Human Insulin Receptor cDNA Research Articles

Targeting Insulin Receptor with a Novel Internalizing Aptamer

Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers.

Chromosomal localization of the hormone sensitive lipase (LIPE) and insulin receptor (INSR) genes in pigs.

Using rat hormone sensitive lipase (LIPE) and human insulin receptor (INSR) cDNA probes, the LIPE gene was assigned to chromosome 6p11-q21 and the INSR gene to chromosome 2q11-q21 in pigs by in situ hybridization. In humans, these two genes are located on the q and p arms of chromosome 19, respectively. The present results provide the first in situ hybridization mapping data for porcine chromosome 2.

A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. This autophosphorylation stimulates the tyrosine kinase activity of the receptor toward intracellular substrates involved in the transmission of the signal. The discovery of pharmacological agents that specifically activate the tyrosine kinase activity of the insulin receptor will be of great importance for the treatment of insulin-resistant or insulin-deficient patients. We have developed a procedure based on Bioluminescence Resonance Energy Transfer (BRET) to monitor the activation state of the insulin receptor. Human insulin receptor cDNA, was fused to either Renilla luciferase or yellow fluorescent protein coding sequences. Fusion insulin receptors were partially purified by wheat-germ lectin chromatography from HEK-293 cells co-transfected with these constructs. The conformational change induced by insulin on its receptor could be detected as an energy transfer (BRET signal) between Renilla luciferase and yellow fluorescent protein. BRET signal paralleled insulin-induced autophosphorylation of the fusion receptor. Dose-dependent effects of insulin, insulin-like growth factor 1 and epidermal growth factor on BRET signal were in agreement with known pharmacological properties of these ligands. Moreover, an antibody, which activated the autophosphorylation of the receptor, had similar effects on BRET signal. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and could, therefore, be used in high-throughput screening for the discovery of molecules with insulin-like properties.

Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase

Fetuin has been identified earlier as the bovine homolog of the human plasma protein, α 2-Heremans Schmid grycoprotein (α 2-HSG). Although bovine fetuin shares over 70% ammo acid sequence similarity with α 2-HSG and rat fetuin, no common function(s) have been identified. We report that immunoaffinity purified bovine fetuin acts as an inhibitor of insulin receptor tyrosine kinase activity (IR-TKA) with half-maximal inhibition at 1.5 μM. In vitro, bovine fetuin (1.5 μM) blocked insulin-induced autophosphorylation of the human IR completely and the half-maximal inhibitory effect was observed at 0.5 μM. Incubation of HIRcB cells (ratl fibroblasts transfected with wild-type human insulin receptor cDNA) with bovine fetuin (1.5 μM) inhibited insulin-induced tyrosine phosphorylation of the IR β-subunit by 40%. In addition, bovine fetuin (2 μM) completely blocked insulin-stimulated DNA synthesis in H-35 rat hepatoma cells. Our results, together with earlier reports on rat fetuin and human α 2-HSG, indicate a common IR-TK inhibitory function for fetuin homologs.

Insulin receptor overexpression in 184B5 human mammary epithelial cells induces a ligand‐dependent transformed phenotype

To determine the role of the insulin receptor overexpression in breast epithelial cell transformation, the 184B5 human breast epithelial cell line was transfected with human insulin receptor cDNA. In two cell lines transfected with and overexpressing human insulin receptors (IR) (223.8 and 184.5 ng IR/10(6) cells), but not in untransfected cells, insulin binding and tyrosine kinase activity were elevated, and insulin induced a dose-dependent increase in colony formation in soft agar.

Protein Kinase Activity of the Insulin Receptor Is Essential for Insulin-Regulated Gene Expression.

Two Chinese hamster ovary (CHO) cell lines stably transfected with human insulin receptor cDNA, CHO-wt and CHO-mut, which express an equivalent number of normal and kinase-defective human insulin receptors, respectively, were used to assess the roles of insulin receptor tyrosine kinase activity in insulin-regulated gene expression. The effect of insulin on gene-33-promoter-driven chloramphenicol acetyltransferase (CAT), RSVLTR-driven beta-galactosidase (pRSVLTR-betagal) and SV40 late-promoter-driven hepatitis B surface antigen (pMLSV(2)HBsAg) were examined in CHO-wt and CHO-mut cells. Insulin-stimulated gene 33 promoter is 10- to 50-fold more effective in CHO-wt cells than that in parental CHO cells. However, no enhancement of insulin sensitivity of gene 33 promoter in CHO-mut cells relative to parental CHO cells was found. Similar phenomena were also observed, in that insulin regulated pRSVLTR-betagal and pMLSV(2)HBsAg in these three CHO lines. Our data indicated that the protein kinase activity of the insulin receptor is essential for the stimulatory activity of insulin toward the activities of different promoters. Copyright 1994 S. Karger AG, Basel

Muscle-specific expression of human insulin receptor in transgenic mice.

