1 inhibition of insulin receptor function occurs via direct interaction with the receptor alpha-subunit.

Plasma cell membrane glycoprotein-1 (PC-1) inhibits insulin receptor (IR) tyrosine kinase activity and subsequent cellular signaling. PC-1 content is elevated in fibroblasts, muscle, and adipose tissue from insulin-resistant subjects, and its elevation correlates with in vivo insulin resistance. In vitro, when PC-1 is transfected and overexpressed in cultured cells, it inhibits IR tyrosine kinase activity. To determine the mechanism whereby PC-1 regulates the IR, we studied how PC-1 interacts with this protein. Overexpression of PC-1 in MCF-7 cells inhibited tyrosine kinase activity of the IR, but not of the IGF-I receptor. When the IR was immunocaptured by specific IR monoclonal antibodies, PC-1 was associated with this receptor. In contrast, after specific immunocapture, PC-1 was not associated with the IGF-I receptor. We next studied HTC cells that were overexpressing an IR alpha-subunit mutant. This IR mutant binds insulin but has a deletion in the tyrosine kinase regulatory domain located in amino acids 485-599. In contrast to normal IRs, PC-1 did not associate with this mutant and did not affect tyrosine kinase activity. To determine whether decreasing PC-1 expression would reverse the inhibition of tyrosine kinase activity, we treated MCF-7 cells overexpressing PC-1 with a monoclonal antibody to PC-1. This treatment decreased PC-1 levels; concomitantly, IR tyrosine kinase activity increased. In contrast, IGF-I receptor tyrosine kinase activity was not increased. These studies indicate, therefore, that PC-1 may inhibit the IR by interacting directly with a specific region in the IR alpha-subunit. These studies also raise the possibility that monoclonal antibodies to PC-1 could be a new treatment for insulin resistance.

Sequence and analysis of promoter region of human insulin-receptor gene.

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.

Protection against oxidative stress-induced insulin resistance in rat L6 muscle cells by mircomolar concentrations of alpha-lipoic acid.

In diabetic patients, alpha-lipoic acid (LA) improves skeletal muscle glucose transport, resulting in increased glucose disposal; however, the molecular mechanism of action of LA is presently unknown. We studied the effects of LA on basal and insulin-stimulated glucose transport in cultured rat L6 muscle cells that overexpress GLUT4. When 2-deoxy-D-glucose uptake was measured in these cells, they were more sensitive and responsive to insulin than wild-type L6 cells. LA, at concentrations < or = 1 mmol/l, had only small effects on glucose transport in cells not exposed to oxidative stress. When cells were exposed to glucose oxidase and glucose to generate H2O2 and cause oxidative stress, there was a marked decrease in insulin-stimulated glucose transport. Pretreatment with LA over the concentration range of 10-1,000 pmol/l protected the insulin effect from inhibition by H2O2. Both the R and S isomers of LA were equally effective. In addition, oxidative stress caused a significant decrease (approximately 50%) in reduced glutathione concentration, along with the rapid activation of the stress-sensitive p38 mitogen-activated protein kinase. Pretreatment with LA prevented both of these events, coincident with protecting insulin action. These studies indicate that in muscle, the major site of insulin-stimulated glucose disposal, one important effect of LA on the insulin-signaling cascade is to protect cells from oxidative stress-induced insulin resistance.

Mechanisms of insulin-induced insulin-receptor downregulation. Decrease of receptor biosynthesis and mRNA levels.

The influence of insulin on the downregulation of its receptor was studied in AR42J cultured pancreatic acinar cells, a cell line that has been demonstrated to be metabolically responsive to insulin. Downregulation induced by insulin was time and dose dependent. After a 20-h incubation with 1 microM insulin, Scatchard analysis revealed approximately 80% loss of insulin receptors. Studies of receptor half-life indicated that treatment with insulin accelerated the degradation of both the alpha- and beta-subunits of the insulin receptor by 30-60%. In addition, biosynthetic-labeling studies indicated that insulin inhibited the biosynthesis of the insulin-receptor precursor by greater than 30%. This decreased biosynthesis of the precursor was associated with decreased production of mature receptor subunits. Poly(A)+ RNA was extracted from control cells and cells treated for 24 h with 100 nM insulin. Slot blots and Northern transfers revealed that insulin induced an approximately 50% decrease in insulin-receptor mRNA levels. Therefore, these studies indicate that insulin may diminish the concentration of its receptors in target cells by at least two mechanisms: acceleration of receptor degradation and inhibition of receptor biosynthesis at the level of mRNA.

