Insulin Receptor Autophosphorylation Research Articles

Deletion of C-terminal 113 amino acids impairs processing and internalization of human insulin receptor: Comparison of receptors expressed in CHO and NIH-3T3 cells

We have studied the structure and the function of a truncated human insulin receptor in which 113 amino acids (aa 1231–1343) at the C-terminus of the β-subunit were deleted. In this study, wild-type and truncated insulin receptors were expressed by stable transfection in NIH-3T3 cells and CHO cells. The mutation impairs post-translational processing of the insulin receptor; proteolytic cleavage is retarded, and degradation of the truncated receptor is accelerated. Furthermore, insulin-stimulated autophosphorylation of the mutant insulin receptor is impaired. This is associated with a defect in insulin-stimulated endocytosis. Finally, in NIH-3T3 cells, the mutant insulin receptor failed to mediate the mitogenic effects of insulin. In CHO cells, transfection of insulin receptor cDNA (either wild-type or mutant) did not alter mitogenic response to insulin. It has previously been shown that deletion of 43 amino acids at the C-terminus of the β-subunit did not affect insulin receptor tyrosine kinase activity. Our data suggest that the structural domain located 43–113 amino acids from the C-terminus appears to have several functional roles. First, the domain appears to promote folding of receptor into the optimal conformation for post-translational processing. Second, the presence of this domain appears to promote the stability of the receptor β-subunit in intact cells. Finally, perhaps as a consequence of the effects upon the stability of the receptor, this domain is required in intact cells for insulin-stimulated autophosphorylation and signal transmission.

Inhibition of the activity of protein tyrosine phosphate 1C by its SH2 domains.

Full-length protein tyrosine phosphatase 1C (PTP1C), the catalytic domain of PTP1C (delta PTP1C), and the N-terminal SH2 domain truncated PTP1C (delta NPTP1C) were overexpressed in Escherichia coli and purified to near homogeneity. Various phosphorylated states of the synthetic phosphotyrosyl peptide TRDIYETDYYRK (IRP), corresponding to the major insulin receptor autophosphorylation sites, were used as substrates for the PTPs. There was no indication for selective dephosphorylation of any of the three phosphotyrosyl residues from the triphosphotyrosyl IRP. Kinetic studies were carried out using all seven different phosphotyrosyl IRPs. Saturation kinetics were observed for PTP1C using the triphosphotyrosyl IRP only. In contrast, for delta PTP1C, saturation was achieved for all seven phosphotyrosyl IRPs. The best substrate for delta PTP1C was the triphosphotyrosyl IRP possessing a Km of approximately 1.6 microM, about 3-4-fold lower than either the mono- or diphosphotyrosyl IRPs. However, in contrast to delta PTP1C, PTP1C had a 22-fold lower affinity for triphosphotyrosyl IRP. Furthermore, deletion of a single N-terminal SH2 domain increased the affinity of the enzyme for the triphosphotyrosyl IRP to a value similar to that obtained with delta PTP1C. The pH optima for all three enzyme constructs were very similar and could not account for the observed change in substrate affinity between the three enzymes. These results suggest that the SH2 domain of PTP1C exerts an inhibitory effect on its PTP activity.

An in-frame insertion in exon 3 and a nonsense mutation in exon 2 of the insulin receptor gene associated with severe insulin resistance in a patient with Rabson-Mendenhall syndrome

We have studied the structure and function of the insulin receptor in a patient (PK) with severe insulin resistance and Rabson-Mendenhall syndrome. Insulin binding to cultured fibroblasts from PK was almost not detectable and insulin-induced insulin receptor autophosphorylation and glucose uptake was abolished. The structure of the receptor gene was analysed by sequencing amplified products of the 22 exons with the flanking intron regions directly as well as after subcloning in pUCBM20 plasmids. Two mutant alleles of the insulin receptor gene were detected. One allele contains in-frame 12 additional base pairs in exon 3 coding for the amino acids Leu-His-Leu-Val located between Asp-261 and Leu-262 in the receptor's extracellular domain, being the first report of an insertion mutation of the insulin receptor gene. In the other allele Arg-86 in exon 2 is changed into a stop codon. Therefore, PK is compound heterozygous at the insulin receptor locus. Direct cDNA sequencing indicates that both mutant alleles are expressed in the patient's fibroblasts. Studies of the parents' fibroblasts revealed that PK inherited the insertion mutation from the father and the nonsense mutation from the mother. Insulin binding to fibroblasts of the mother was reduced (63% of control cells) and hormone binding to the father's cells shows a larger reduction (37% of control cells), but less severe than the patient's cells (11% of control). This investigation provides further evidence that the Rabson-Mendenhall syndrome is causally related to mutations in the insulin receptor gene.

Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level.

The insulin-dependent tyrosine kinase activity (TKA) of the insulin receptor (IR) plays an essential role in insulin signaling. Thus, dysregulation of IR-TKA might be an important element in the states of insulin resistance. A phosphorylated rat hepatic glycoprotein (pp63) acting as an inhibitor of IR-TK has been described. In search of the human homolog of pp63, we isolated a cDNA clone from a human liver lambda gt11 cDNA library. DNA sequence analysis reveals identity with the mRNA product of a human gene AHSG encoding a serum protein, alpha 2-Heremans Scmid-glycoprotein (alpha 2HSG), with heretofore unknown physiological function. Northern blot analysis demonstrates a 1.8-kilobase mRNA in human liver and HepG2 hepatoma cells. alpha 2HSG, purified from human serum, specifically inhibits insulin-stimulated IR autophosphorylation in vitro and in vivo as well as exogenous substrate tyrosine phosphorylation. alpha 2HSG also inhibits both insulin-induced tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 subunit of phosphatidylinositol-3 kinase in H-35 hepatoma cells. alpha 2HSG inhibits insulin-dependent mitogenesis, but does not affect insulin-stimulated induction of the metabolic enzyme tyrosine aminotransferase. alpha 2HSG does not compete with insulin for binding to IR. Finally, the action of alpha 2HSG is specific toward the IR-TK; its effect does not extend to insulin-like growth factor-I-stimulated TKA. Our results allow us to assign a biochemical function for human alpha 2HSG, namely regulation of insulin action at the IR-TK level.

Specific phosphorylation of membrane proteins of Mr 44,000 and Mr 32,000 by the autophosphorylated insulin receptor from the hepatopancreas of the shrimp Penaeus monodon (Crustacea: Decapoda).

The insulin receptor, purified from the hepatopancreas of the shrimp Penaeus monodon, is a hydrophobic heterodimer of subunits with relative masses (Mr) of 70,000 and 58,000, as estimated by FPLC on Superose 12 and SDS-PAGE. Only the subunit of Mr 70,000 was autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the hepatopancreas of the shrimp Penaeus monodon. Proteins of Mr 44,000 and Mr 32,000 on the plasma membrane from the hepatopancreas of the shrimp Panaeus monodon were phosphorylated by the autophosphorylated insulin receptor from the shrimp hepatopancreas, but not by that from the human placenta. The detergent, Triton X-100, caused noticeable enhancement of the autophosphorylation of both shrimp and human insulin receptors.

Anti-phosphoserine and Anti-phosphothreonine Antibodies Modulate Autophosphorylation of the Insulin Receptor but Not EGF Receptor

We examined the effect of anti-phosphothreonine and anti-phosphoserine antibodies on insulin receptor autophosphorylation. These antibodies did not affect insulin binding activity of the receptor. These antibodies, however, inhibited insulin-stimulated autophosphorylation of insulin receptor, while did not affect EGF-stimulated autophosphorylation of EGF receptor. The inhibition was reversed by adding large amounts of phosphoserine or phosphothreonine. These data suggest that phosphoserine and phosphothreonine on insulin receptor play an important role in insulin-induced conformational change of the receptor.

Streptavidin blotting: a sensitive technique to study cell surface proteins; application to investigate autophosphorylation and endocytosis of biotin-labeled insulin receptors.

Covalent attachment of biotin provides a useful method to label cell surface proteins. Subsequent to biotinylation, the protein can be purified by immunoprecipitation with a specific antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer to a membrane by electroblotting, the biotinylated protein can be detected by probing with labeled streptavidin. This technique has been used to investigate recombinant human insulin receptors expressed on the surface of murine NIH-3T3 cells. Biotinylation of the extracellular domain with an impermeant reagent did not impair the ability of an antibody directed against an epitope in the intracellular domain to immunoprecipitate insulin receptors. In contrast, biotinylation reduced the avidity of a polyclonal antibody directed against the extracellular domain of the receptor. Nevertheless, by increasing the concentration of the antireceptor antibody, it was possible to successfully immunoprecipitate the biotinylated receptor. Furthermore, biotinylated receptors retained the ability to bind insulin and undergo insulin-stimulated autophosphorylation and internalization. The use of enzyme-labeled streptavidin enables the use of chemiluminescence techniques to detect the receptors, thus obviating the need to employ radioactivity. Just as the technique is useful to study cell surface insulin receptors, it can be adapted to investigate other cell surface receptors and proteins.

