Activation of phosphatidylinositol-3-kinase by insulin is mediated by both A and B human insulin receptor types.

Activation of a phosphatidylinositol-3-kinase (PI-3-kinase) is one of the earliest consequences of insulin binding to the receptor. The human insulin receptor exists in two isoforms which differ in the length of the alpha-subunit (HIR-A = 719 aa, HIR-B = 731 aa). To test whether both isoforms transduce an insulin signal on PI-3-kinase we used rat-1-fibroblasts expressing HIR-A or HIR-B. We found that insulin stimulates 32P incorporation into PIP through both HIR-A and HIR-B to a similar extent (approx. 8-10 fold).     

Stimulation of phospholipase C activity by insulin is mediated by both isotypes of the human insulin receptor.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B. We studied whether both insulin receptor isotypes are able to mediate an insulin signal to phospholipase C. Plasma membranes were prepared from rat-1 fibroblasts transfected either with HIR-A or HIR-B and insulin stimulated PIP-hydrolysis was determined. We found that insulin stimulates PIP-hydrolysis in a similar dose dependent manner and to a similar extent in plasma membranes expressing HIR-A and HIR-B. These data suggest that both receptor isoforms are equally able to activate phospholipase-C.     

The two isotypes of the human insulin receptor (HIR-A and HIR-B) follow different internalization kinetics.

Human insulin receptor (HIR) is expressed in two isoforms which differ in the C-terminal end of the alpha-subunit (HIR-A = -12 aa, HIR-B = +12 aa). We studied internalization kinetics of HIR-A and HIR-B in Rat1 fibroblasts. Internalized receptors were quantified by 125I-insulin binding after cell trypsinisation and solubilization, surface receptors were determined by 125I-insulin binding to intact cells and by chemical crosslinking with B26-125I-insulin. HIR-A and HIR-B show different kinetics of receptor internalization. While in HIR-A cells the maximum of internalization (approx. 65% of total) is reached after 10 min followed by a high recycling rate (approx. 80% of internalized receptors after 20 min), the internalization in HIR-B cells reaches a maximum (approx. 60% of total) after 15 min without detectable recycling within 30 min. The data show that the different alpha-subunits of both receptor types determine different velocities of internalization and determine whether a fast recycling occurs.     

Distinct alpha-subunit structures of human insulin receptor A and B variants determine differences in tyrosine kinase activities.

Human insulin receptor isoforms (HIR-A and -B) differ in their alpha-subunit structures which result from alternatively spliced precursor mRNAs. This structural difference causes distinct binding affinities for insulin. To determine the impact of the structural difference on receptor signaling, we characterized the tyrosine kinase activity of HIR-A and HIR-B in vitro and determined the insulin stimulated beta-subunit phosphorylation and tyrosine kinase activation in the intact cell. When 32P incorporation in beta-subunits of equal amounts of isolated HIR-A and HIR-B was measured, an increased 32P incorporation in tyrosine residues of the beta-subunit of HIR-B (2.5-fold) compared to that of HIR-A was found after in vitro insulin stimulation. This was paralleled by an increased rate of phosphorylation (2.0-fold) or poly(GluNa,Tyr 4:1). In vitro analysis of Km values for ATP were similar for HIR-A (Km = 14.3 microM +/- 3.8) and HIR-B (Km = 20.2 microM +/- 8.6), whereas the Vmax of HIR-B was significantly increased (HIR-A Vmax = 5.5 mumol/60 min micrograms-1 +/- 1.4, HIR-B Vmax = 42.5 mumol/60 min micrograms-1 +/- 19.2). HPLC analysis of tryptic beta-subunit phosphopeptides revealed identical patterns, suggesting that the difference in kinase activities is not due to an alteration of the phosphorylation-activation cascade within the beta-subunit. However, when cleavage of the alpha-subunit by short-time trypsinization was used to activate the tyrosine kinase, the differences in 32P incorporation between HIR-A and HIR-B were abolished.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.     

Association of the insulin-receptor variant Met-985 with hyperglycemia and non-insulin-dependent diabetes mellitus in the Netherlands: a population-based study.

One of the characteristics of non-insulin-dependent diabetes mellitus (NIDDM) is the presence of insulin resistance. Most NIDDM patients have a normal sequence of the insulin receptor, indicating that, if insulin-receptor mutations contribute to the development of NIDDM, they will be present only in a minor fraction of the NIDDM population. The goal of the present study was to examine whether insulin-receptor mutations contribute to the development of NIDDM. We examined 161 individuals with NIDDM and 538 healthy controls from the population-based Rotterdam study for the presence of mutations in the insulin-receptor gene by SSCP. A heterozygous mutation changing valine-985 into methionine was detected in 5.6% of diabetic subjects and in 1.3% of individuals with normal oral glucose tolerance test. Adjusted for age, gender, and body-mass index, this revealed a relative risk for diabetes of 4.49 (95% confidence interval 1.59-12.25) for Met-985 carriers. When the total study group was analyzed, the prevalence of the mutation increased with increasing serum glucose levels (test for trend P < .005). We conclude that the Met-985 insulin-receptor variant associates with hyperglycemia and represents a risk factor for NIDDM.     

