Mechanism of insulin receptor kinase inhibition in non-insulin-dependent diabetes mellitus patients. Phosphorylation of serine 1327 or threonine 1348 is unaltered.

The tyrosine kinase activity of insulin receptor isolated from the skeletal muscle of NIDDM patients has previously been found to be decreased compared with the activity of receptor from nondiabetic subjects but the mechanism underlying this defect is unknown. Phosphorylation of receptor serine/threonine residues has been proposed to exert an inhibitory influence on receptor tyrosine kinase activity and Ser 1327 and Thr 1348 have been identified as specific sites of phosphorylation in the insulin receptor COOH terminal domain. To address the potential negative regulatory role of phosphorylation of these residues in vivo, we assessed the extent of phosphorylation of each site in insulin receptor isolated from the skeletal muscle of 12 NIDDM patients and 13 nondiabetic, control subjects. Phosphorylation of Ser 1327 and Thr 1348 was determined using antibodies that specifically recognize insulin receptor phosphorylated at these sites. In addition, a phosphotyrosine-specific antibody was used to monitor receptor tyrosine phosphorylation. The extent of insulin-induced tyrosine autophosphorylation was decreased in receptor isolated from diabetic versus nondiabetic muscle, thus confirming earlier reports. In contrast, there was no significant difference in the extent of phosphorylation of either Ser 1327 or Thr 1348 in receptor isolated from diabetic or nondiabetic muscle as assessed by immunoprecipitation (Ser 1327: 5.6 +/- 1.6% diabetics vs. 4.7 +/- 2.0% control; Thr 1348: 3.8 +/- 1.0% diabetics vs. 3.2 +/- 1.2% control). Moreover, within each group there was no correlation between the level of tyrosine kinase activity and the extent of serine/threonine phosphorylation. It is concluded that the stoichiometry of serine/threonine phosphorylation of insulin receptor in vivo is low, and that increased phosphorylation of Ser 1327 or Thr 1348 is not responsible for the decreased insulin receptor tyrosine kinase activity observed in the skeletal muscle of NIDDM patients.

Modulation of insulin receptor signaling. Potential mechanisms of a cross talk between bradykinin and the insulin receptor.

Insulin resistance of the skeletal muscle plays a key role in the development of the metabolic endocrine syndrome and its further progression to type II diabetes. Impaired signaling from the insulin receptor to the glucose transport system and to glycogen synthase is thought to be the cause of skeletal muscle insulin resistance. An incomplete activation of the insulin receptor tyrosine kinase, which is found in type II diabetes, appears to contribute to the pathogenesis of the signaling defect. Available data suggest that the impaired tyrosine kinase function of the insulin receptor is not due to an inherited defect but rather is caused by a modulation of insulin receptor function. We used rat-1 fibroblasts and NIH-3T3 cells stably overexpressing human insulin receptor and 293 cells transiently overexpressing human insulin receptor to characterize conditions modulating the signaling function of the insulin receptor kinase. Using these cell models, we could demonstrate that activation of different protein kinase C (PKC) isoforms by high glucose levels or phorbol esters causes a rapid inhibition of the receptor tyrosine kinase activity. This effect is most likely mediated through serine phosphorylation of the receptor beta-subunit. It can be prevented by PKC inhibitors and the new oral antidiabetic agent thiazolidindione. The data suggest that PKC might be an important negative regulator of insulin receptor function. Because we have recently shown that bradykinin activates different isoforms of PKC in these cell types, an inhibitory cross talk between the bradykinin receptor and the insulin receptor through PKC activation seemed possible. However, we were unable to observe an insulin receptor tyrosine kinase inhibition through bradykinin, suggesting that different isoforms of PKC are activated by hyperglycemia and bradykinin. On the other hand, a modulation of bradykinin signals by insulin could be demonstrated in these cells. Bradykinin-induced tyrosine phosphorylation of proteins of approximately 130 and 70 kDa was inhibited by insulin treatment of rat-1 fibroblasts. These data suggest that signals from the insulin receptor modify signaling from the bradykinin receptor to tyrosine phosphorylation of different cellular proteins.

Troglitazone prevents glucose-induced insulin resistance of insulin receptor in rat-1 fibroblasts.