Streptavidin blotting: a sensitive technique to study cell surface proteins; application to investigate autophosphorylation and endocytosis of biotin-labeled insulin receptors

Streptavidin blotting: a sensitive technique to study cell surface proteins; application to investigate autophosphorylation and endocytosis of biotin-labeled insulin receptors

Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect.

Previous studies have shown that the combination of vanadate and H2O2 generates peroxide(s) of vanadate (pervanadate) that is able to mimic insulin in stimulating lipogenesis or protein synthesis and inhibiting lipolysis in rat adipocytes. Here we report that pervanadate is a potent trigger of 3-O-methylglucose transport in rat adipocytes, with an effective concentration of 5 microM and a maximum at 20 microM. Moreover, pervanadate produced an additional activation of approximately 60% on glucose influx in cells treated with maximally activating concentrations of insulin. Vanadate was ineffective in potentiating insulin-stimulated glucose uptake. Quercetin, a bioflavonoid that inhibits insulin receptor tyrosine kinase, blunted this effect of pervanadate. Treatment of adipocytes with pervanadate inhibited protein phosphotyrosyl phosphatase activity of cell extracts in a dose-dependent manner, with an ID50 of 5 microM and complete inhibition at 80 microM. In contrast, vanadate (1-800 microM) did not appreciably inhibit cell phosphotyrosyl phosphatases. The inhibitory effect of pervanadate correlated with the increase in protein phosphotyrosine accumulation, as determined by Western blotting with antiphosphotyrosine antibodies. The most prominent phosphotyrosine-containing band detected in pervanadate-treated adipocytes was that of autophosphorylated insulin receptor, identified by immunoblotting or immunoprecipitation with antiinsulin receptor antibodies. The addition of insulin to pervanadate-treated adipocytes (20 microM) caused a further increase (approximately 70%) in receptor autophosphorylation. In a cell-free system using partially purified insulin receptor devoid of tyrosine phosphatase activity, pervanadate did not stimulate the receptor autophosphorylation or interfere with the stimulating effect of insulin. These results suggest that 1) pervanadate triggers glucose uptake by increasing autophosphorylation of insulin receptor, preventing its dephosphorylation; 2) under physiological conditions, cellular protein phosphotyrosyl phosphatase activity is high, thereby significantly opposing insulin-mediated hexose transport; and 3) pervanadate has the unique ability to markedly increase maximal cell responsiveness in stimulating glucose transport achieved at a saturating insulin concentration. These findings suggest a possible clinical application in the management of glucose uptake in pathological conditions of insulin resistance and hyperinsulinemia.

The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity.

Glucose transporter recycling in rat adipose cells. Effects of potassium depletion.

Depletion of intracellular potassium (K+) induced a 4-fold increase in basal and 1 microM phorbol-12-myristate-13-acetate (PMA)-stimulated 3-O-methylglucose transport in rat adipose cells. K+ depletion had no effect on the maximum insulin (0.7 microM)-stimulated transport rate but enhanced the sensitivity to insulin 3-fold (EC50 = 0.05 versus 0.15 nM) by a mechanism that did not result from changes in the insulin receptor binding, autophosphorylation, or tyrosine kinase activity. Western blotting analysis revealed that K+ depletion induced a 2.2-fold increase in GLUT4 in plasma membranes from basal cells, enhanced the PMA-stimulated GLUT4 translocation by 4-fold, and increased the 5-fold insulin-stimulated GLUT4 translocation by 15%, indicating the presence of an inactive GLUT4 intermediate. The time course for insulin's stimulation of transport activity was accelerated by K+ depletion (t1/2 = 3 versus 1.5 min). Conversely, the reversal of transport activity, on removal of insulin, was delayed (t1/2 = 11 versus 22 min). The corresponding t1/2 values for the loss of GLUT4 were 22 min in control cells and 40 min in K(+)-depleted cells, again indicating the existence of an inactive intermediate. Photolabeling intact cells with the impermeant, exofacial photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 - yloxy)-2-propylamine in the continuous presence of insulin revealed that K+ depletion had no effect on the GLUT4 externalization rate but halved the rate of internalization. K+ depletion elicited entirely analogous effects on the recycling of insulin-like growth factor II/mannose 6-phosphate receptor, strongly supporting the involvement of a coated pit mechanism in the recycling of GLUT4 transporters. An inactive conformation of GLUT4 has been detected in plasma membranes from insulin-stimulated cells, which is enhanced by K+ depletion, suggesting a limitation in the adipose cells' capacity to express active GLUT4 transporters.

