Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation.

Some patients with extreme insulin resistance have mutations in their insulin receptor gene. We previously identified five such mutations located in the extracellular domain of the insulin receptor (Asn-->Lys15, His-->Arg209, Phe-->Val382, Lys-->Glu460, and Asn-->Ser462) and studied the effects of these mutations upon posttranslational processing, insulin binding, and tyrosine autophosphorylation. We now characterize the ability of these mutant receptors to mediate biological actions of insulin in transfected NIH-3T3 fibroblasts. All cell lines expressing mutant receptors showed marked impairment in insulin-stimulated c-jun expression and thymidine incorporation when compared with cells expressing wild-type human insulin receptors. The most severe impairment was seen in cells expressing the Val382 mutant (a mutation which causes an intrinsic defect in receptor autophosphorylation). These cells had insulin responses similar to the untransfected cells (used as a negative control). In contrast, cells expressing the Lys15 mutant have the ability to achieve a normal level of maximal autophosphorylation but require an abnormally high concentration of insulin to do so (as the result of decreased insulin binding affinity). These cells show a higher basal rate and much lower insulin stimulation of both c-jun expression and thymidine incorporation when compared with the cells expressing the wild-type human insulin receptors. This pattern is also seen in the cells expressing the other mutants with normal autophosphorylation (Arg209, Glu460, and Ser462). Although the most severe defects in insulin action are seen with the mutation which has an intrinsic defect in receptor autophosphorylation, the ability to undergo normal autophosphorylation does not seem to preclude mutations from impairing the ability of receptors to mediate some of the actions of insulin.     

Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain.

Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain-binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane.     

A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells.     

A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation.

Mutations of the insulin receptor gene have been shown to cause insulin-resistant diabetes in patients with genetic forms of insulin resistance. We have previously reported that a mutation substituting valine for Phe382 in the alpha-subunit of the insulin receptor is associated with impaired transport of the mutant receptor to the plasma membrane (Accili, D., Frapier, C., Mosthaf, L., McKeon, C., Elbein, S. C., Permutt, M. A., Ramos, E., Lander, E. S., Ullrich, A., and Taylor, S. I. (1989) EMBO J. 8, 2509-2517). In this study, we demonstrate that the Val382 mutation impairs the ability of insulin to activate receptor autophosphorylation. Furthermore, the Val382 receptor has reduced activity to phosphorylate other peptide substrates in the presence of insulin. Nevertheless, when the Val382 mutant and wild-type receptors are mixed together, the wild-type human insulin receptor is able to phosphorylate the Val382 mutant receptor, thereby activating the tyrosine kinase activity of the mutant receptor. Thus, the conformational change caused by the Val382 mutation compromises the ability of the receptor to transmit a signal across the plasma membrane. Furthermore, our observations suggest that receptor phosphorylation by an intermolecular mechanism (i.e. transphosphorylation) may play a role in mediating the action of insulin upon the target cell.     

Phosphorylation state and biological function of a mutant human insulin receptor Val996

Chinese hamster ovary (CHO) cell transfectants that expressed human insulin receptors whose glycine 996 was substituted by valine were studied. Receptor processing and insulin binding were unaffected by this mutation; however, this mutant insulin receptor had little or no tyrosine kinase activity. Nevertheless, the Val996 mutant exhibited seryl and threonyl phosphorylation in both the basal and insulin-stimulated state in intact cells. This is in contrast to the Lys----Ala1018 tyrosine kinase deficient mutant (Russell, D. S., Gherzi, R., Johnson, E. L., Chou, C-K., and Rosen, O. M. (1987) J. Biol. Chem. 262, 11833-11840). Cells expressing the normal human receptor were 10-fold more sensitive to insulin than the untransfected CHO cells with respect to phosphorylation of a cellular substrate (pp 185) on tyrosyl residues, glucose incorporation into glycogen, thymidine incorporation into DNA, and phosphorylation of ribosomal protein S6. Cells expressing the mutant receptor exhibited the same insulin sensitivity as the untransfected CHO cells. Insulin was rapidly internalized in cells expressing the normal human receptor and the number of receptors expressed on the cell surface was decreased in response to exposure to insulin. However, little insulin was internalized in cells expressing the mutant receptor, and the number of receptors on the cell surface was not significantly diminished in response to exposure to insulin. It is concluded that despite the occurrence of seryl and threonyl phosphorylations, post-receptor effects of insulin described above are not mediated by the tyrosine kinase-deficient receptor, Val996.     