Variations in skeletal muscle insulin signaling are thought to have important effects on in vivo glucose homeostasis. To address the role of the insulin receptor in insulin action in muscle, we overexpressed human insulin receptors in the skeletal muscle of transgenic mice. A muscle-specific transgene (TMPE/HIR) was constructed by using promotor and enhancer elements derived from the rat MLC1/3 locus coupled to the intact protein-coding region of the human insulin-receptor cDNA. After testing the transgene for expression in cultured C2C12 myotubes, six founder mice transgenic for TMPE/HIR were generated. We determined that one line of mice had significant expression of human insulin-receptor mRNA in skeletal muscle. The analysis of several tissues from these mice by immunoprecipitation of labeled insulin receptors with a human-specific antireceptor antibody, revealed exclusive expression of human insulin receptors in skeletal muscle. Using both human-specific and non--species-specific anti-insulin receptor antibodies, we developed two immunoassays capable of quantitating the relative amounts of human and total insulin receptors in muscle. Compared with nontransgenic littermate controls, the total number of insulin receptors was increased by 30% in heterozygous transgenics and 68% in homozygotes. Human insulin-receptor protein contributed substantially to the total insulin-receptor pool present in transgenic muscle (42% for heterozygotes, 61% for homozygotes). Intraperitoneal glucose and insulin tolerance tests were performed with homozygous transgenic and nontransgenic littermate mice. Results with both approaches were significantly different for the two groups of mice, suggesting that the modest increase in insulin receptors in the muscle of transgenic mice causes a direct increase in insulin responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of insulin receptor level with both insulin action and breakdown of a potential insulin mediator precursor; studies in CHO cell-lines transfected with insulin receptor cDNA

A phosphatidylinositol-glycan (Pl-glycan) has been described previously that may serve as the precursor for a mediator of some of insulins actions. The present study further addresses the potential relevance of this compound by correlating its breakdown with other insulin actions in Chinese hamster ovary (CHO) cells which express different levels of insulin receptor. Comparisons were drawn between parent CHO cells expressing 3 · 10 3 receptors/cell and two cell-lines transfected with human insulin receptor cDNA, that expressed 600 (CHO.TH) and 300 (CHO.T) times the parent receptor level. A Pl-glycan was isolated from all cells that incorporated [ 3H]glucosamine, [ 3H]galactose and [ 3H]inositol and was rapidly turned over upon insulin stimulation. Maximal turnover by insulin of approx. 20% was achieved in each cell line consistent with the fibroblastic nature of these cells. The effect of increased insulin receptor expression was to increase the sensitivity of the PI-glycan response to insulin. Increasing receptor number from 3 · 10 3 to 0.88 · 10 6 also increased the sensitivity of response of other insulin actions measured in this study, namely activation of pyruvate dehydrogenase (PDH), glucose utilization and transport. Thus turnover of the PI-glycan is linked closely to both metabolic actions of insulin and to cell surface insulin receptor expression further supporting its potential role in insulin action.

Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases.

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.

Targeted inactivation of the insulin receptor gene in mouse 3T3-L1 fibroblasts via homologous recombination.

To study the role of the insulin receptor in determining adipocyte differentiation of the mouse cell line 3T3-L1, we have introduced a mutation that inactivates the insulin receptor gene by homologous recombination. In two independent clones, inactivation of one allele of the insulin receptor gene was associated with a 50-70% reduction in the number of insulin receptors. In addition, both clones were markedly impaired in their ability to differentiate into adipocytes. The defect in adipocyte-specific differentiation was corrected by expression of transfected human insulin receptor cDNA. These data suggest that the insulin receptor may play an important role in promoting differentiation of 3T3-L1 cells into adipocytes in vitro.

