Functionally distinct insulin receptors generated by tissue-specific alternative splicing.

Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.     

Comparison of the functional insulin binding epitopes of the A and B isoforms of the insulin receptor

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA; the B isoform has 12 additional amino acids (718-729) encoded by exon 11 of the gene. The isoforms have been reported to have different ligand binding properties. To further characterize their insulin binding properties, we have performed structure-directed alanine-scanning mutagenesis of a major insulin binding site of the receptor, formed from the receptor L1 domain (amino acids 1-470) and amino acids 705-715 at the C terminus of the alpha subunit. Alanine mutants of each isoform were transiently expressed as recombinant secreted extracellular domain in 293 cells, and their insulin binding properties were evaluated by competitive binding assays. Mutation of Arg(86) and Phe(96) of each isoform resulted in receptors that were not secreted. The Kds of unmutated receptors were almost identical for both isoforms. Several new mutations compromising insulin binding were identified. In L1, mutation of Leu(37) decreased affinity 20- to 40-fold and mutations of Val(94), Glu(97), Glu(120), and Lys(121) 3 to 10-fold for each isoform. A number of mutations produced differential effects on the two isoforms. Mutation of Asn(15) in the L1 domain and Phe(714) at the C terminus of the alpha subunit inactivated the A isoform but only reduced the affinity of the B isoform 40- to 60-fold. At the C terminus of the alpha subunit, mutations of Asp(707), Val(713), and Val(715) produced 7- to 16-fold reductions in affinity of the A isoform but were without effect on the B isoform. In contrast, alanine mutations of Tyr(708) and Asn(711) inactivated the B isoform but only reduced the affinities of the A isoform 11- and 6-fold, respectively. In conclusion, alanine-scanning mutagenesis of the insulin receptor A and B isoforms has identified several new side chains contributing to insulin binding and indicates that the energetic contributions of certain side chains differ in each isoform, suggesting that different molecular mechanisms are used to obtain the same affinity.     

The insulin receptor isoform exon 11- (IR-A) in cancer and other diseases: a review.

The insulin receptor plays a vital role in mediating the actions of insulin. These include metabolic and mitogenic effects. This review will focus on the role of the insulin receptor isoforms in normal development and the pathogenesis of certain cancers and type 2 diabetes. There are two insulin receptor isoforms arising from the alternative splicing of exon 11 resulting in either the exon 11+ (IR-B) isoform (including 12 amino acids encoded by exon 11) or the exon 11- (IR-A) isoform. The isoforms have different affinities for insulin, IGF-II and IGF-I with the exon 11- isoform binding both insulin and IGF-II with high affinities. Interestingly, differential expression of the insulin receptor isoforms has been demonstrated in disease. Several cancer cell types that also overexpress IGF-II preferentially express the exon 11- isoform. Activation of the exon 11- insulin receptor by IGF-II and insulin results in mitogenic effects and a potentiation of the cancer phenotype. Also hyperinsulinemia has been associated with increased risk of cancer. Differential expression of the insulin receptor isoforms has also been demonstrated in type 2 diabetes although there is some discrepancy in the literature as to which isoform is expressed.     

Hybrid receptors formed by insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) have low insulin and high IGF-1 affinity irrespective of the IR splice variant

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.     

Binding properties of chimeric insulin receptors containing the cysteine-rich domain of either the insulin-like growth factor I receptor or the insulin receptor related receptor.

We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.     

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron

Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

胰岛素受体变异性剪接与肿瘤和2型糖尿病

    胰岛素受体基因第11号外显子因为变异性剪接而形成两种胰岛素受体,两者与配体胰岛素、胰岛素样生长因子的结合力以及分别诱导的信号传导通路、发挥的生物学效应存在显著差异。这种差异不仅可能是导致胰岛素抵抗、2型糖尿病的重要原因,也会影响肿瘤细胞的生长、增殖、抗凋亡。虽然具体的调节机制尚不明确,但高胰岛素血症及高血糖等代谢因素是影响胰岛素受体变异性剪接的重要原因,同时基因序列敲除试验证实,胰岛素受体基因水平的改变会影响胰岛素受体的变异性剪接。           

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.     