Production of inhibitor of insulin-receptor tyrosine kinase in fibroblasts from patient with insulin resistance and NIDDM.

Although non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in insulin action, the molecular basis of this resistance is unknown. We studied fibroblasts from a markedly insulin-resistant patient with NIDDM but without acanthosis nigricans. Her fibroblasts were resistant to insulin when alpha-aminoisobutyric acid uptake was measured. Fibroblasts from this patient demonstrated normal insulin-receptor content as measured by both insulin-receptor radioimmunoassay and by Scatchard analysis. However, when compared with nondiabetic control subjects, insulin-receptor kinase assays of wheat-germ-purified receptors prepared from her fibroblasts showed very low basal and no insulin-stimulated tyrosine kinase activity. The insulin receptor was then removed from the wheat-germ fraction by monoclonal antibody affinity chromatography. This insulin-receptor-deficient fraction inhibited both basal and insulin-stimulated tyrosine kinase activity of highly purified insulin receptors. When the specificity of this inhibition was tested, less inhibition was seen with insulinlike growth factor I-receptor tyrosine kinase, and even less inhibition was seen with the proto-oncogene p60c-src tyrosine kinase. Thus, these studies indicate that fibroblasts from an insulin-resistant patient with NIDDM produce a relatively specific glycoprotein inhibitor of insulin-receptor tyrosine kinase. Therefore, these studies raise the possibility that this inhibitor may play an important role in the insulin resistance seen in this patient.

Regulation of insulin-receptor mRNA levels by glucocorticoids.

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulin-receptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.

Skeletal muscle content of membrane glycoprotein PC-1 in obesity. Relationship to muscle glucose transport.

Membrane glycoprotein PC-1, an inhibitor of insulin signaling, produces insulin resistance when overexpressed in cells transfected with PC-1 cDNA. In the present study, we determined whether PC-1 plays a role in the insulin resistance of skeletal muscle in obesity. Rectus abdominus muscle biopsies were taken from patients undergoing elective surgery. Subjects included both NIDDM patients (n = 14) and nondiabetic patients (n = 34) across a wide range of BMI values (19.5-90.1). Insulin-stimulated glucose transport was measured in incubated muscle strips, and PC-1 content, enzymatic activity, and insulin receptor content were measured in solubilized muscle extracts. Increasing BMI correlated with both an increase in the content of PC-1 in muscle (r = 0.55, P < 0.001) and a decrease in insulin stimulation of muscle glucose transport (r = -0.58, P = 0.008). NIDDM had no effect on either PC-1 content or glucose transport for any given level of obesity. Insulin stimulation of muscle glucose transport was negatively related to muscle PC-1 content (r = -0.68, P = 0.001) and positively related to insulin receptor content (r = 0.60, P = 0.005). Multivariate analysis indicated that both skeletal muscle PC-1 content and insulin receptor content, but not BMI, were independent predictors of insulin-stimulated glucose transport. Muscle PC-1 content accounted for 42% and insulin receptor content for 17% of the variance in glucose transport values. These studies raise the possibility that increased expression of PC-1 and a decreased insulin receptor content in skeletal muscle may be involved in the insulin resistance of obesity.

Small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells.