Expression of inducible membrane-anchored insulin receptor kinase enhances deoxyglucose uptake.

We have transfected mouse L cells with a recombinant membrane-anchored insulin receptor kinase (called MARK), under the transcriptional control of a glucocorticoid-responsive element. The transfected construct includes only 15 extracellular residues, with the transmembrane and intracellular kinase domains of the human insulin receptor cDNA (amino acid residues -27 to 12 and 915 to 1343 (see 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), predicted Mr = 56,000 with signal sequence, 53,000 without). Transfected cells which are exposed to dexamethasone express two proteins of approximate Mr = 54,000 which (a) react with anti-peptide antisera raised to human insulin receptor sequences, (b) localize to the membrane fraction, and (c) possess ligand (insulin)-independent tyrosine kinase activity. In extracts of steroid-treated MARK cells, a phosphotyrosine-containing protein of Mr = 185,000 is detected, which corresponds in size to a known endogenous substrate for the insulin receptor. Control studies were performed with the nontransfected parent line, and with L cells transfected with an inactive human insulin receptor protein tyrosine kinase. Dexamethasone induced no change in the proteins detected by an anti-phosphotyrosine antibody in the two control lines. In studies of deoxyglucose uptake, dexamethasone stimulated increase in deoxyglucose uptake 2-fold in the MARK cells compared to the same cells studied in the absence of dexamethasone. Dexamethasone had no effect in the control cell lines. These studies demonstrate that a membrane-anchored insulin receptor kinase, devoid of virtually the entire extracellular domain of the insulin receptor, is sufficient to induce enhanced deoxyglucose uptake.

Characterization of the growth of murine fibroblasts that express human insulin receptors: I. The effect of insulin in the absence of other growth factors

The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle.

Characterization of the growth of murine fibroblasts that express human insulin receptors: II. Interaction of insulin with other growth factors

The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin ⩾ 10% fetal calf serum > PDGF > IGF-1 ⪢ EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum > PDGF > EGF ⪢ IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1–10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.

Expression of Atypical and Classical Insulin Receptors in Chinese Hamster Ovary Cells Transfected with Cloned cDNA for the Human Insulin Receptor*

Human placenta and IM-9 lymphocytes contain subpopulations of atypical insulin receptors which differ from classical insulin receptors in their higher binding affinity for insulin-like growth factors I and II (IGF-I and IGF-II). Both types of insulin receptors may be derived from different but related genes, or may represent alternative post-translational modifications of the same gene product. To test these possibilities, we have examined the IGF binding characteristics of the human insulin receptors expressed in Chinese hamster ovary (CHO) cells which had been stably transfected with cloned human insulin receptor cDNA (CHO-T cells). The parent CHO cells contained 3 x 10(3) rodent insulin receptors/cell, and the CHO-T cells, 2.0 x 10(6) human insulin receptors/cell. Competition binding studies showed that the binding of [125I]IGF-I and [125I]multiplication stimulating activity (MSA/rat IGF-II) to parent CHO cells was primarily to type I and II IGF receptors, which cross-react poorly or not at all with insulin. However, competition binding studies with CHO-T cells showed that [125I]IGF-I binding was displaced 60-70%, and [125I]MSA binding, 50-55%, by low concentrations of insulin (20 ng/ml) and no further by higher concentrations of insulin (500 ng/ml). The insulin-insensitive IGF binding sites corresponded to the rodent type I and II IGF receptors; the insulin-sensitive IGF binding sensitive sites resembled the human atypical insulin receptors in that they bound IGF-I and MSA with moderately high affinity and reacted with insulin, MSA, and IGF-I in that order of potency. Atypical insulin receptors were also demonstrated by insulin-sensitive [125I]IGF-I and [125I]MSA binding to solubilized CHO-T proteins adsorbed to microtiter wells coated with monoclonal antibodies specific for the human insulin receptor. These results suggest that atypical human insulin receptors are generated by differential post-translational processing of the same gene product as classical human insulin receptors.

Anti-(insulin receptor) monoclonal antibody-stimulated tyrosine phosphorylation in cells transfected with human insulin receptor cDNA

The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.