Altered expression of the two naturally occurring human insulin receptor variants in isolated adipocytes of non-insulin-dependent diabetes mellitus patients.

The human insulin receptor gene is expressed in two variant isoforms which differ by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the extracellular alpha-subunit as a consequence of alternative splicing of exon 11. Expression of the two variant isoforms is regulated in a tissue-specific manner. In this study, we have measured the levels of the two receptor variants in isolated adipocytes from 10 non-insulin-dependent diabetes mellitus (NIDDM) and 11 normal subjects using an immunological assay, based on the ability of a human anti-receptor autoantibody to discriminate between HIR-A and HIR-B. Results indicate that levels of HIR-B variant are increased in NIDDM patients.     

Evolution of the insulin receptor family and receptor isoform expression in vertebrates

The molecular phylogeny of the vertebrate insulin receptor (IR) family was reconstructed under maximum likelihood (ML) to establish homologous relationships among its members. A sister group relationship between the orphan insulin-related receptor (IRR) and the insulin-like growth factor 1 receptor (IGF1R) to the exclusion of the IR obtained maximal bootstrap support. Although both IR and IGF1R were identified in all vertebrates, IRR could not be found in any teleost fish. The ancestral character states at each position of the receptor molecule were inferred for IR, IRR + IGF1R, and all 3 paralogous groups based on the recovered phylogeny using ML in order to determine those residues that could be important for the specific function of IR. For 18 residues, ancestral character state of IR was significantly distinct (probability >0.95) with respect to the corresponding inferred ancestral character states both of IRR + IGF1R and of all 3 vertebrate paralogs. Most of these IR distinct (shared derived) residues were located on the extracellular portion of the receptor (because this portion is larger and the rate of generation of IR shared derived sites is uniform along the receptor), suggesting that functional diversification during the evolutionary history of the family was largely generated modifying ligand affinity rather than signal transduction at the tyrosine kinase domain. In addition, 2 residues at positions 436 and 1095 of the human IR sequence were identified as radical cluster-specific sites in IRR + IGF1R. Both Ir and Irr have an extra exon (namely exon 11) with respect to Igf1r. We used the molecular phylogeny to infer the evolution of this additional exon. The Irr exon 11 can be traced back to amphibians, whereas we show that presence and alternative splicing of Ir exon 11 seems to be restricted exclusively to mammals. The highly divergent sequence of both exons and the reconstructed phylogeny of the vertebrate IR family strongly indicate that both exons were acquired independently by each paralog.     

GluR3 flip and flop: differences in channel opening kinetics

Ample evidence from earlier studies of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR3 included, suggests that alternative splicing not only enriches AMPA receptor diversity but also, more importantly, creates receptor variants that are functionally different. However, it is not known whether alternative splicing affects the receptor channel opening that occurs in the microsecond time domain. Using a laser-pulse photolysis technique combined with whole-cell recording, we characterized the channel opening rate process for two alternatively spliced variants of GluR3, i.e., GluR3flip and GluR3flop. We show that the alternative splicing that generates flip and flop variants of GluR3 receptors regulates the channel opening process by controlling the rate of channel closing but not the rate of channel opening or the glutamate binding affinity. Specifically, the flop variant closes its channel almost 4-fold faster than the flip variant. We therefore propose that the function of the flip-flop sequence module in the channel opening process of AMPA receptors is to stabilize the open channel conformation, presumably by its pivotal structural location. Furthermore, a comparison of the flip isoform among all AMPA receptor subunits, based on the magnitude of the channel opening rate constant, suggests that GluR3 is kinetically more similar to GluR2 and GluR4 than to GluR1.     

Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR

The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.     

Glucose regulation of insulin receptor affinity in primary cultured adipocytes

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.