[Show abstract][Hide abstract] ABSTRACT: Sirtuins catalyze NAD(+)-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.
Preview · Article · Mar 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: TNFalpha plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCdelta, a novel PKC isoform, is known to participate in proximal TNFalpha signaling events. However, it has remained unclear whether PKCdelta plays a role in distal TNFalpha signaling events. Here we demonstrate that PKCdelta is activated by TNFalpha in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCdelta and JNK, we used pharmacological and genetic inhibitors of NFkappaB. We found that inhibition of NFkappaB attenuated PKCdelta and JNK activations. Further analysis revealed that ER stress contributes to TNFalpha-stimulated PKCdelta and JNK activations. To investigate the role of PKCdelta in TNFalpha action, we used 29-mer shRNAs to silence PKCdelta expression. A reduction of ~90% in PKCdelta protein levels reduced TNFalpha-stimulated stress kinase activation, including JNK. Further, PKCdelta was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCdelta was necessary for induction of the UPR. Indeed, a reduction in PKCdelta protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCdelta does not globally regulate the UPR. Next, the role of PKCdelta in TNFalpha mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCdelta expression. We found that a reduction in PKCdelta protein levels reversed the TNFalpha-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFalpha-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFkappaB and ER stress contribute in part to PKCdelta activation; 2) PKCdelta plays a key role in the propagation of the TNFalpha signal; and 3) PKCdelta contributes to TNFalpha-induced inhibition of insulin signaling events.
No preview · Article · Sep 2009 · Cellular Signalling
[Show abstract][Hide abstract] ABSTRACT: The neurohormone ovary ecdysteroidogenic hormone I (OEH I), originally identified from Aedes aegypti (L.), has an effect on mosquito ecdysteroidogenesis. Would there be a similar presence and function in other adult flies, e.g., black blow fly, Phormia regina (Meigen)? One pair of median neurosecretory cells (MNCOEH I) and one pair of lateral neurosecretory cells (LNCOEH I) were immunopositive to OEH I antiserum in the brain of sugar-fed and liver-fed female P. regina. In addition, two pairs of neurosecretory cells (SOGNCOEH I) positive to this antiserum were found in the suboesophageal ganglion only in adult female P. regina at 16, 20, 23, 28, 36, and 72 h after a liver meal. In addition to the positive pairs of MNCOEH I and LNCOEH I in females, there were four more pairs of positive neurons (MSNFNCOEH I) in the region between the MNCOEH I and the esophageal foramen in P. regina males, but not in females. The presence of OEH I in male flies needs further attention as to the function of this hormone in male dipterans. We also found substances positive to Drosophila melanogaster Meigen insulin receptor antiserum located in the corpus cardiacum and the nerves going to the corpus allatum of adult female P. regina at 6 and 30 h after the liver meal, but not to the brain.
Full-text · Article · Jan 2009 · Annals of the Entomological Society of America
[Show abstract][Hide abstract] ABSTRACT: A high-resolution time series of transcript abundance was generated to describe global expression dynamics in response to nutrition in Drosophila. Nonparametric change-point statistics revealed that within 7 h of feeding upon yeast, transcript levels changed significantly for approximately 3,500 genes or 20% of the Drosophila genome. Differences as small as 15% were highly significant, and 80% of the changes were <1.5-fold. Notably, transcript changes reflected rapid downregulation of the nutrient-sensing insulin and target of rapamycin pathways, shifting of fuel metabolism from lipid to glucose oxidation, and increased purine synthesis, TCA-biosynthetic functions and mitochondria biogenesis. To investigate how nutrition coordinates these transcriptional changes, feeding-induced expression changes were compared with those induced by the insulin-regulated transcription factor dFOXO in Drosophila S2 cells. Remarkably, 28% (995) of the nutrient-responsive genes were regulated by activated dFOXO, including genes of mitochondrial biogenesis and a novel homolog of mammalian peroxisome proliferator-gamma coactivator-1 (PGC-1), a transcriptional coactivator implicated in controlling mitochondrial gene expression in mammals. These data implicate dFOXO as a major coordinator of the transcriptional response to nutrients downstream of insulin and suggest that mitochondria biogenesis is linked to insulin signaling via dFOXO-mediated repression of a PGC-1 homolog.
