A cascade of tyrosine autophosphorylation in the beta-subunit activates the phosphotransferase of the insulin receptor

We identified the major autophosphorylation sites in the insulin receptor and correlated their phosphorylation with the phosphotransferase activity of the receptor on synthetic peptides. The receptor, purified from Fao hepatoma cells on immobilized wheat germ agglutinin, undergoes autophosphorylation at several tyrosine residues in its beta-subunit; however, anti-phosphotyrosine antibody (alpha-PY) inhibited most of the phosphorylation by trapping the initial sites in an inactive complex. Exhaustive trypsin digestion of the inhibited beta-subunit yielded two peptides derived from the Tyr-1150 domain (Ullrich, A, Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) called pY4 and pY5. Both peptides contained 2 phosphotyrosyl residues (2Tyr(P], one corresponding to Tyr-1146 and the other to Tyr-1150 or Tyr-1151. In the absence of the alpha-PY additional sites were phosphorylated. The C-terminal domain of the beta-subunit contained phosphotyrosine at Tyr-1316 and Tyr-1322. Removal of the C-terminal domain by mild trypsinolysis did not affect the phosphotransferase activity of the beta-subunit suggesting that these sites did not play a regulatory role. Full activation of the insulin receptor during in vitro assay correlated with the appearance of two phosphopeptides in the tryptic digest of the beta-subunit, pY1 and pY1a, that were inhibited by the alpha-PY. Structural analysis suggested that pY1 and pY1a were derived from the Tyr-1150 domain and contained 3 phosphotyrosyl residues (3Tyr(P] corresponding to Tyr-1146, Tyr-1150, and Tyr-1151. The phosphotransferase of the receptor that was phosphorylated in the presence of alpha-PY at 2 tyrosyl residues in the Tyr-1150 domain was not fully activated during kinase assays carried out with saturating substrate concentrations which inhibited further autophosphorylation. During insulin stimulation of the intact cell, the 3Tyr(P) form of the Tyr-1150 domain was barely detected, whereas the 2Tyr(P) form predominated. We conclude that 1) autophosphorylation of the insulin receptor begins by phosphorylation of Tyr-1146 and either Tyr-1150 or Tyr-1151; 2) progression of the cascade to phosphorylation of the third tyrosyl residue fully activates the phosphotransferase during in vitro assay; 3) in vivo, the 2Tyr(P) form predominates, suggesting that progression of the autophosphorylation cascade to the 3Tyr(P) form is regulated during insulin stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of insulin receptor autophosphorylation in signal transduction.

We have examined the role of autophosphorylation in insulin signal transmission by oligonucleotide directed mutagenesis of seven potential tyrosine autophosphorylation sites in the human insulin receptor. Chinese hamster ovary cells transfected with these receptors were analyzed for insulin stimulated 2-deoxyglucose uptake, thymidine incorporation, endogenous substrate phosphorylation, and in vitro kinase activity. We found that phosphorylation on tyrosine residues 953, 1316, and 1322 were not necessary for receptor-mediated signal transduction. Mutation of tyrosine 960 reduced but did not abolish the signaling capabilities of the receptor. Finally, the simultaneous mutation of tyrosine residues 1146, 1150, and 1151 (the numbering system is that of Ullrich et al. (Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) resulted in a biologically inactive receptor, suggesting that the insulin receptor can be inactivated by removal of key autophosphorylation sites.     

Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.

In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.     

The insulin receptor activation process involves localized conformational changes.

The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.     

Mutation of the two carboxyl-terminal tyrosines results in an insulin receptor with normal metabolic signaling but enhanced mitogenic signaling properties

Our previous studies have shown that the deletion of the insulin receptor carboxyl terminus impairs metabolic, but augments mitogenic, signaling (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J. M. (1988) J. Biol. Chem. 263, 8904-8911; Thies, R.S., Ulrich, A., and McClain, D. A. (1989) J. Biol. Chem. 264, 12820-12825). To explore further the regulatory role of the insulin receptor carboxyl terminus, a mutant insulin receptor was constructed in which the two tyrosines (Y1316 and Y1322) on the carboxyl terminus were replaced with phenylalanines. Rat 1 fibroblasts expressing high levels of this mutant receptor (Y/F2 cells) exhibited normal insulin binding and normal insulin internalization. The absence of the two tyrosines in the carboxyl terminus did not affect the phosphotransferase activity of the beta-subunit and insulin-stimulated glucose transport. However, the Y/F2 cells showed markedly enhanced sensitivity for insulin-stimulated DNA synthesis. Dose-response curves for both insulin-stimulated thymidine uptake and 5-bromo-2-deoxyuridine incorporation in the Y/F2 cell lines were shifted to the left (4-10-fold) compared with those observed in the cells expressing similar numbers of wild type receptors. Thus, the two tyrosines of the insulin receptor carboxyl terminus do not modulate the kinase function of the insulin receptor, although they are autophosphorylated in native receptors. Moreover, these tyrosines are not necessary for stimulation of glucose transport. On the other hand, these results suggest that the two carboxyl-terminal tyrosine residues exert an inhibitory effect on mitogenic signaling in native insulin receptors.     