Troglitazone (CS045), a compound belonging to the thiazolidine diones, is being tested as a new oral antidiabetic agent. Evidence exists from animal studies and clinical trials with non-insulin-dependent diabetes mellitus patients that Troglitazone might reduce insulin resistance. The molecular mechanism of this effect is not understood. In this study, we investigated whether Troglitazone might interfere with the mechanism of glucose-induced insulin resistance. Several studies indicate that hyperglycemia reduces the kinase activity of the insulin receptor in different cell types. This effect is paralleled by translocation of several protein kinase C (PKC) isoforms, and it can be prevented by PKC inhibitors, which suggests that glucose-induced receptor desensitization is mediated by activation of PKC. We studied the effect of hyperglycemia on the insulin receptor kinase activity and its modulation by Troglitazone in rat-1 fibroblasts that stably overexpress the human insulin receptor. Before stimulation with insulin (10(-7) M), cells were acutely exposed to hyperglycemic conditions in the absence or presence of Troglitazone (0.01-2 micrograms/ml). The insulin receptor was solubilized from a plasma membrane fraction or whole cell lysates, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted against antiphosphotyrosine and anti-insulin receptor beta-subunit (CT 104) antibodies. Acute hyperglycemia (25 mM glucose) induced a significant inhibition of the insulin receptor kinase (IRK) activity within 30 min (inhibition to 30 +/- 12.5% of maximal insulin-stimulated beta-subunit phosphorylation, n = 9, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute hyperglycemia provides an insulin-independent inducer for GLUT4 translocation in C2C12 myotubes and rat skeletal muscle.

GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381-3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10(-7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose +/- insulin (1.6 x 10(-9) mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme alpha 1 Na(+)-K(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis of Phe381 and Phe382 in the extracellular domain of the insulin receptor: effects on receptor biosynthesis, processing, and ligand-dependent internalization.

Mutations of the extracellular domain of the insulin receptor impair processing and transport of receptors to the plasma membrane. We have previously reported that a mutation substituting Val for Phe382 in the alpha-subunit of the insulin receptor impairs intracellular processing and insulin-induced autophosphorylation of the mutant receptor. In this investigation, we have generated two independent mutations of amino acids Phe381 and Phe382 of the insulin receptor: Val for Phe381 and Leu for Phe382. These substitutions cause a slight impairment of intracellular processing and transport of the mutant receptors. Furthermore, insulin-dependent internalization of the mutant receptors is unaffected by these mutations. Thus, of the three substitutions studied to date, Val for Phe382 is the only mutation of the Phe381-Phe382 sequence that causes a major defect in post-translational processing of the receptor.

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

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

Nutritionally induced insulin resistance and receptor defect leading to beta-cell failure in animal models.

Animals with genetically or nutritionally induced insulin resistance and Type 2 diabetes comprise two groups: those with resilient beta-cells, e.g., ob/ob mice or fa/fa rats, capable of longstanding compensatory insulin hypersecretion and those with labile beta-cells in which the secretion pressure leads to beta-cell degranulation and apoptosis, e.g., db/db mice and Psammomys gerbils (sand rats). Psammomys features low insulin receptor density; on a relatively high energy diet it becomes hyperinsulinemic and hyperglycemic. In hyperinsulinemic clamp the hepatic glucose production is only partially suppressed by insulin, even in the normoglycemic state. The capacity of insulin to activate muscle and liver receptor tyrosine kinase is nearly abolished. GLUT4 content and mRNA are markedly reduced. Hyperinsulinemia was also demonstrated to inhibit insulin signaling and glucose transport in several other species. Among the factors affecting the insulin signaling pathway, phosphorylation of serine/threonine appears to be the prominent cause of receptor malfunction as inferred from the finding of overexpression of PKC epsilon isoforms in the muscle and liver of Psammomys. The insulin resistance syndrome progressing in animals with labile beta-cells to overt diabetes and beta-cell failure is a "thrifty gene" characteristic. This is probably also true for human populations emerging from food scarcity into nutritional affluence, inappropriate for their metabolic capacity. Thus, the nutritionally induced hyperinsulinemia, associated with PKC epsilon activation may be looked upon from the molecular point of view as "PKC epsilon overexpression syndrome."

