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.     

Proteolytic activity in the insulin receptor

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.     

Regulation of biological functions by an insulin receptor monoclonal antibody in insulin receptor beta-subunit mutants.

We investigated the effects of MA-5, a human-specific monoclonal antibody to the insulin receptor alpha-subunit, on transmembrane signaling in cell lines transfected with and expressing both normal human insulin receptors and receptors mutated in their beta-subunit tyrosine kinase domains. In cell lines expressing normal human insulin receptors, MA-5 stimulated three biological functions: aminoisobutyric acid (AIB) uptake, thymidine incorporation, and S6 kinase activation. Under conditions where these biological functions were stimulated, there was no detectable stimulation of receptor tyrosine kinase. We then combined the use of this monoclonal antibody with cells expressing insulin receptors with mutations in the beta-subunit tyrosine kinase domain; two of ATP binding site mutants V1008 (Gly----Val) and M1030 (Lys----Met) and one triple-tyrosine autophosphorylation site mutant F3 (Tyr----Phe at 1158, 1162, and 1163). In cells expressing V1008 receptors, none of the three biological functions of insulin was stimulated. In cells expressing M1030 receptors, AIB uptake was stimulated to a small, but significant, extent whereas the other two functions were not. In cells expressing F3 receptors, AIB uptake and S6 kinase activation, but not thymidine incorporation, were fully stimulated. The data suggest, therefore, that (1) activation of insulin receptor tyrosine kinase may not be a prerequisite for signaling of all the actions of insulin and (2) there may be multiple signal transduction pathways to account for the biological actions of insulin.     

Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least 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 Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

Antipeptide antibodies toward the extracellular domain of insulin receptor beta-subunit

In order to investigate structure and function of beta-subunit extracellular portion, four polyclonal antibodies (AP1, AP2, AP3 and AP4) toward peptides comprised in this region were generated. None of them recognizes native human and rat insulin receptor both in vitro and in whole cells. Two antibodies, AP1 and AP2, immunoprecipitate isolated (DTT-reduced) human beta-subunits and bind to human IM-9 cell after alpha-subunit tryptic cleavage. Only AP1 recognizes rat beta-subunit both in vitro and in trypsin treated rat FAD cells. These findings suggest that: (i) the extracellular portion of the insulin receptor beta-subunit is partially covered by the alpha-subunit in human and rat native insulin receptors; (ii) human and rat beta-subunit extracellular domains are different, at least in the amino acid sequence corresponding to residues 785-796 of the human insulin receptor.     

Nucleotide-dependent binding of the GTPase domain of the signal recognition particle receptor beta-subunit to the alpha-subunit

The signal recognition particle (SRP) receptor (SR) is a heterodimer of two polypeptides (SRalpha and SRbeta) that each contain a GTP-binding domain. The GTP-binding domain in the peripheral membrane SRalpha subunit has a well defined role in regulating targeting of SRP-ribosome-nascent chain complexes to the translocon. The only well established function for the transmembrane SRbeta subunit is anchoring SRalpha on the endoplasmic reticulum membrane. Deletion of the amino-terminal transmembrane domain of SRbeta did not affect receptor dimerization, but revealed a cryptic translocation signal that overlaps the GTPase domain. We demonstrate that the domain of SRalpha that binds SRbeta does so by binding directly to the nucleotide-bound form of the GTPase domain of SRbeta. An SRbeta mutant containing an amino acid substitution that allows the GTPase domain to bind XTP dimerized with SRalpha most efficiently in the presence of XTP or XDP, but not ATP. Our results suggest an additional level of regulation of SRP receptor function based on regulated dissociation of the receptor subunits.     

Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity

The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the alpha-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Sch?ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). To investigate the insulin binding properties of dimerized alpha-subunits, we have reintroduced the domains containing alpha-alpha disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4-8 pm) similar to what was obtained for the solubilized holoreceptor (14-24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the alpha-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.     

Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.