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.     

An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport.

In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited.     

Substitution of lysine for asparagine at position 15 in the alpha-subunit of the human insulin receptor. A mutation that impairs transport of receptors to the cell surface and decreases the affinity of insulin binding

Mutations in the insulin receptor gene can compromise the ability of the receptor to mediate insulin action. Previously, in investigations of a patient with a genetic form of insulin resistance, we have identified a mutant allele encoding an insulin receptor in which lysine is substituted for asparagine at position 15 of the alpha-subunit. In the present study, we have characterized the Lys15-mutant receptor expressed by transfection of mutant cDNA into NIH-3T3 cells. The Lys15-mutation causes at least two defects in insulin receptor function. First, the mutation retards the post-translational processing of the receptor and impairs transport of the receptor to the plasma membrane, thereby reducing the number of receptors on the cell surface. Second, the mutation causes a 5-fold reduction in the affinity of the receptor to bind insulin. These two defects combine to render the target cell resistant to normal physiological concentrations of insulin. It seems likely that both functional defects associated with the Lys15-mutation can be explained by assuming that the mutation distorts the three-dimensional structure of the receptor. Presumably, the abnormal conformation interferes with the transport of the receptor through the endoplasmic reticulum and Golgi, and also inhibits the binding of insulin to its binding site on the receptor.     

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation.

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell line, most of the p21ras was rapidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109). To determine whether this process also occurs in cells expressing physiologically relevant numbers of insulin receptors, insulin stimulated Ras.GTP formation was quantitated in Chinese hamster ovary (CHO)-derived cell lines expressing varying numbers of insulin receptors. In the parental CHO9 cells, expressing only 5.10(3) insulin receptors, insulin stimulation for 3 min increased Ras.GTP levels with 10%. Upon increasing the number of insulin receptors in these cells, Ras.GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. These data show that receptor overexpression is not a prerequisite for insulin-stimulated Ras.GTP formation. The yield of Ras.GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CHO9- and NIH3T3-derived cell lines, respectively. These values argue against signal-amplifying processes between the insulin receptor and p21ras. To determine whether receptor autophosphorylation is required for Ras.GTP formation, NIH3T3 cells overexpressing insulin receptors were stimulated with a monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser1200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects in CHO cells, were used. In both cases, no Ras.GTP formation was observed. Furthermore, Rat-1-derived cell lines expressing mutant p21ras, which is permanently in the active GTP-bound form, still responded to insulin by increasing the glucose uptake. These results support our hypothesis that Ras.GTP formation is activated by the tyrosyl-phosphorylated insulin receptor and suggest that an active Ras.GTP complex does not mediate metabolic signaling.     

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.     

Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor

DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding. We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A. J., Langley, E., Wilson, E. M., and Vidal, J. (1995) J. Biol. Chem. 270, 5251-5257). In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast. We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene. In the deletion mutant, the AR did not bind hormone to the same extent as the wild type. Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important. Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant. By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function. A model for the mechanism of DnaJ-like protein action is discussed.     

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.     

Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild- type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.     

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.     

The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor.

