Antibody-induced down-regulation of a mutated insulin receptor lacking an intact cytoplasmic domain

Insulin receptor down-regulation was studied in various Chinese hamster ovary (CHO) cell lines expressing transfected human insulin receptor cDNAs. In addition to a cell line expressing the normal receptor (CHO.T line), three lines expressing mutated receptors were studied: the CHO.T-t line, which expresses a receptor with a degraded cytoplasmic domain due to the removal of the C-terminal 112 amino acids, and the CHO.YF1 and CHO.YF3 lines, in which important autophosphorylation sites of the receptor kinase (tyrosines-1162 and -1163) have been replaced by phenylalanine. A monoclonal anti-receptor antibody, but not insulin itself, was found to down-regulate cell surface receptor levels in all four cell lines by 60-80% after 18-h treatment at 37 degrees C. Down-regulation of the CHO.T and CHO.T-t receptors occurred at similar antibody concentrations and with a similar time course, although the maximum level of CHO.T-t down-regulation (60%) was generally lower than the level of CHO.T down-regulation (80%). Pulse-chase labeling of these two cell types with [35S]methionine revealed that antibody treatment of both CHO.T and CHO.T-t cells resulted in a similar increase in the rate of degradation of mature receptor subunits. These results indicate that antibody-induced down-regulation of the insulin receptor in these cells can occur in the absence of various autophosphorylation sites of the receptor and that the mechanism of antibody-induced down-regulation is different from that for insulin.     

Insulin receptor tyrosine residues 1162 and 1163 control insulin stimulation of myristoyl-diacylglycerol generation and subsequent activation of glucose transport

Chinese hamster ovary (CHO) transfectants expressing human insulin receptors that were mutated at tyrosines 1162 and 1163 (CHO-Y2 cells) exhibit decreased insulin stimulation of both receptor tyrosine kinase and 2-deoxyglucose uptake compared with transfectants expressing wild-type human insulin receptors (CHO-R cells). We now provide evidence that insulin stimulation of myristoyl-diacylglycerol (DAG) production is also markedly impaired in CHO-Y2 cells; this is manifested as a decreased responsiveness and sensitivity to insulin as compared with CHO-R and parental CHO cells. Further, we report that (i) the concentration-response curves of insulin-stimulated myristoyl-DAG production and 2-deoxyglucose uptake were superimposable within each of the three cell lines. (ii) The insulin-induced increase in myristoyl-DAG production preceded that in 2-deoxyglucose uptake, and the time course was altered for both responses in CHO-Y2 cells. (iii) Insulin also increased the phosphorylation of a 40-kDa protein known to be a substrate for protein kinase C, but to a much lesser extent in CHO-Y2 cells than in CHO-R cells. (iv) Exogenously added 1,2-dimyristoyl-glycerol and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) again stimulated both the phosphorylation of the 40-kDa protein and 2-deoxyglucose uptake, but in contrast to insulin, they elicited the same level of response in both CHO-R and CHO-Y2 cells. (v) Finally, in protein kinase C-depleted CHO-R cells, insulin and PMA stimulation of 40-kDa protein phosphorylation as well as PMA stimulation of 2-deoxyglucose uptake were completely abolished whereas insulin-stimulated 2-deoxyglucose uptake was only partially decreased. Taken together, these results suggest that insulin stimulation of 2-deoxyglucose uptake involves myristoyl-DAG production and, at least in part, protein kinase C activation, all three of these processes being controlled by receptor tyrosines 1162 and 1163.     

Analysis of the order of autophosphorylation of human insulin receptor tyrosines 1158, 1162 and 1163.

Insulin receptor tyrosines 1158, 1162 and 1163 are the most rapidly autophosphorylated residues following insulin binding. Although progression of these tyrosines from a bis- to tris-phosphorylated state leads to activation of the receptor tyrosine kinase towards added substrates, rather paradoxically, a receptor with a Y1158F mutation has been reported to be capable of normal activation. In the present study we demonstrate that autophosphorylation of the insulin receptor probably initiates on either of tyrosines 1158 and 1162 while autophosphorylation of tyrosine 1163 occurs predominantly late in the autophosphorylation cascade. Our results are compatible with tyrosines 1162 and 1163 being the major determinants of kinase activity and explain why wild-type insulin receptors only become active after all three of tyrosines 1158, 1162 and 1163 have been phosphorylated.     