We measured rates of protein synthesis in vivo in subcellular fractions (soluble, myofibrillar and stromal fractions) of the heart and the gastrocnemius from rats after fasting or under hypoxic conditions (i.e. atmospheres containing 5% or 10% O2). Such interventions are known to inhibit protein synthesis under some circumstances. The recovery of tissue protein after fractionation was 80-100%. The proportions of protein present in the soluble and stromal fractions were different in the two muscles. The rates of protein synthesis in the myofibrillar and stromal fractions were less than those for total mixed tissue protein, whereas the rate for soluble protein was greater. Both fasting and moderate hypoxia (10% O2 for 24 h) inhibited protein synthesis in the gastrocnemius. In this tissue, the synthesis of the myofibrillar fraction was apparently the most sensitive to inhibition, and this resulted in some significant increases in the soluble-fraction/myofibrillar-fraction protein-synthesis rate ratios. In the heart, fasting inhibited protein synthesis, but moderate hypoxia (10% O2 for 24 h) did not. The rate of protein synthesis in the cardiac myofibrillar fraction was again more sensitive to fasting than were the rates in the other fractions, but it was not as sensitive as that in the gastrocnemius. Under severely hypoxic conditions (5% O2 for 1 or 2 h), protein synthesis was decreased in all fractions in both tissues. These results suggest that the rates of protein synthesis in these relatively crude subcellular fractions vary.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

The inhibitory effect of 2-deoxyglucose on insulin receptor autophosphorylation does not depend on known serine phosphorylation sites or other conserved serine residues of the receptor beta-subunit.

Hyperglycemia induces insulin resistance in diabetic patients. It is known that supraphysiological levels of D-glucose or 2-deoxyglucose inhibit the insulin receptor and it is speculated that this effect is mediated by serine phosphorylation of the insulin receptor beta-subunit and other proteins of the insulin signaling chain. To test this hypothesis we prepared point mutations of the human insulin receptor where serine was exchanged to alanine at 16 different positions, either at known phosphorylation sites or at positions which are conserved in different tyrosine kinase receptors. These receptor constructs were expressed in HEK 293 cells and the effect of 2-deoxyglucose (25 mM) on insulin (100 nM) induced receptor autophosphorylation was studied. 2-Deoxyglucose consistently inhibits insulin stimulated autophosphorylation of all constructs to the same degree as observed in wild-type human insulin receptor. The data suggest that none of the chosen serine positions are involved in 2-deoxyglucose induced receptor inhibition.     

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.     

Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation

This report describes the use of an antibody directed against the carboxyl terminus of the insulin receptor beta subunit to assess the fate of the insulin receptor protein over the time course of insulin-induced receptor down-regulation. The insulin receptor beta subunit is lost from the cellular membranes of insulin-treated 3T3-C2 fibroblasts with a time course superimposable with the insulin-induced loss of cellular insulin binding activity. Concomitant with the time-dependent loss of the intact beta subunit from the membranes, a 61,000-Da fragment of the insulin receptor beta subunit accumulates in the cytosol of the cells in a time-dependent manner. The insulin-induced loss of the intact beta subunit from the cellular membranes is inhibited by cycloheximide. Chloroquine and the thiol protease inhibitors leupeptin and E-64 inhibit the insulin-induced loss of the intact beta subunit from the membranes and induce an accumulation of the intact subunit in the membranes. However, in the presence of leupeptin, E-64, or chloroquine, the insulin-induced loss of insulin binding activity occurs normally. These data indicate that down-regulation results in the loss of the intact beta subunit from the cellular membranes with the production of a fragment of the beta subunit in the cytosol. The protease responsible for the generation of the fragment is a thiol protease which requires acidic conditions. Since the insulin-induced proteolysis of the beta subunit can be totally inhibited under conditions where the insulin-induced loss of insulin binding activity proceeds normally, the proteolysis of the beta subunit is a process which is separate and distinguishable from the insulin-induced loss of insulin binding activity.     

Adipocyte insulin receptor. Generation of a cryptic domain of the alpha-subunit during internalization of hormone-receptor complexes.

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.     

Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.     

Insulin stimulates proteolysis of the alpha-subunit, but not the beta-subunit, of its receptor at the cell surface in rat liver.