[Show abstract][Hide abstract] ABSTRACT: Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCdelta and PKCtheta correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCdelta, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCdelta or wild-type PKCdelta followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCdelta was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1-glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCdelta acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCdelta on at least 18 sites. The importance of three of the PKCdelta phosphorylation sites in IRS-1 was shown in vitro by a 75-80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCdelta modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
Full-text · Article · Mar 2004 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: The serine/threonine kinase Akt/PKB plays key roles in the regulation of cell growth, survival, and metabolism. It remains unclear, however, whether the functions of individual Akt/PKB isoforms are distinct. To investigate the function of Akt2/PKBbeta, mice lacking this isoform were generated. Both male and female Akt2/PKBbeta-null mice exhibit mild growth deficiency and an age-dependent loss of adipose tissue or lipoatrophy, with all observed adipose depots dramatically reduced by 22 weeks of age. Akt2/PKBbeta-deficient mice are insulin resistant with elevated plasma triglycerides. In addition, Akt2/PKBbeta-deficient mice exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure. In contrast, female Akt2/PKBbeta-deficient mice remain mildly hyperglycemic and hyperinsulinemic until at least one year of age. Thus, Akt2/PKBbeta-deficient mice exhibit growth deficiency similar to that reported previously for mice lacking Akt1/PKBalpha, indicating that both Akt2/PKBbeta and Akt1/PKBalpha participate in the regulation of growth. The marked hyperglycemia and loss of pancreatic beta cells and adipose tissue in Akt2/PKBbeta-deficient mice suggest that Akt2/PKBbeta plays critical roles in glucose metabolism and the development or maintenance of proper adipose tissue and islet mass for which other Akt/PKB isoforms are unable to fully compensate.
Full-text · Article · Aug 2003 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: Insulin receptor substrates (IRS) 1 and 2 are phosphorylated on serine/threonine (Ser/Thr) residues in quiescent cells (basal phosphorylation), and phosphorylation on both Ser/Thr and tyrosine residues is increased upon insulin stimulation. To determine whether basal Ser/Thr phosphorylation of IRS proteins influences insulin receptor catalyzed tyrosine phosphorylation, recombinant FLAG epitope-tagged IRS-1 (F-IRS-1) and IRS-2 (F-IRS-2) were expressed, purified, and subjected to both dephosphorylation and hyperphosphorylation prior to phosphorylation by the insulin receptor kinase. As expected, hyperphosphorylation of F-IRS-1 and F-IRS-2 by GSK3beta decreased their subsequent phosphorylation on tyrosine residues by the insulin receptor. Surprisingly, however, dephosphorylation of the basal Ser/Thr phosphorylation sites impaired subsequent phosphorylation on tyrosine, suggesting that basal Ser/Thr phosphorylation of F-IRS-1 and F-IRS-2 plays a positive role in phosphorylation by the insulin receptor tyrosine kinase. Dephosphorylation of basal Ser/Thr sites on F-IRS-1 also significantly reduced tyrosine phosphorylation by the IGF-1 receptor. However, dephosphorylation of F-IRS-2 significantly increased phosphorylation by the IGF-1 receptor, suggesting that basal phosphorylation of IRS-2 has divergent effects on its interaction with the insulin and IGF-1 receptors. Phosphorylation of endogenous IRS-1 and IRS-2 from 3T3-L1 adipocytes was modulated in a similar manner. IRS-1 and IRS-2 from serum-fed cells were hyperphosphorylated, and dephosphorylation induced either by serum deprivation or by alkaline phosphatase treatment after immunoprecipitation led to an increase in tyrosine phosphorylation by the insulin receptor. Dephosphorylation of IRS-1 and IRS-2 immunoprecipitated from serum-deprived cells, however, resulted in inhibition of tyrosine phosphorylation by the insulin receptor. These data suggest that Ser/Thr phosphorylation can have both a positive and a negative regulatory role on tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IGF-1 receptors.