The regulatory role of known tyrosine autophosphorylation sites of the insulin receptor kinase domain. An assessment by replacement with neutral and negatively charged amino acids.

Autophosphorylation of the insulin receptor has been previously documented to activate the phosphotransferase activity of the receptor from 20- to 200-fold. Biochemical studies have correlated activation of the receptor kinase with the autophosphorylation of tyrosines residues 1158, 1162, and 1163. To further assess the role of these 3 tyrosines in the activation process, we have studied the effect of their substitution with either the neutral amino acids phenylalanine or alanine or with the negatively charged amino acids aspartate and glutamate. In several other proteins, it has been shown that substitution of phosphorylated residues with negatively charged amino acids can mimic the phosphorylation state of the protein. In agreement with previous studies, tyrosines at positions 1162 and 1163 were found to be crucial in the kinase activation process. In contrast, mutant receptors with tyrosine 1158 changed to either phenylalanine or aspartate were still activated to the same extent as the wild-type receptor. An increased basal exogenous kinase activity was observed upon replacement of tyrosines 1162 and 1163 with, in increasing order of potency, aspartate = glutamate less than alanine = phenylalanine. These results indicate that phosphorylation of tyrosines 1162/1163 but not 1158 play a critical role in the activation of the receptor kinase and that the mechanism of activation of the receptor kinase by autophosphorylation is more complex than just an introduction of a cluster of negative charges in this region of the receptor. In addition, the finding of an increased basal kinase activity in receptors lacking tyrosines 1162 and 1163 could explain the reported ability of this receptor to mediate certain biological responses.     

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.     

Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity

The internalization and degradation of insulin was assessed in Chinese hamster ovary cell lines expressing either the wild-type receptor or mutated receptors lacking kinase activity. The mutated receptors included receptors which differed from the wild-type receptor by a single amino acid (substitution of an arginine for lysine at position 1030, a site critical for ATP binding) as well as receptors which had a deletion of 112 amino acids at the carboxyl terminus. Cells expressing mutated receptors lacking kinase activity were found to internalize and degrade insulin at about half the rate of cells expressing wild-type receptors with kinase activity. Moreover, insulin was found incapable of inducing the internalization of the mutated receptors, whereas it could stimulate the internalization of the wild-type receptor. Finally, the constitutive rate of receptor internalization was found to be the same for the mutant and wild-type receptors. These results implicate the intrinsic tyrosine-specific kinase activity of the insulin receptor in the ligand-induced, but not the constitutive, internalization of this receptor.     

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.     

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.     

Differential role of insulin receptor autophosphorylation sites 1162 and 1163 in the long-term insulin stimulation of glucose transport, glycogenesis, and protein synthesis.

The long-term regulatory effect of insulin on glucose transport activity and glucose transporter expression was examined in Chinese hamster ovary (CHO) transfectants that overexpress either human insulin receptors of the wild type (CHO-R cells) or human insulin receptors mutated at two major autophosphorylation sites, Tyr1162 and Tyr1163 (CHO-Y2 cells). Previous studies showed that, when acutely stimulated by insulin, CHO-Y2 cells exhibit decreased receptor kinase activity along with decreased signaling of several pathways, including that for glucose transport, as compared with CHO-R cells. We now report the following. (i) When treated for 24 h with insulin (10(-10) to 10(-6) M), CHO-R and CHO-Y2 cells displayed closely similar concentration-dependent increases in 2-deoxyglucose uptake. In both transfectants, the maximal insulin-induced increase (approximately 3.5-fold) in uptake was cycloheximide-sensitive and was paralleled by equivalent increases in the levels of GLUT-1 immunoreactive protein and mRNA. (ii) By contrast, under similar conditions, CHO-Y2 cells exhibited a marked decrease in their response to insulin for [U-14C]glucose incorporation into glycogen (decreased sensitivity and maximal responsiveness) and for [U-14C]leucine incorporation into protein (decreased sensitivity) as compared with CHO-R cells. (iii) After a 24-h treatment with 10(-7) M insulin, CHO-R (but not CHO-Y2) cells showed a decreased ability to respond to a subsequent acute insulin stimulation of either receptor exogenous kinase activity or 2-deoxyglucose uptake as compared with respective untreated controls. These results indicate that (i) insulin receptors mutated at Tyr1162 and Tyr1163 retain normal signaling of the long-term stimulatory effect of insulin on glucose transport activity and GLUT-1 expression, but not on glycogenesis and overall protein synthesis; (ii) these three insulin signaling pathways may be triggered by distinct domains of the insulin receptor beta-subunit; and (iii) wild-type (but not twin-tyrosine mutant) receptors undergo negative regulation by chronic insulin treatment for subsequent signaling of acute biological actions of insulin.     