Insulin receptors in rat liver plasma membranes contain two alpha- and two beta-subunits held together by interchain disulphide bonds ([alpha beta]2 receptors). Affinity-labelled receptors were digested with chymotrypsin or elastase and then exposed to dithiothreitol before solubilization from membranes and SDS/polyacrylamide-gel electrophoresis. This resulted in partial reduction and isolation of Mr-225,000 alpha beta, Mr-200,000 alpha 1 beta, Mr-165,000 alpha beta 1 and Mr-145,000 alpha 1 beta 1 receptor halves containing intact (alpha, beta) or degraded (alpha 1, beta 1) subunits. The ability to identify half-receptor complexes containing intact or degraded subunits made it possible to assay each subunit simultaneously for insulin-induced proteolysis in isolated plasma membranes or during perfusion of rat liver in situ with insulin. In liver membranes, insulin binding increased the fraction of receptors containing degraded alpha-subunits to about one-third of the total population during 2 h of incubation at 23 degrees C. beta-Subunit proteolysis increased only minimally during this time. Plasma membranes isolated from livers perfused with insulin at 37 degrees C contained degraded alpha-subunits but only intact beta-subunits, showing that insulin induced cell-surface proteolysis of the binding, but not the kinase, domain of its receptor. Since previous observations [Lipson, Kolhatkar & Donner (1988) J. Biol. Chem 263, 10495-10501] have shown that receptors containing degraded alpha-subunits are internalized but do not recycle, it is possible that cell-surface degradation may play a role in the regulation of insulin-receptor number in hepatic tissue. Proteolysis of the beta-subunit is not a likely mechanism by which receptor-kinase activity may be attenuated under physiological conditions.     

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)     

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.     

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.     

The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus.

We studied the structure and function of the human insulin receptor (IR) and a mutant which lacked the last 43 amino acids of the beta-subunit (IR delta ct). This deletion removed tyrosine (Tyr1322, Tyr1316) and threonine (Thr1336) phosphorylation sites. In Chinese hamster ovary (CHO) cells, insulin binding to the mutant receptor was normal, and [35S]methionine labeling indicated that both the IR and IR delta ct were processed normally; however, the beta-subunit of IR delta ct was 5 kDa smaller than that of the IR. The time course of insulin-stimulated autophosphorylation of the partially purified IR delta ct was normal, but the maximum autophosphorylation was reduced 20-30%. Tryptic phosphopeptide mapping confirmed the absence of the C-terminal phosphorylation sites and indicated that phosphorylation of the regulatory region (Tyr1146, Tyr1150, Tyr1151) occurred normally; kinase activity of the IR and IR delta ct was activated normally by insulin-stimulated autophosphorylation. In the intact CHO cells, insulin-stimulated serine and threonine phosphorylation of the IR delta ct was reduced 20%, suggesting that most Ser/Thr phosphorylation sites are located outside of the C terminus. During insulin stimulation, the wild-type and mutant insulin receptor activated the phosphatidylinositol 3-kinase. Moreover, insulin itself or human-specific anti-insulin receptor antibodies stimulated glycogen and DNA synthesis equally in both CHO/IR and CHO/IR delta ct cells. These data suggest that the C terminus plays a minimal role in IR function and signal transmission in CHO cells.     

The beta-subunit of human choriogonadotropin interacts with the exodomain of the luteinizing hormone/choriogonadotropin receptor and changes its interaction with the alpha-subunit.

Human CG (hCG) consists of a common alpha-subunit and a hormone-specific beta-subunit. Similarly, its receptor is also composed of two domains, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). hCG initially binds the exodomain of the receptor after which the resulting hCG/exodomain complex is thought to interact with the endodomain. This secondary interaction is considered responsible for signal generation. Despite the importance, it is unclear which hormone subunit interacts with the exodomain or the endodomain. As a step to determine the mechanisms of the initial and secondary interactions and signal generation, we investigated the interaction of the hormone-specific beta-subunit in hCG with the receptor's exodomain. A photoactivable hCG derivative consisting of the wild-type alpha-subunit and a photoactivable beta-subunit derivative was prepared and used to label the exodomain. The analysis and immunoprecipitation of photoaffinity labeled exodomain demonstrate that the beta-subunit in hCG makes the direct contact with the exodomain.