Insulin leads to a parallel translocation of PI-3-kinase and protein kinase C zeta.

Protein kinase C consists of a family of at least 12 isoforms which exhibit clear differences in their cofactor dependence and responsiveness to phospholipids. Insulin effects on PKC translocation/activation are now clearly established but responsiveness to this hormone was observed so far only for the classical PKC-isoforms alpha and beta. While activation of the classical PKC's requires Ca2+ and occurs mainly through Diacylglycerol (DAG), stimulation of the atypical isoform PKC-zeta appears to function through a different mechanism involving PI-3-kinase activation. In the present study we used rat-1 fibroblasts stably over-expressing human insulin receptor to investigate whether insulin can activate PKC-zeta and whether such an effect might be related to insulin's effect on PI-3-kinase. After stimulation of the cells with insulin (10(-7) mol/l) for one to ten minutes, a rapid translocation of PKC-zeta to the plasma membrane was detectable, as determined by immunoblotting of plasma membrane proteins with antibodies against PKC-zeta. In parallel immunoblots applying antibodies against the regulatory subunit of PI-3-kinase (p85), an insulin-induced translocation of p85 was detectable within one minute after stimulation. The translocation of p85 was associated with an increase in PI-3-kinase activity at the plasma membrane. The data show that insulin stimulates translocation of PKC-zeta in rat-1 fibroblasts. The parallel kinetics of PI-3-kinase translocation/activation and PKC-zeta translocation are compatible with the idea that the insulin effect on PKC-zeta is transduced through PI-3-kinase activation.

A viral enhancer element specifically active in human haematopoietic cells.

One particular class of DNA regulatory elements, the enhancers or activators, can, relatively independently of distance and orientation, dramatically increase the transcriptional activity of homologous and heterologous promoters located in cis (see refs 1-3 for reviews, also refs 4-6). Sequence differences between various heterologous enhancers may explain their apparent host- and/or tissue-specific action. Furthermore, differences in the transcriptional control elements may contribute to viral tropism. At least for murine leukaemia virus isolates, thymotropism and leukaemogenicity have been attributed to alterations within the viral long terminal repeat, which harbours their enhancers and other transcriptional control elements. We report here the identification of a viral enhancer element possessing a very restricted tissue range. The enhancer is active in all human cells of the haematopoetic system tested, but not in cells of fibroblast or epithelial origin.

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.

Modulation of insulin receptor signalling: significance of altered receptor isoform patterns and mechanism of hyperglycaemia-induced receptor modulation.

Insulin resistance of the skeletal muscle plays a key role in the development of the metabolic endocrine syndrome and its further progression to non-insulin dependent diabetes (NIDDM). Available data suggest that insulin resistance is caused by an impaired signal from the insulin receptor to the glucose transport system and to glycogen synthase. The impaired response of the insulin receptor tyrosine kinase which is found in NIDDM appears to contribute to the pathogenesis of the signalling defect. The reduced kinase activation is not caused by mutations within the insulin receptor gene. We investigated two potential mechanisms that might be relevant for the abnormal function of the insulin receptor in NIDDM, i.e. changes in the expression of the receptor isoforms and the effect of hyperglycaemia on insulin receptor tyrosine kinase activity. The insulin receptor is expressed in two different isoforms (HIR-A and HIR-B). We found that HIR-B expression in the skeletal muscle is increased in NIDDM. However, the characterisation of the functional properties of HIR-A and HIR-B revealed no difference in their tyrosine kinase activity in vivo. The increased expression of HIR-B might represent a compensatory event. In contrast, hyperglycaemia might directly inhibit insulin-receptor function. We have found that in rat-1 fibroblasts which overexpressing human insulin receptor an inhibition of the tyrosine kinase activity of the receptor may be induced by high glucose levels. This appears to be mediated through activation of certain protein kinase C isoforms which form stable complexes with the insulin receptor and modulate the tyrosine kinase activity of the insulin receptor through serine phosphorylation of the receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of insulin signalling in non-insulin-dependent diabetes mellitus: significance of altered receptor isoform patterns and mechanisms of glucose-induced receptor modulation.