All five functional domains of the low density lipoprotein (LDL) receptor were assembled in their modern form more than 350 million years ago, as revealed from the sequence of two cloned cDNAs from the frog Xenopus laevis. The two cDNAs appear to represent duplicated copies of the LDL receptor gene that arose when the entire genome of Xenopus duplicated approximately 30 million years ago. Both frog LDL receptors bound Xenopus LDL with high affinity and human LDL with lower affinity when expressed in monkey COS cells. The receptors also showed high affinity for rabbit beta-migrating very low density lipoprotein and canine apoE-HDLc, both of which contain apolipoprotein E. Each of the seven cysteine-rich repeats in the ligand binding domain of the Xenopus receptors resembles its counterpart in the human, indicating that these repeats had already acquired their independent structures by the time of amphibian development. The cytoplasmic tail of both Xenopus receptors is 86% identical to the human, including the FDNPVY sequence necessary for internalization in coated pits. The attainment of a fully developed receptor structure in Xenopus suggests that earlier forms of the receptor may exist in animals that are older than amphibians. An accompanying paper demonstrates that expression of both Xenopus receptor genes is controlled by a sterol regulatory element that closely resembles the human sequence (Mehta, K.D., Brown, M.S., Bilheimer, D.W., and Goldstein, J.L. (1991) J. Biol. Chem. 266, 10415-10419).     

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.     

Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction

We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.     

The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum

We here describe a mutant low density lipoprotein receptor gene that produces a shortened receptor protein lacking three domains: the region of clustered O-linked carbohydrates, the membrane-spanning region, and the cytoplasmic tail. The defect is attributable to a single nucleotide substitution that creates a premature termination codon at amino acid 660, eliminating 180 residues from the mature protein. The truncated protein retains only two domains: a complete ligand-binding region (residues 1-292) and a partial epidermal growth factor precursor homology region (residues 293-659). The termination codon occurs in the middle of a cysteine-rich sequence that is part of the epidermal growth factor precursor homology domain. The mutant protein is present in markedly reduced amounts and may be translated at a reduced rate. After synthesis, most of the receptor remains within the cell for several hours with its N-linked carbohydrate in an unprocessed endoglycosidase H-sensitive form. This finding suggests that the shortened receptor leaves the endoplasmic reticulum at an abnormally slow rate, which is likely attributable to abnormal folding of the truncated protein. The mutation creates a new restriction site for the enzyme HinfI, thus permitting diagnosis by Southern blotting of genomic DNA. Two copies of this mutant gene were present in each of four unrelated Arab patients with homozygous familial hypercholesterolemia (three from Lebanon and one from Syria). We believe that this mutation, hereafter referred to as the "Lebanese allele," is responsible for the extraordinarily high incidence of familial hypercholesterolemia in Lebanon.     

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.     

Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor

Recently, we have described a COOH-terminal deletion mutation of the human insulin receptor (HIR delta CT) that exhibits normal insulin-mediated kinase activity and endocytosis, but is inefficient in stimulating glucose transport and glycogen synthase (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; Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this paper, we report that despite this defect in metabolic signaling, the truncated receptor exhibits augmented mitogenic activity compared to normal receptors. These results were verified in three independently isolated clones of Rat 1 fibroblasts transfected with the HIR delta CT cDNA. The increase in insulin sensitivity of mitogenic stimulation was proportional to the number of HIR delta CT receptors expressed on the cells. By contrast, only the cells with normal receptors and none of the HIR delta CT clones exhibit increased sensitivity for a metabolic action of insulin, the stimulation of glucose uptake. Stimulation of cells by other mitogens and autoradiographic analysis confirm that the enhanced mitogenic effects seen in HIR delta CT cells are attributable only to the presence of the truncated insulin receptors. These receptors mediate the tyrosine phosphorylation of a number of cellular proteins, and the pattern of these phosphorylations differs quantitatively from that seen in cells with normal receptors. We conclude: 1) The COOH terminus plays a role in signaling metabolic actions of insulin, perhaps through its recognition of substrates for the receptor kinase. 2) By contrast, the COOH terminus is an inhibitory regulator of mitogenesis, and removal of the terminal 43 amino acids converts the receptor from a moderately active growth signaler to a very active one. 3) The changes seen in biologic activities of the HIR delta CT receptor are associated with quantitative changes in substrate phosphorylation by the receptor kinase.