Insulin and IGF-1 increase mitogenesis and glucose metabolism in the multiple myeloma cell line, RPMI 8226.

Insulin receptors and insulin-like growth factor-1 (IGF-1) receptors are present in circulating human B lymphocytes (B cells) and certain B cell malignancies, but no function has been attributed to either receptor. We report a human myeloma cell line, RPMI 8226, that exhibits insulin and IGF-1-dependent receptor and substrate tyrosine phosphorylation as well as hormone-responsive cellular metabolism. Competitive hormone-binding analysis revealed that the cell line expressed approximately 4 x 10(3) high affinity insulin binding sites and 1.1 x 10(4) high affinity IGF-1 binding sites per cell. The Kd of the insulin-binding sites for insulin was 0.32 nM. The Kd of high affinity IGF-1 binding sites for IGF-1 was 0.89 nM. Insulin receptor autophosphorylation was maximum at 200 nM as was tyrosine phosphorylation of the 180-kDa cytosolic receptor substrate. Insulin-dependent activation of phosphatidylinositol 3-kinase paralleled receptor phosphorylation. In contrast, IGF-1 produced its maximum effects at 200 nM for receptor phosphorylation and 20 nM for substrate phosphorylation and PI 3-kinase activation. In growth synchronized cells, IGF-1 and insulin at 200 nM increased DNA synthesis by 122 +/- 18% and 101 +/- 27%, respectively. IGF-1 increased DNA synthesis 88 +/- 21% at 2 nM and the effect of insulin at 2 nM was 34 +/- 12%. Flux through the glycolytic pathway was also increased by insulin and IGF-1. At 200 and 2 nM, insulin increased production of lactate by 33 +/- 9% and 19 +/- 11%, respectively. IGF-1 increased lactate production 47 +/- 3% and 23 +/- 3% at identical hormone concentrations. Finally, in two additional myeloma cell lines, U266 (human) and Ag8.653 (mouse), insulin and IGF-1 increased tyrosine phosphorylation of receptor beta-subunit (95 kDa), the prominent 180-kDa substrate (pp185), and several other substrates. Thus, functional insulin and IGF-1 receptors are present in myeloma cell lines. Through these receptors, insulin and IGF-1 regulate mitogenesis and glucose metabolism, and may be important in potentiating plasma cell malignancy.

Insulin and IGF-I receptor signaling in cultured neurons.

The data presented in this chapter are summarized in the schematic shown in Figure 9. Insulin binds to and stimulates autophosphorylation of neuronal insulin receptors, whereas, IGF-I and IGF-II binds to and stimulate autophosphorylation of neuronal IGF-I receptors. IGF-II is also capable of binding to the insulin receptor. Whether or not it activates the insulin receptor kinase remains to be clarified. Activated insulin and IGF-I receptor kinases phosphorylate a 70-kDa protein at early times in culture. This protein may mediate some actions of insulin, but we speculate that there are other intermediary proteins involved in the transduction pathway resulting in the activation of S6 kinase and PKC epsilon. The stimulation of S6 kinase by insulin and IGF-I may be associated with the translational activation of protein synthesis by these peptides. The stimulation of PKC epsilon appears to be a necessary step in the transcriptional regulation of the c-fos gene by insulin and IGF-I. The regulation of neuronal protein synthesis at a translational step and the initiation of transcriptional programs regulated by AP-1 represent two mechanisms by which insulin and IGFs alter neuronal growth and differentiation.