Insulin resistance in skeletal muscle plays a key role in the development of the metabolic-endocrine syndrome and its further progression to non-insulin-dependent diabetes mellitus (NIDDM). Available data suggest that insulin resistance is caused by impaired signalling from the insulin receptor to the glucose transport system and to glycogen synthase. The impaired response of the insulin receptor tyrosine kinase, which is found in NIDDM, appears to contribute to the pathogenesis of the signalling defect. The reduced kinase activation is not caused by mutation of the receptor. Two potential mechanisms were investigated that might be relevant to the abnormal function of the insulin receptor in NIDDM. That is, changes of the receptor isoforms and the effect of hyperglycaemia. The insulin receptor is expressed in two different isoforms (HIR-A and HIR-B). HIR-B expression in skeletal muscle is increased in NIDDM. Characterization of the functional properties of HIR-B, however, revealed that increased HIR-B expression did not cause impaired tyrosine kinase activity, but more probably represented a compensatory event. In contrast, hyperglycaemia is able to inhibit insulin receptor function. In a rat-1 fibroblast cell line overexpressing human insulin receptor, inhibition of the tyrosine kinase activity of the receptor can be induced by high glucose levels. This effect appears to be mediated through activation of certain protein kinase C isoforms, which are able to form stable complexes with the insulin receptor and modulate its tyrosine kinase activity through serine phosphorylation of the receptor beta-subunit. This mechanism might also be relevant in human skeletal muscle and thereby contribute to the pathogenesis of insulin resistance.

Altered expression of insulin receptor types A and B in the skeletal muscle of non-insulin-dependent diabetes mellitus patients.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B, which are generated by alternative splicing of a primary gene transcript and differ by a 12-amino acid insertion sequence in the alpha-subunit. The two receptor isoforms bind insulin with different affinities and are differentially expressed in human tissues. We report here a tissue-specific alteration of the insulin receptor RNA splice pattern in non-insulin-dependent diabetes mellitus (NIDDM) patients. Whereas skeletal muscle of healthy individuals contains exclusively high-affinity HIR-A-encoding RNA, we consistently find low-affinity HIR-B RNA expression in NIDDM muscle tissue at levels similar to HIR-A.

Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.

Insulin signaling is inhibited by micromolar concentrations of H(2)O(2). Evidence for a role of H(2)O(2) in tumor necrosis factor alpha-mediated insulin resistance.

Both hyperglycemia and tumor necrosis factor alpha (TNFalpha) were found to induce insulin resistance at the level of the insulin receptor (IR). How this effect is mediated is, however, not understood. We investigated whether oxidative stress and production of hydrogen peroxide could be a common mediator of the inhibitory effect. We report here that micromolar concentrations of H(2)O(2) dramatically inhibit insulin-induced IR tyrosine phosphorylation (pretreatment with 500 microM H(2)O(2) for 5 min inhibits insulin-induced IR tyrosine phosphorylation to 8%), insulin receptor substrate 1 phosphorylation, as well as insulin downstream signaling such as activation of phosphatidylinositol 3-kinase (inhibited to 57%), glucose transport (inhibited to 36%), and mitogen-activated protein kinase activation (inhibited to 7.2%). Both sodium orthovanadate, a selective inhibitor of tyrosine-specific phosphatases, as well as the protein kinase C inhibitor Gö6976 reduced the inhibitory effect of hydrogen peroxide on IR tyrosine phosphorylation. To investigate whether H(2)O(2) is involved in hyperglycemia- and/or TNFalpha-induced insulin resistance, we preincubated the cells with the H(2)O(2) scavenger catalase prior to incubation with 25 mM glucose, 25 mM 2-deoxyglucose, 5.7 nM TNFalpha, or 500 microM H(2)O(2), respectively, and subsequent insulin stimulation. Whereas catalase treatment completely abolished the inhibitory effect of H(2)O(2) and TNFalpha on insulin receptor autophosphorylation, it did not reverse the inhibitory effect of hyperglycemia. In conclusion, these results demonstrate that hydrogen peroxide at low concentrations is a potent inhibitor of insulin signaling and may be involved in the development of insulin resistance in response to TNFalpha.