In type 2 diabetes, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. To study a new class of antidiabetic agents, we compared two small, nonpeptide molecules that activate insulin receptor (IR) beta-subunit tyrosine kinase activity: Merck L7, a direct IR agonist, and Telik's TLK16998, an IR sensitizer. In rat hepatoma cells (HTCs) that overexpress the IR (HTC-IR), IR autophosphorylation was directly activated by L7 in the absence of insulin. TLK16998 did not directly activate IR autophosphorylation, but it enhanced IR autophosphorylation in the presence of insulin. Tyrosine phosphorylation of an endogenous 185-kDa IR substrate was also significantly enhanced by both Merck L7 alone and TLK16998 plus insulin. Adding TLK16998 to L7 produced synergistic effects, further indicating that these two compounds act on the IR through separate mechanisms. We next studied HTC-IR(Delta485-599) cells, which overexpress a mutant IR with a deletion in the alpha-subunit connecting domain that does not undergo autophosphorylation in response to insulin binding. L7 was able to directly activate autophosphorylation of the deletion mutant IR in these cells, whereas TLK16998 had no effect. Compounds were then tested in three other cell models of impaired IR function. Both TLK16998 and Merck L7 improved IR autophosphorylation in cells with diminished IR signaling due to either treatment with tumor necrosis factor-alpha or overexpression of membrane glycoprotein PC-1. However, in TPA (tetradecanoylphorbol acetate)-treated cells, TLK16998 but not Merck L7 was able to significantly reverse the impaired insulin-stimulated IR autophosphorylation. In summary, these two classes of IR activators selectively increased IR function in a variety of insulin-resistant cell lines.

Tissue-specific antibodies against the fibroblast insulin receptor in a patient with lupus nephritis and hypoglycemia.

We recently reported that the serum from a patient with lupus nephritis, insulin resistance, and hypoglycemia contains multiple populations of antibodies directed at the human insulin receptor. In the present study, we found a subpopulation of antibodies (eluted from a protein A-Sepharose affinity column at pH 4.3) directed at the human fibroblast insulin receptor. When tested against human placental membranes, IM-9 lymphocytes, circulating monocytes and erythrocytes, and isolated adipocytes, the antibody subpopulation did not compete with 125I-insulin for binding to its receptor. In contrast, the antibody subpopulation competed with 125I-insulin for binding to the human fibroblast insulin receptor. This antibody subpopulation stimulated [3H]alpha-aminoisobutyric acid [( 3H]AIB) uptake to these cells. Unlike the effect of insulin, however, this regulation of transport was not antagonized by a mouse monoclonal antibody to the human insulin receptor that inhibits 125I-insulin binding. These studies indicate, therefore, that a tissue-specific antibody subpopulation can occur spontaneously in patients with antibodies to the human insulin receptor. Furthermore, they indicate the presence of anti-insulin receptor autoantibodies specifically directed against a tissue that is not primarily involved in glucose metabolism.

Progestins induce down-regulation of insulin-like growth factor-I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II.

Insulin-like growth factor-I (IGF-I) receptors are present in breast cancer cells and may play a role in breast cancer cell growth. We have studied the effect of progestins on IGF-I receptors in T47D human breast cancer cells. T47D cells constitutively express high levels of progesterone receptors and are a model for studying the regulation of cellular functions by progestins. Treatment of T47D cells with either progesterone or the synthetic progestin promegestone (R5020) decreased IGF-I receptor content by approximately 50%, as measured by Scatchard analysis and receptor biosynthesis studies. In contrast to progestins, estradiol, dexamethasone, and dihydrotestosterone did not influence IGF-I receptor content. No effect of R5020 was seen after 12 h of incubation, a near-maximal effect was seen after 24 h, and greatest effects were seen after 72 h. R5020 decreased IGF-I receptor mRNA abundance, indicating that progestins acted at the level of gene expression. However, progestins also increased the secretion of IGF-II, a ligand for the IGF-I receptor. In contrast to IGF-II, T47D cells did not express IGF-I. The addition of exogenous IGF-II to T47D cells down-regulated both IGF-I receptor binding and IGF-I receptor mRNA abundance. This study indicates, therefore, that progestins regulate IGF-I receptors in breast cancer cells and suggests that this regulation occurs via an autocrine pathway involving enhanced IGF-II secretion.

Regulating insulin-receptor-gene expression by differentiation and hormones.

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin and insulin-like growth factor I regulate the same biological functions in HEP-G2 cells via their own specific receptors.