Role of the rate of internalization of the agonist-receptor complex on the agonist-induced down-regulation of the lutropin/choriogonadotropin receptor.

The extent of agonist-induced down-regulation of the LH/CG receptor (LHR) in human kidney 293 cells transfected with the rat LHR (rLHR) is much lower than in two Leydig tumor cell lines (MA-10 and R2C) that express the rodent LHR endogenously. This difference can not be attributed to differences in the recycling of internalized receptors, or in the replenishment of new receptors at the cell surface. It can be correlated, however, with the half-life of internalization of the bound agonist, which is approximately 60 min in Leydig tumor cells and about 100 min in transfected 293 cells. To determine whether the rate of internalization of the bound agonist affects down-regulation, we compared these two parameters in 293 cells expressing four rLHR mutants that enhance internalization and three mutants that impair internalization. We show that all four mutations of the rLHR that enhanced internalization enhanced down-regulation, while only one of the three mutations that impaired internalization impaired down-regulation. In addition, cotransfections of 293 cells with the rLHR-wt and three constructs that enhanced internalization (G protein-coupled receptor kinase 2, beta-arrestin, and arrestin-3) increased down-regulation, while a related construct (visual arrestin) that had no effect on internalization also had no effect on down-regulation. We conclude that the rate of internalization of the agonist-LHR complex is the main determinant of the extent of down-regulation of the LHR.     

Replacement of the human insulin receptor transmembrane and cytoplasmic domains by corresponding domains of the oncogene product v-ros leads to accelerated internalization, degradation, and down-regulation

Internalization, degradation, and insulin-induced down-regulation of insulin receptors were studied comparatively in transformed Chinese hamster ovary (CHO) cell lines, CHO.T and CHO.IR.ros, respectively expressing either the wild-type human insulin receptor (hIR) or a mutated hybrid receptor in which the transmembrane and cytoplasmic domains of hIR were replaced by corresponding domains of the transforming protein p68gag-ros (v-ros) of avian sarcoma virus UR2. At 37 degrees C, degradation of insulin receptors photoaffinity labeled on the cell surface (440 kDa) was most rapid for the hybrid hIR.ros (t1/2 1.0 +/- 0.1 h), intermediate for the wild-type hIR (t1/2 2.7 +/- 0.5 h), and slowest for the endogenous CHO insulin receptors (t1/2 3.7 +/- 0.7 h). Initial intracellular accumulation of the hIR.ros hybrid was also most rapid, reaching maximal amounts in 20 min following which the receptors disappeared rapidly from the intracellular compartment. In contrast, intracellular accumulation of the receptors in the CHO.T and CHO cells was slower, reaching maximal amounts in 60 min, and rapid disappearance of the receptors from the intracellular compartment did not occur. Chloroquine, a lysosomotropic agent, inhibited degradation of both the wild-type hIR and the chimeric hIR.ros and increased their intracellular accumulation. However, the chloroquine effect was much more marked for the hIR.ros receptors whose intracellular accumulation was increased by greater than 300% (in comparison with approximately 60% increase for the wild-type hIR), demonstrating marked intracellular degradation of the hybrid hIR.ros at chloroquine-sensitive sites. Insulin-induced down-regulation of the cell surface hIR.ros (52% loss in 3 h) was also more marked than the wild-type hIR (approximately 30% loss in 3 h). Thus, in the hybrid hIR.ros receptor, which was shown previously to exhibit insulin-stimulated autophosphorylation and kinase activity but not insulin-stimulated metabolic function, the capacity for internalization and down-regulation is not only preserved but is also markedly accelerated. These findings suggest that 1) the postreceptor coupling mechanisms mediating insulin-induced receptor internalization, degradation, and down-regulation are different from those mediating metabolic functions; and 2) v-ros may contain the structural information directing accelerated receptor catabolism.     