Effect of pioglitazone on insulin receptors of skeletal muscles from high-fat-fed rats

A new oral agent, 5-[4-[2-(5-ethyl-12-pyridyl)ethoxy]-benzyl]-2,4-thiazolidinedione, or pioglitazone, has been developed for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). We examined its effectiveness in high-fat-fed rats resistant to insulin. Administration of the agent (10 mg · kg −1 · d −1) for 2 weeks resulted in decreases in hyperlipidemia and hyperinsulinemia, indicating that insulin sensitivity had increased in vivo in high-fat-fed rats. To clarify the mechanism of the drug, we examined insulin binding and kinase activity of insulin receptors from muscles of both untreated and treated high-fat-fed rats. Pioglitazone treatment did not change insulin binding in high-fat-fed rats, but increased insulin-stimulated autophosphorylation of insulin receptors to the level of control animals. Kinase activity toward an exogenous substrate, poly Glu 4-Tyr 1, in pioglitazone-treated high-fat-fed rats was also increased to the level of control animals. These results suggest that pioglitazone increases insulin sensitivity by activating tyrosine kinase activity of receptors in high-fat-fed rats, and this drug appears to be a useful one with a new mode of action for the treatment of NIDDM with insulin resistance.

Why Does Insulin Resistance Develop During Maturation?

We compared skeletal muscle glucose uptake between young and mature rats. Hindlimb perfusions at insulin concentrations of 0, 100, 250, or 10,000 microU/mL were performed on male Sprague-Dawley rats at 5 weeks or 4 months of age. Basal glucose uptake, and glucose uptake at all insulin concentrations were significantly lower in the 4-month-old mature rats (p < .05). This difference was most pronounced at maximally stimulating insulin concentrations. Skeletal muscle insulin receptor binding, autophosphorylation, and tyrosine kinase activity did not differ between young and mature rats. Surprisingly, GLUT-4 glucose transporter content was significantly higher in several muscles of the mature rats (p < .05). Therefore, the decline in insulin-stimulated glucose uptake in hindlimbs of mature rats cannot be explained by decreased activity of these steps in the glucose transport system.

Insulin receptor autophosphorylation. II. Determination of autophosphorylation sites by chemical sequence analysis and identification of the juxtamembrane sites.

Autophosphorylation sites of the human insulin receptor were identified by microsequence analysis of 19 discrete tryptic [32P]phosphopeptides, purified from the autophosphorylated cytoplasmic kinase domain (CKD). Seventeen phosphopeptides were generated by cleavage at Arg and/or Lys, but two phosphopeptides were generated reproducibly by anomalous cleavages. Two new sites were identified in the juxtamembrane region of the intact insulin receptor beta-subunit (the amino terminus of the CKD), including phosphotyrosines 965 and 972. Three sites in the central region, including phosphotyrosines 1158, 1162, and 1163, were identified from six phosphopeptides; tyrosine at 1158 was the least phosphorylated. Monophosphopeptides contained phosphotyrosine at either residue 1158 or 1163, but not at 1162. Bisphosphorylation included phosphotyrosines only at 1162 and 1163. The two autophosphorylation sites near the carboxy terminus were found in seven phosphopeptides, including phosphotyrosines at 1328 and 1334. When mapped by reverse-phase high-performance liquid chromatography, these phosphopeptides eluted in the order central domain, first; carboxy-terminal region, second; and juxtamembrane (amino-terminal) domain, last.

Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain

Kinetic analysis of autophosphorylation was done using a non-Michaelis-Menten kinetic model. This model describes autophosphorylation in terms of a fast reaction phase, a slow reaction phase, and a partition function for the two phases. Kinetic parameters determined by this new approach show that insulin stimulates autophosphorylation by promoting (1) a 10-fold increase in the rate constant for the fast phase of the reaction and (2) a 2-fold increase in the partition function favoring the fast phase. Insulin did not significantly affect the binding constant for ATP in this fast phase. Kinetic parameters obtained for the cytoplasmic kinase domain were similar to those obtained for the native insulin receptor in the absence of insulin. The insulin receptor has three subdomains encompassing its seven autophosphorylation sites. The juxtamembrane sites react primarily in the slow kinetic phase, favored by the absence of stimulation and low ATP concentrations. The carboxy-terminal and central autophosphorylation subdomains react primarily in the fast kinetic phase, favored by raising the ATP concentration and/or the presence of insulin. These observations demonstrate that: (1) both ATP and insulin regulate reaction in each autophosphorylation subdomain, (2) insulin stimulation occurs predominantly in the central and carboxy-terminal regions, and (3) autophosphorylation observed with the cytoplasmic kinase domain was similar to native insulin receptor in the absence of insulin. These findings are consistent with conclusions based on the kinetic analysis of autophosphorylation.