The receptors for insulin and insulin-like growth factor I (IGF-I) are closely related molecules, with an extracellular binding domain and an intracellular tyrosine kinase domain. The interaction of insulin and IGF-I with their respective receptors activates the receptor kinase domain, leading to the biological actions of the hormones. Since insulin generally regulates metabolic events and IGF-I generally regulates growth events, it is believed that structural differences in the tyrosine kinase domains of the two respective receptors may elicit different biological responses via different transmembrane signaling mechanisms. We studied the regulation of glycogen metabolism and amino acid uptake in human cultured HEP-G2 hepatoma cells, which have distinct receptors for both insulin and IGF-I. The receptor specificity of these responses was probed with specific monoclonal antibodies to both the insulin and IGF-I receptors. Stimulation of both [3H]glucose incorporation into glycogen and alpha-[3H]aminoisobutyric acid uptake by insulin was half-maximal at concentrations of 1-5 nmol/L. These effects were blocked by the insulin receptor monoclonal antibody MA-10, but not by the IGF-I receptor antibody alpha IR-3. Stimulation of both functions by IGF-I was half-maximal at concentrations of 1-5 nmol/L, and these effects were inhibited by alpha IR-3, but not by MA-10. These studies indicate that in HEP-G2 cells both insulin and IGF-I, via their own receptors, stimulate the same biological responses.

Inhibitors of insulin receptor tyrosine kinase in fibroblasts from diverse patients with impaired insulin action: evidence for a novel mechanism of postreceptor insulin resistance.

Insulin resistance is a major feature of noninsulin-dependent diabetes mellitus. This resistance appears to involve molecules, apart from the insulin receptor, that are capable of altering its function. Previously, we reported that dermal fibroblasts from a female patient with insulin resistance and noninsulin-dependent diabetes produced an inhibitor of insulin receptor tyrosine kinase activity. We have now studied inhibitors in fibroblasts from four additional patients (one male and three females) with severe insulin resistance. Although clinical features were diverse, these patients had in common normal fasting glucose values, with fasting and postprandial hyperinsulinemia. The fibroblast insulin receptor content was within the normal range, but both basal and insulin-stimulated tyrosine kinase activity in fibroblast extracts were markedly decreased compared to those in extracts of fibroblasts from nondiabetic subjects. Studies revealed that these fibroblasts contained a glycoprotein inhibitor of insulin receptor tyrosine kinase activity. This inhibitor was not found in extracts of either similar insulin-resistant patients with normal insulin receptors or insulin-resistant patients with insulin receptor abnormalities. The inhibitor was not adsorbed with antiserum to either tyrosine phosphatases or fetuin. These studies thus suggest that one or more unique inhibitors of insulin receptor tyrosine kinase are present in fibroblasts of certain patients with severe insulin resistance. The presence of insulin receptor tyrosine kinase inhibitors in target cells, therefore, may constitute a novel mechanism of postreceptor insulin resistance.

A soluble PC-1 circulates in human plasma: relationship with insulin resistance and associated abnormalities.

An increased tissue content of PC-1, an inhibitor of insulin receptor signaling, may play a role in insulin resistance. Large scale prospective studies to test this hypothesis are difficult to carry out because of the need for tissue biopsies. The aim of this study was to investigate whether PC-1 is measurable in human plasma and whether its concentration is related to insulin sensitivity. A soluble PC-1, with mol wt and enzymatic activity similar to those of tissue PC-1, was measurable in human plasma by a specific enzyme-linked immunosorbent assay and was inversely correlated to skeletal muscle PC-1 content (r = -0.5; P < 0.01). The plasma PC-1 concentration was decreased (P < 0.05) in insulin-resistant (22.7 +/- 3.0 ng/mL; n = 25) compared to insulin-sensitive (36.7 +/- 4.5; n = 25) nondiabetic subjects and was correlated negatively with the waist/hip ratio (r = -0.48; P < 0.001) and mean blood pressure (r = -0.3; P < 0.05) and positively with high density lipoprotein/total cholesterol (r = 0.38; P < 0.01) and both the M value and the plasma free fatty acid level decrement at clamp studies (r = 0.28; n = 50; P = 0.05 and r = 0.43; n = 22; P < 0.05, respectively). A plasma PC-1 concentration of 19 ng/mL or less identified a cluster of insulin resistance-related alterations with 75% accuracy. In conclusion, PC-1 circulates in human plasma, and its concentration is related to insulin sensitivity. This may help to plan studies aimed at understanding the role of PC-1 in insulin resistance.