Protein kinase C isoforms beta 1 and beta 2 inhibit the tyrosine kinase activity of the insulin receptor.

Downregulation of insulin receptor tyrosine kinase (IRK) activity yields to impaired insulin signalling and contributes to the pathogenesis of cellular insulin resistance. Activation of protein kinase C (PKC) by different agents is associated with an inhibition of IRK activity in various cell types. There is evidence that this effect on IRK activity might be mediated through phosphorylation of specific serine residues of the insulin receptor beta-subunit. Neither the domains of the IRK where inhibiting serine phosphorylation occurs nor the PKC isoform responsible for IRK inhibition have been identified. PKC consists of a family of at least 12 isoforms. The aim of the present study was to determine which PKC isoform might be capable of IRK inhibition. The human insulin receptor and the PKC isoforms alpha, beta 1, beta 2, gamma, delta, epsilon, eta, theta and zeta were overexpressed in human embryo kidney fibroblasts (HEK 293 cells) in order to answer this question. PKCs were activated by preincubation with the phorbolester (TPA) (10(-7) mol/l) following insulin stimulation of the cells. When the IRK was coexpressed with the PKC isoforms beta 1 and beta 2, a 50 +/- 15.7 and 45 +/- 10.1% inhibition of tyrosine autophosphorylation of IRK was observed while coexpression with the other isoforms did not significantly modify IRK autophosphorylation. The data suggest that the PKC isoforms beta 1 and beta 2 might be candidates for insulin receptor inhibition.

Impact of mutations at different serine residues on the tyrosine kinase activity of the insulin receptor.

Insulin binding to its receptor activates a cascade of signaling events which are initiated by tyrosine autophosphorylation of the receptor and activation of the tyrosine kinase activity towards the insulin receptor substrates. In addition to phosphorylation at tyrosine residues a serine phosphorylation of the insulin receptor is observed. Neither the functional significance of serine phosphorylation of the receptor nor the location of relevant regulatory sites has been determined exactly so far. We studied potential functions of serine residues in human insulin receptor (HIR) with respect to its ability to undergo insulin stimulated autophosphorylation. Using site directed mutagenesis of HIR we exchanged serine to alanine at 13 different positions in the HIR beta-subunit. Sites were chosen according to the criteria of known serine phosphorylation sites (1023/25, 1293/94, 1308/09), conserved positions in hIR, hIGF-1 receptor, hIRR, and dIR (962, 994, 1037, 1055, 1074/78, 1168, 1177/78/82, 1202, 1263, 1267). All HIR mutants were expressed in HEK 293 cells and basal and insulin stimulated autophosphorylation were determined. We found that the exchange of serine to alanine at position 994 and at position 1023/25 increased insulin stimulated receptor autophosphorylation significantly (147% +/- 12% and 129% +/- 6% of control, p < 0.01, n = 7), while all other exchanges did not significantly alter insulin stimulated HIR autophosphorylation. The data suggest that the serine residues at position 994 as well as 1023/25 might be part of inhibitory domains of the insulin receptor.

Cell-type-specific control elements of the lymphotropic papovavirus enhancer.

Lymphotropic papovavirus (LPV) exhibits a highly restricted host range, in which only cells of primate B-lymphocyte origin are permissive for infection. Its enhancer element contributes to this tropism, since transcriptional potentiation is confined to cells of the hematopoietic lineage. Nuclear extracts from B and T cells, but not from HeLa cells, contain protein factors that interact specifically with the LPV 63-base-pair enhancer repeat, as demonstrated by DNase I footprinting and gel retardation experiments. Within the repeat three sequence motifs were identified: the core motif, the Pu box, and a novel element named T motif. Functional analysis demonstrated that these motifs as well as some sequences upstream of the repeat contribute to the optimal activity of the enhancer. There are clear differences between the patterns of binding of the B and T lymphocyte nuclear proteins to the enhancer which are also reflected in the transcriptional activity of the enhancer in both cell types. Furthermore, the activity of the LPV enhancer and its interaction with nuclear proteins seem to be regulated during B-cell differentiation.

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.