A defective intramolecular autoactivation cascade may cause the reduced kinase activity of the skeletal muscle insulin receptor from patients with non-insulin-dependent diabetes mellitus

The insulin receptor purified from skeletal muscle of patients with non-insulin-dependent diabetes mellitus (NIDDM) displayed a 25-55% reduction in insulin-stimulated autophosphorylation and tyrosyl-specific phosphotransferase activity relative to controls. This decrease was not explained by alterations of muscle fiber composition, insulin binding affinity or capacity, or the Km values for ATP; the lower kinase activity was entirely attributed to a decrease in the Vmax of the enzyme. Phosphorylation sites in the beta-subunit of the control and diabetic receptor were identified by tryptic digestion and reverse-phase high performance liquid chromatography. Autophosphorylation occurred primarily in two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C terminus containing Tyr-1316 and 1322. Autophosphorylation of the regulatory region at all three tyrosyl residues (tris-phosphorylation) appears to be necessary to activate the receptor kinase (White, M. F., Shoelson, S. E., Stepman, E. W., Keutmann, H. & Kahn, C. R. (1988) J. Biol. Chem. 263, 2969-2980). The receptor from NIDDM patients showed a decreased level of tris-phosphorylation of the regulatory region which was closely associated (r2 = 0.97) with the decreased kinase activity. In contrast, weak associations were found between kinase activity and the bis-phosphorylated forms of the regulatory region (r2 = 0.51) and the C terminus (r2 = 0.35). Therefore, the reduced formation of the tris-phosphorylated regulatory region in the diabetic receptors suggests that a defective autophosphorylation cascade leading to tris-phosphorylation of the regulatory region may cause, in part, the reduced insulin-stimulated kinase activity of the insulin receptor in muscle of NIDDM patients.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

HPC-1 is associated with synaptotagmin and omega-conotoxin receptor.

Monoclonal antibodies were produced that recognize a membrane protein of 35,000 Da (p35) expressed in brain and adrenal medulla. They immunoprecipitated 50% of omega-conotoxin (omega-CgTX) receptor, a putative N-type calcium channel, solubilized from rat brain. Anti-synaptotagmin (p65) antibodies also immunoprecipitate omega-CgTX receptor (Leveque, C., Hoshino, T., David, P., Shoji-Kasai, Y., Leys, K., Omori, A., Lang, B., El Far, O., Sato, K., Martin-Moutot, N., Newsom-Davis, J., Takahashi, M., and Seagar, M.J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3625-3629); however, immunoprecipitation by anti-p35 antibodies and anti-synaptotagmin antibodies was not additive. Furthermore, both p35 and synaptotagmin were recovered in the immunoprecipitates with anti-synaptotagmin and anti-p35 antibodies, respectively, indicating that a population of omega-CgTX receptor exists as a ternary complex with synaptotagmin and p35. A cDNA coding p35 was isolated from a rat brain cDNA library by immuno-screening, and the primary structure of the protein was revealed to be identical to that of HPC-1 (Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem. 267, 10613-10619). HPC-1 has a putative transmembrane segment at the C terminus and four heptad motifs, which may be involved in protein-protein interaction. These results suggest that HPC-1 may play a role in neurotransmitter release from nerve terminals by associating with omega-CgTX-sensitive N-type calcium channel and synaptotagmin.     

Identification of two pairs of spatially approximated residues within the carboxyl terminus of secretin and its receptor

The carboxyl-terminal domains of secretin family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within secretin and the prototypic secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant secretin receptors (V13M and V16M) and sequence analysis. We also developed a new probe incorporating a photolabile Bpa at position 26 of secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC(50) = 72 +/- 6 pm) and bound to the secretin receptor specifically and with high affinity (K(i) = 10.3 +/- 2.4 nm). It covalently labeled the secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36). These new insights, along with our recent report of contact between residue 6 within the amino-terminal half of secretin and this same amino-terminal region of this receptor (Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.     