Human autoantibodies directed against the human, but not the rat, insulin receptor.

Prior studies with monoclonal antibodies produced against the human insulin receptor in mice revealed that these antibodies may be species specific. Whether species-specific antibodies to the insulin receptor occur spontaneously in patients, however, has not been previously investigated. Recently, we found that the serum immunoglobulin G from a patient with lupus nephritis, insulin resistance, and hypoglycemia contained multiple subpopulations of antibodies directed at the human insulin receptor. We report herein that one such subpopulation has a high affinity for the human insulin receptor. This antibody subpopulation at 10 nM half-maximally inhibited [125I]insulin binding to human IM-9 lymphocytes, circulating erythrocytes and monocytes, isolated adipocytes, and placenta membranes. In contrast, this antibody subpopulation did not inhibit [125I]insulin binding to isolated rat adipocytes and hepatocytes, even at concentrations as high as 100 nM. These studies indicate that species-specific antibodies can occur spontaneously in patients with antiinsulin receptor antibodies.

Role of PC-1 in the etiology of insulin resistance.

Defects in insulin receptor tyrosine kinase activity have been demonstrated in tissues from insulin resistant subjects, but mutations in the insulin receptor gene are rare. Therefore, other molecules that are capable of modulating the insulin receptor most likely play a major role in insulin resistance. In cultured fibroblasts from an insulin resistant patient with Type 2 diabetes, we first identified membrane glycoprotein PC-1 as an inhibitor of the insulin receptor tyrosine kinase activity. PC-1 is overexpressed in fibroblasts from other insulin resistant subjects, both with and without Type 2 diabetes. PC-1 is a large class II exoprotein whose function is unknown. Studies in muscle and fat of insulin resistant subjects two primary tissues for insulin activation, reveal that elevated levels of PC-1 are inversely correlated with decreased insulin action both in vivo and in vitro. Transfection and expression of PC-1 in cultured cells demonstrate that overexpression of PC-1 produces impairments in insulin receptor tyrosine kinase activity, and the subsequent cellular responses to insulin. These studies indicate, therefore, that PC-1 is a major factor in the etiology of insulin resistance, and is a potential new therapeutic target for anti-diabetic therapy.

Evidence that insulin plus ATP may induce a conformational change in the beta subunit of the insulin receptor without inducing receptor autophosphorylation.

The effect of insulin and ATP on insulin receptor beta subunit conformation was studied in vitro with radioiodinated monoclonal antibodies directed at several regions of the receptor beta subunit. Insulin plus ATP inhibited their binding to the receptor. The greatest inhibitory effect of insulin and ATP was seen with antibody 17A3 which recognizes a domain of the beta subunit that is near the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163. ATP alone inhibited 17A3 binding with a one-half maximal ATP inhibitory concentration of 186 +/- 7 microM. Insulin at concentrations as low as 100 pM potentiated the effect of ATP; at 100 nM where insulin had its maximal effect, insulin lowered the one-half maximal inhibitory concentration of ATP to 16 +/- 6 microM. At 1 mM CTP, GTP, ITP, TTP, and AMP were without effect in either the presence or absence of insulin; in contrast, ADP was inhibitory in the presence of insulin. Of major interest was adenyl-5'-yl imidodiphosphate (AMP-PNP). This nonhydrolyzable analog of ATP inhibited 17A3 binding, and the effect of AMP-PNP (like ATP) was potentiated by insulin. Two insulin receptor beta subunit mutants then were studied. Mutant receptor F3, where the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163 were changed to phenylalanines, bound to 17A3; antibody binding was inhibited by insulin and ATP in a manner similar to normal receptors. In contrast, mutant receptor M1030, where the lysine in the ATP binding site at residue 1030 was changed to methionine, bound 17A3, but unlike either normal receptors or F3 receptors, the binding of 17A3 was not inhibited by insulin and ATP. Therefore, these studies raise the possibility that, in vivo, ATP binding in the presence of insulin may induce a conformational change in the insulin receptor beta subunit which in turn signals some of the biological effects of insulin.