Regulation of the Insulin Receptor Kinase by Hyperinsulinism

A murine fibroblast cell line transfected with human insulin receptor cDNA, NIH 3T3 HIR3.5, was observed to display insulin-induced down-regulation of insulin-binding activity in a time- and concentration-dependent manner. Maximal inhibition of insulin-binding activity (54%) occurred within 16 h of exposure to 100 nM insulin in vivo, where in vivo refers to intact cells in tissue culture. The decrease in cellular insulin-binding activity was the consequence of a decrease in the number of cell-associated insulin receptors as determined by Scatchard analysis of insulin binding, 125I-insulin affinity cross-linking, and Western blotting of the insulin receptor beta subunit. Acute insulin treatment in vivo (1-60 min) resulted in the activation of the insulin receptor protein tyrosine kinase as determined by in vitro phosphorylation of glutamic acid:tyrosine (4:1), where in vitro refers to broken cell preparations. This acute in vivo insulin activation of the insulin receptor tyrosine kinase resulted in a greater stimulation (1.4-1.9-fold) of tyrosine kinase activity in the glutamic acid:tyrosine (4:1) assay than the maximal stimulation produced by insulin treatment in vitro. In contrast, long term (24 h) insulin treatment in vivo resulted in a 50-70% decrease in intrinsic protein tyrosine kinase activity of the insulin receptors compared with that of acutely activated (1 min) insulin receptors. Under these conditions, the insulin receptor protein kinase activity remained insulin independent in the in vitro substrate kinase assay. Surprisingly, the insulin-independent activated (1 min in vivo insulin-treated) and uncoupled (24 h in vivo insulin-treated) insulin receptors displayed similar stoichiometries of 32P incorporation into the beta subunit by in vitro autophosphorylation when compared with the control insulin receptors, ranging from 1.5 to 1.8 mol of phosphate incorporated/mol of insulin receptor. Phosphoamino acid analysis demonstrated that the phosphoserine/phosphothreonine content of in vivo 32P-labeled insulin receptors increased markedly within a 1-h exposure to insulin in vivo, whereas insulin-induced receptor desensitization was not apparent until 10-24 h after exposure to insulin. These data suggest that insulin treatment in vivo results initially in the activation of the insulin receptor kinase followed by a subsequent uncoupling of protein kinase activity. This insulin-induced desensitization of the insulin receptor kinase does not correlate with the extent of beta subunit serine/threonine phosphorylation.

Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts.

The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated approximately 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor beta-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin, with a half-maximal effect at approximately 3 nM insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only approximately 30% that of insulin. Two antibodies (25-49 and 83-14) showed a similar concentration dependence to insulin in their metabolic effects and in the inhibition of 125I-labeled insulin binding to cells. The other two antibodies (83-7 and 18-44) were somewhat less potent and did not inhibit insulin binding. None of the antibodies significantly increased insulin receptor phosphorylation at concentrations up to 100 nM, which at least in the case of 25-49 and 83-14 was sufficient for full receptor occupancy. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.

Molecular and Clinical Characterization of an Insertional Polymorphism of the Insulin-Receptor Gene

A restriction-fragment-length polymorphism (RFLP) detected with the human insulin-receptor cDNA and the enzyme Sac I has been reported to be associated with non-insulin-dependent diabetes mellitus (NIDDM) in White and Black populations and segregated with diabetes in two small pedigrees with maturity-onset diabetes of the young. A size difference of approximately 500 base pairs (bp) was demonstrated between the alleles of this and several other RFLPs that mapped to the same 300-bp region near the transmembrane coding region of the cDNA beta-chain, thus suggesting the presence of an insertion in this region that could affect insulin-receptor function. Genomic DNA fragments containing this RFLP were cloned from an individual heterozygous for the putative insertion, and the differing fragments of the two alleles were sequenced. The presence of a 400-bp insertion was thus confirmed and was demonstrated to be entirely within an intron. No significant coding-region differences from published cDNA sequences were detected in four exons sequenced from the region of the insertional allele. The sequenced regions included multiple Alu repeat sequences. The RFLP was unusual in that the larger allele consisted of an additional Alu repeat sequence that included a new Pst I site. Because the nature and location of the insertion did not suggest a role in insulin-receptor function, the association of this RFLP with NIDDM and hyperinsulinemia was reexamined in a small sample of Whites. No association could be demonstrated, and the insertion also failed to segregate with NIDDM in five White pedigrees.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Molecular cloning of syndecan, an integral membrane proteoglycan.

We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).