Autophosphorylation of the insulin receptor in vitro. Designation of phosphorylation sites and correlation with receptor kinase activation

Chemical degradation and antipeptide antibodies were used to study alterations in the structure and function of the human placental insulin receptor following autophosphorylation in vitro. Antibodies elicited to residues 1143-1162 (P2) of the human insulin proreceptor immunoprecipitated the native, phosphorylated receptor but not the unphosphorylated receptor. Since this antibody recognizes both forms of the receptor on immunoblots, it was concluded that the accessibility of the P2 domain to the antibody is increased by in vitro autophosphorylation. Chemical cleavage at either tryptophan or methionine residues followed by immunoprecipitation with antipeptide antibodies was used to map the in vitro autophosphorylation sites of the beta subunit of the insulin receptor. Two phosphorylated fragments were resolved. One, recognized by antibody elicited to amino acid residues 1328-1343 (P5), is derived from the carboxyl terminus of the beta subunit and includes tyrosine 1316. The other, recognized by antibody to P2, is located in a domain that includes tyrosine 1150. The rate of phosphorylation of this latter site correlates with the rate of activation of the insulin receptor kinase during in vitro autophosphorylation. The results support the following conclusions: autophosphorylation alters the conformation of the beta subunit of the insulin receptor; autophosphorylation in vitro leads to phosphorylation of tyrosine residues near the carboxyl terminus of the protein and in the P2 domain that includes tyrosine 1150; activation of the insulin receptor kinase correlates with autophosphorylation of the domain containing tyrosine 1150.     

Control of insulin receptor autophosphorylation by polypeptide substrates: inhibition and stimulation by interaction with the catalytic subunit.

Autophosphorylation of purified insulin receptor, in the absence of insulin, was stimulated by selected polypeptide substrates. In the presence of 1 microM insulin these peptides inhibited autophosphorylation. Stimulation was observed with reduced [S-(carboxamidomethyl)cysteinyl]lysozyme (RCAM-lysozyme) and three peptides generated by CNBr cleavage, V8 proteinase digestion and/or chemical modification. We also generated two peptide substrates from RCAM-lysozyme which did not stimulate receptor autophosphorylation and were very weak inhibitors. As a control peptide, the simple substrate angiotensin inhibited receptor autophosphorylation in the absence or presence of insulin. However, stimulatory peptide, but not insulin, significantly shifted the concentration dependence for inhibition by angiotensin. The stimulatory peptides also increased autophosphorylation of the cloned cytoplasmic domain of the kinase [R-BIRK; Villalba, M., Wente, S. R., Russell, D. S., Ahn, J., Reichelderfer, C. F., & Rosen, O. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7848]. Therefore, stimulation occurs by interaction with the cytoplasmic process of the beta-subunit and not through interaction with the insulin binding alpha-subunit of the native receptor. Autophosphorylation was analyzed by mapping 32P-labeled tryptic phosphopeptides from the beta-subunit and from R-BIRK. Nearly identical phosphopeptide maps were found, comparing first, basal R-BIRK and basal native receptor, second, peptide- and insulin-stimulated native receptor, and third, peptide-stimulated R-BIRK and insulin-stimulated native receptor. Therefore, R-BIRK functions as a basal-state enzyme and can be stimulated in an insulin-like manner. On the basis of these observations, stimulation by insulin and by peptides yields similar functional results, but by apparently different mechanisms.     

Identification of insulin receptor tyrosine residues autophosphorylated in vitro

To identify the autophosphorylation sites on the human insulin receptor (IR), partially purified human IR was incubated in vitro in the presence of insulin and manganese [gamma-32P]ATP so as to achieve near-maximal activation of the histone 2b kinase activity. Approximately 70% of all beta subunit [32P]phosphotyrosine resides on two tryptic peptide segments identified by microsequencing as IR precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, R., Petruzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated peptide 5) and 1315-1329 (tyrosine at 1316, 1322, designated peptide 8), which were recovered in approximately equal amounts. Half of the remaining unidentified [32P]phosphotyrosine residues reside on another tryptic peptide of Mr 4000-5000. Assignment of [32P]phosphotyrosine to specific residues required subdigestion and Edman degradation of 32P peptides covalently coupled to solid supports. Peptide 5 was recovered in triple and double phosphorylated forms in a molar ratio of about 2:1. Tyr-1146 contained 32P in both forms of peptide 5; in the double phosphorylated form, phenylthiohydantoin-[32P]phosphotyrosine was recovered at both Tyr-1150 and Tyr-1151, in a ratio of about 1:2. Thus, the double phosphorylated peptide 5 is presumably a mixture of Tyr-P-1146/1150 and Tyr-P-1146/1151, predominantly the latter. Peptide 8 was recovered only as the double phosphorylated form. We conclude that autophosphorylation of human IR in vitro leads to the phosphorylation of at least 6 of the 13 tyrosine residues on the beta subunit intracellular extension. Five of these tyrosines are clustered in two domains; one domain is in the structurally unique C-terminal tail and contains Tyr-1316 and -1322 which are both phosphorylated. The second domain is located in the segment of the tyrosine kinase region homologous to the major in vitro autophosphorylation site of pp60 v-src and contains Tyr-1146, which is fully phosphorylated, and Tyr-1150 and -1151; although the majority of IR beta subunits exhibit phosphorylation of both tyrosine 1150 and 1151, up to 20-25% of Tyr-1150 remains unphosphorylated at complete kinase activation.     

Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor.

The vitronectin receptor mediates cell adhesion to the extracellular matrix proteins vitronectin, fibrinogen, von Willebrand factor, and thrombospondin in an RGD-dependent manner. We previously demonstrated the direct interaction between the vitronectin receptor and an RGD-containing peptide by photoaffinity labeling the receptor with 125I-sulfosuccinimidyl-2-(p-azido-salicylamido)-1,3'-dithioprop ion ate (SASD)-GRGDSPK (Smith, J. W., and Cheresh, D. A. (1988) J. Biol. Chem. 263, 18726-18731). In that report, we identified amino acid residues 61-203 of the beta-subunit as proximal to the ligand binding site. Here we demonstrate that 125I-SASD-GRGDSPK affinity labels the alpha-subunit of the receptor at least two distinct sites within the region encompassing residues 139-349. Both of these regions are within the putative divalent cation binding region of the alpha-subunit. Collectively, our results suggest that discrete amino-terminal domains of both subunits of the receptor contribute to the structure of the ligand binding domain and furthermore that the ligand and divalent cation binding domains are spatially and functionally linked.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Wheat-germ agglutinin mimics metabolic effects of insulin without increasing receptor autophosphorylation

Expression of insulin metabolic effects can be obtained by anti-receptor antibodies without activation of the tyrosine kinase activity [O'Brien R. M., Soos M. A. and Siddle K. (1987) EMBO J. 6, 4003-4010; Forsayeth J. R., Caro J. F., Sinha M. K., Maddux B. A. and Goldfine I. D. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 34,448-34,514; Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J. and Rossi B. (1988) EMBO J. 7, 4111-4117; Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E. and Goldfine I. D. (1989) J. biol. Chem. 264, 2438-2444; Soos M. A., O'Brien R. M., Brindle N. P. J., Stigter J. M., Okamoto A. K., Whittaker J. and Siddle K. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5217-5221.]. Recently, we have proposed that receptor cross-linking is sufficient in itself to stimulate glycogen synthesis, even if aggregation was performed on receptors mutated on Tyr 1162 and Tyr 1163 and thus devoid of tyrosine kinase activity [Debant A., Ponzio G., Clauser E., Contreres J. O. and Rossi B. (1989) Biochemistry 28, 14-17]. The aim of this study was to gain information on the involvement of receptor clustering in the expression of the different insulin biological effects. To this end, we studied the mimetic effects of wheat-germ agglutinin, which is likely to induce receptor aggregation without interacting with the receptor protein moiety. Wheat-germ agglutinin failed to promote DNA synthesis, whereas the lectin behaved as a potent mimicker of insulin on tyrosine aminotransferase activity and amino-acid transport. However, this stimulatory effect did not parallel the activation of receptor autophosphorylation. Our data reinforce the idea that the expression of the metabolic effects of insulin are not strictly dependent on a general tyrosine kinase activation.     

Identification of functional insulin receptors on membranes from an insulin-producing cell line (RINm5F).

Insulin receptors on RINm5F cell membranes (an insulin-producing rat pancreatic cell line) were studied. To study the insulin receptor alpha-subunit, 125I-labelled photoreactive insulin was covalently bound to the membranes in the absence or presence of unlabelled insulin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions showed specific labelling of an Mr 130 000 protein. The receptor beta-subunit was studied by using a cell-free phosphorylation assay. Analysis under reducing conditions showed a phosphoprotein of Mr 95 000 whose level of phosphorylation was selectively increased by insulin, and which was specifically immunoprecipitated by antibodies to the insulin receptor. Further, covalent hormone-receptor complexes purified with anti-insulin antibodies were able to undergo autophosphorylation, indicating the existence of operational receptor subunit arrangements. RINm5F cell insulin receptors (and, by analogy, possibly those of native B-cells) thus display structural and functional integrity comparable with those of conventional insulin target cells.     

The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit.

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.