Polymorphism and signalling of melatonin receptors.

Melatonin receptors belong to the superfamily of G protein-coupled receptors. Cloning of Mel1c receptors expressed in Xenopus skin revealed the existence of a polymorphism for these receptors. Heterologous expression of the two allelic isoforms, called Mel1c(alpha) and Mel1c(beta), indicated functional differences in their signalling properties. Both isoforms are coupled to the cAMP and cGMP pathways. However, the alpha isoform is preferentially coupled to the cAMP pathway, whereas the beta isoform couples preferentially to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between the two isoforms are localized within intracellular receptor regions potentially involved in G protein coupling. Allelic isoforms were also observed for Mel1a receptors expressed in ovine pars tuberalis, suggesting that polymorphism is a general feature of the melatonin receptor family. We also evaluated the potential of the two human melatonin receptor subtypes, Mel1a and Mel1b, to modulate the cGMP pathway. Melatonin inhibited intracellular cGMP levels in a dose-dependent manner in HEK293 cells transfected with the human Mel1b receptor. This was not the case for HEK293 cells transfected with the human Mel1a receptor. In conclusion, our results indicate that the expression of receptor subtypes and isoforms may permit differential signalling between melatonin receptors.     

Cloning and localization of hFP(S): a six-transmembrane mRNA splice variant of the human FP prostanoid receptor

FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.     

Decreased agonist, but not antagonist, binding to the naturally occurring Thr92Lys variant of the h5-HT7(a) receptor

In the present study on transfected human embryonic kidney (HEK)293 cells, we aimed at establishing whether expression of the naturally occurring Thr92Lys variation of the Gs-coupled h5-HT7(a) receptor leads to changes of ligand binding properties, of agonist-evoked cAMP formation and/or of antagonist-mediated blockade of the latter. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulated [3H]cAMP accumulation in whole cells were determined. Saturation binding experiments in membranes of transiently transfected cells expressing either the wild-type or the variant receptor revealed a single binding site in both cases and no difference in Bmax between both receptor isoforms. In competition binding experiments in membranes of stably transfected cells, the Thr92Lys variant exhibited a 2.8-11 times lower binding affinity of the ligands 5-hydroxytryptamine (5-HT), 5-CT, 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4yl)-1H-indole (RU24969), (+/-)-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and sumatriptan compared to the wild-type receptor. However, the variant did not differ from the wild-type with respect to the binding properties of the antagonists (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolodine-1-sulfonyl)phenol hydrochloride (SB-269970), risperidone, mesulergine and clozapine. In agreement with the decreased binding affinity of 5-HT, 5-CT, RU24969 and 8-OH-DPAT for the variant receptor, these agonists were less potent in stimulating [3H]cAMP accumulation in cells stably expressing the Thr92Lys h5-HT7(a) receptor. Sumatriptan did not stimulate cAMP accumulation in spite of its affinity for both receptor isoforms pointing to a putative weak antagonistic property of this drug at the h5-HT7 receptor. SB-269970 and clozapine were equipotent at both the variant and the wild-type receptor in producing a rightward shift of the 5-HT concentration-response curve for its stimulant effect on [3H]cAMP accumulation. In view of, e.g., the purported involvement of the 5-HT7 receptor in the regulation of circadian rhythm, it may be concluded that the decrease in affinity of 5-HT and other 5-HT receptor agonists at the (Thr92Lys) h5-HT7 receptor may be associated with changes of sleep physiology and of actions of new 5-HT7 receptor agonists designed to treat circadian dysregulation.     

A novel murine PDGF-D splicing variant results in significant differences in peptide expression and function

Platelet-derived growth factor (PDGF) is a potent mesenchymal cell mitogen and chemoattractant involved in the pathogenesis of fibroproliferative diseases. There are four known PDGF ligand isoforms designated A-D, two of which, C and D, were only recently discovered. We have identified a splicing variant in the PDGF-D isoform that occurs in mice, but not in humans. The presence of the splicing variant in murine PDGF-D appears to be due to an aberration in the splicing site at the junction of exons 5 and 6. The splicing variant results in a deletion predicted to have significant effects on peptide activity since it results in the deletion of bases within the cysteine knot domain that are important for peptide dimerization and receptor binding. It is important to appreciate differences between murine and human PDGF gene expression because PDGF is a key mitogen in the pathogenesis of fibrosis and mice are commonly employed as models for human disease.     

Expression and modulation of CD44 variant isoforms in humans

CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.     

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.