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.     

Mechanisms of internalization and recycling of the chemokine receptor, CCR5.

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C(3) exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner.     

Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines

The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF-dependent receptor endocytosis.     

The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation

We have previously demonstrated that the human insulin receptor, mutated in the ATP-binding domain of the beta-subunit, is kinase-defective and fails to mediate multiple post-receptor actions of insulin in stably transfected Chinese hamster ovary cells (Chou, C.-K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, O. M. (1987) J. Biol. Chem. 262, 1842-1847). This study addresses the role of protein-tyrosine kinase activity in insulin-mediated receptor down-regulation. Although the mutant insulin proreceptor was properly processed and able to bind insulin like the wild-type human receptor, it differed from the latter in the following respects: 1) it failed to mediate internalization of surface-bound radiolabeled ligand; 2) it did not undergo short- or long-term down-regulation in response to 1 microM insulin; 3) it did not exhibit ligand-promoted receptor turnover; and 4) it was not phosphorylated on either tyrosine or serine residues in response to insulin. Although the cells transfected with the mutant receptor failed to respond to insulin-mediated insulin receptor down-regulation, they were able to down-regulate their insulin-like growth factor I receptors in response to insulin-like growth factor I or high concentrations of insulin and were sensitive to monoclonal antibody-induced down-regulation of their insulin receptors. Antibody-mediated receptor internalization alone, however, was unable to mimic at least one action of insulin, thymidine incorporation into DNA, and did not lead to any phosphorylation of the receptor. It is concluded that either the protein-tyrosine kinase activity of the insulin receptor or its phosphorylation state is essential for ligand-mediated receptor down-regulation.     

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.     

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.     

The Human Insulin Receptor Cdna: A New Tool to study the Function of this Receptor

Abstract

The human insulin receptor (hIR) is an integral transmembrane glycoprotein comprised of two α and two β subunits. An immediate consequence of insulin binding to the extracellular α subunit is the autophosphorylation of tyrosine residues on the intracellular domain of the β subunit. The placental hIR cDNA has been cloned and sequenced, providing the primary structural features of the protein.

In order to investigate the functions of the β subunit and particularly the role of autophosphorylation and tyrosine phosphokinase (TPK) activity (a feature shared by other receptors and oncogene proteins) in transmembrane signalling, we designed an expression system of the hIR cDNA in eucaryotic cells. Superexpressing CHO cell lines that contain about 10⁶ functional hIR/cell have been developed. In these cells half maximum stimulation of glucose uptake occurs at 5x 10^-10M insulin, whereas normal CHO cells require 5x 10^-12M insulin. In this expression system we have carried out site-directed mutagenesis experiments in which domains of the molecule have been deleted or particular amino acids have been replaced by others. The replacement of either or both the tyrosine residues 1162 and 1163 compromise an autophosphorylated site that is important for kinase function and the insulin response. Expression of an isolated membrane-bound form of the β-subunit produces a 6 fold increase in glucose uptake. This insulin-independent effect disappears if the twin tyrosines are mutated or if the β subunit is expressed in the cytoplasm. These studies also show that the C terminal 112 amino acid portion of the β subunit is important for the stability of this protein.     

Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.     

Agonist-induced internalization and recycling of the human A(3) adenosine receptors: role in receptor desensitization and resensitization

A(3) adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine-5'-N-methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A(3) adenosine receptor showed a profile typical of these receptors in other cell lines (K:(D) = 1.3+/-0.08 nM; B(max) = 400+/-28 fmol/mg of proteins). The iodinated agonist, bound at 4 degrees C to whole transfected cells, was internalized by increasing the temperature to 37 degrees C with a rate constant of 0.04+/-0.034 min(-1). Agonist-induced internalization of A(3) adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02+/-0.0017 min(-1). Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.