Okadaic acid stimulates IGF-II receptor translocation and inhibits insulin action in adipocytes.

Okadaic acid, an inhibitor of protein phosphatases 2A and 1, stimulates glucose transport in muscle and fat cells, suggesting that serine/threonine phosphorylation steps are involved in the translocation of glucose transporters. Here we have investigated whether such phosphorylation events could also participate in another membrane-associated insulin-stimulated process: insulin-like growth factor II (IGF-II) receptor translocation in adipocytes. Maximally effective concentrations of insulin and okadaic acid stimulated deoxyglucose uptake by 5.5- and 2.5-fold, respectively, whereas IGF-II binding was increased 3.5-fold and 1.5-fold. Subcellular fractionation indicated that the okadaic acid-induced stimulation of IGF-II binding resulted from an increase in the number of IGF-II receptors in the plasma membrane with a concomitant disappearance from the low-density microsomal fraction. These changes occurred in parallel to those observed for the glucose transporter GLUT-4. Both insulin-stimulated glucose transport and IGF-II binding were prevented when cells were pretreated with okadaic acid. To understand the mechanism of this inhibitory effect, insulin receptor autophosphorylation and the tyrosine phosphorylation of endogenous proteins were studied. Insulin induced the tyrosine phosphorylation of its receptor beta-subunit and of proteins at 120 and 185 kDa, whereas okadaic acid alone had no effect. When okadaic acid and insulin were added together, the beta-subunit autophosphorylation was similar to that observed with insulin alone, but the tyrosine phosphorylation of substrates was prevented. Taken together, our data suggest that, in adipocytes, serine/threonine phosphorylation events mimicked by okadaic acid are required for the translocation of IGF-II receptors and glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells

1. 1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4°C) or for 30 sec at 37°C causes activation of the insulin receptor subsequently isolated from the cells. 2. 2. Receptor activation was assessed by increased phosphotyrosine content of the β-subunit of the receptor, and increased autophosphorylation using [ 32P]-ATP. 3. 3. Treatment of the cells for 15 min at 37°C however completely abolished insulin binding and all insulin receptor kinase activity. 4. 4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.

Inhibitory effect of fluoride on insulin receptor autophosphorylation and tyrosine kinase activity

Fluoride is a nucleophilic reagent which has been reported to inhibit a variety of different enzymes such as esterases, asymmetrical hydrolases and phosphatases. In this report, we demonstrate that fluoride inhibits tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle and human placenta. Fluoride inhibited in a similar dose-dependent manner both beta-subunit autophosphorylation and tyrosine kinase activity for exogenous substrates. This inhibitory effect of fluoride was not due to the formation of complexes with aluminum and took place in the absence of modifications of insulin-binding properties of the insulin receptor. Fluoride did not complete with the binding site for ATP or Mn2+. Fluoride also inhibited the autophosphorylation and tyrosine kinase activity of receptors for insulin-like growth factor I from human placenta. Addition of fluoride to the pre-phosphorylated insulin receptor produced a slow (time range of minutes) inhibition of receptor kinase activity. Furthermore, fluoride inhibited tyrosine kinase activity in the absence of changes in the phosphorylation of prephosphorylated insulin receptors, and the sensitivity to fluoride was similar to the sensitivity of the unphosphorylated insulin receptor. The effect of fluoride-on tyrosine kinase activity was markedly decreased when insulin receptors were preincubated with the copolymer of glutamate/tyrosine. Prior exposure of receptors to free tyrosine or phosphotyrosine also prevented the inhibitory effect of fluoride. However, the protective effect of tyrosine or phosphotyrosine was maximal at low concentrations, suggesting the interaction of these compounds with the receptor itself rather than with fluoride. These data suggest: (i) that fluoride interacts directly and slowly with the insulin receptor, which causes inhibition of its phosphotransferase activity; (ii) that the binding site of fluoride is not structurally modified by receptor phosphorylation; and (iii) based on the fact that fluoride inhibits phosphotransferase activity in the absence of alterations in the binding of ATP, Mn2+ or insulin, we speculate that fluoride binding might affect the transfer of phosphate from ATP to the tyrosine residues of the beta-subunit of the insulin receptor and to the tyrosine residues of exogenous substrates.