[Show abstract][Hide abstract] ABSTRACT: Studies in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans have revealed that components of the insulin signaling pathway have been highly conserved during evolution. Genetic analysis in Drosophila suggests that structural conservation also extends to the functional level. Flies carrying mutations that reduce insulin signaling have a growth deficiency phenotype similar to that seen in mice with disruptions of genes encoding insulin-like growth factors (IGFs) or the IGF-I receptor. Recent studies in flies have demonstrated a role for the insulin signaling pathway in the regulation of metabolism, reproduction and lifespan via modulation of central neuroendocrine pathways. Similarly, mice with loss of brain insulin receptors or insulin receptor substrate 2 deficiency exhibit neuroendocrine defects and female infertility. These parallels suggest that the insulin system has multiple conserved roles, acting directly to modulate growth and indirectly, via the neuroendocrine system, to modulate peripheral physiology in response to changes in nutrient availability.
No preview · Article · May 2002 · Trends in Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: The Drosophila melanogaster gene insulin-like receptor (InR) is homologous to mammalian insulin receptors as well as to Caenorhabditis elegans daf-2, a signal transducer regulating worm dauer formation and adult longevity. We describe a heteroallelic, hypomorphic genotype of mutant InR, which yields dwarf females with up to an 85% extension of adult longevity and dwarf males with reduced late age-specific mortality. Treatment of the long-lived InR dwarfs with a juvenile hormone analog restores life expectancy toward that of wild-type controls. We conclude that juvenile hormone deficiency, which results from InR signal pathway mutation, is sufficient to extend life-span, and that in flies, insulin-like ligands nonautonomously mediate aging through retardation of growth or activation of specific endocrine tissue.
[Show abstract][Hide abstract] ABSTRACT: The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin-stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1 adipocytes.
No preview · Article · Jun 2000 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The Drosophila insulin receptor (INR) homolog includes an extension of approximately 400 amino acids at the carboxyl-terminal end of its beta subunit containing several tyrosine-based motifs known to mediate interactions with signaling proteins. In order to explore the role of this extension in INR function, mammalian expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney cells in the absence of the ligand-binding alpha subunits. beta-Myc and betaDelta proteins were constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitated a phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1), whereas INR betaDelta did not, suggesting that the site of interaction was within the carboxyl-terminal extension. IRS-1 was phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase, SHP-2, Raf-1, and JAK2, were not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases conferred an equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal extension. However, INR beta-Myc-expressing cells exhibited enhanced survival relative to parental and betaDelta cells, suggesting that the carboxyl-terminal extension, through its interaction with IRS-1, plays a role in the regulation of cell death.
No preview · Article · Sep 1999 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Drosophila contain an insulin receptor homologue, encoded by the inr gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand- activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene and six independent mutations that lead to a loss of expression or function of the receptor protein were identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for inr in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size.
[Show abstract][Hide abstract] ABSTRACT: The glycosylation of the Drosophila insulin receptor (DIR) has been compared to that of the rat insulin receptor by examining the binding of receptors to the lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat insulin receptors bound and were specifically eluted from all three lectins, only a small fraction of the DIR (< 5%) was retained on wheat germ agglutinin. In contrast, the DIR bound strongly to Concanavalin-A and lentil lectin and was recovered from lentil lectin columns after elution with alpha-methyl-mannoside. The pattern of lectin binding indicates that glycosylation of the DIR and rat insulin receptors differs, with the DIR containing primarily high mannose-type oligosaccharides. After lectin chromatography, the DIR exhibited an elevated level of basal autophosphorylation and kinase activity, which could be restored to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT did not, however, affect ligand-stimulated kinase activity. The ability of low concentrations of DTT to deactivate the DIR kinase suggests that, like the mammalian receptor, beta-subunit thiols may be involved in regulation of conformational changes between activated and unactivated receptor states. Interestingly, DTT-induced deactivation of the DIR was blocked by preincubation with an antipeptide antibody against the carboxy-terminal domain of the DIR. This suggests that the DIR carboxyl terminus undergoes a conformational change during the activation-inactivation cycle of the kinase, which can be sterically hindered by the antibody. Conformational changes in this region of the mammalian receptor have been observed, and these data suggest that features of the insulin receptor activation mechanism have been substantially conserved during evolution.
[Show abstract][Hide abstract] ABSTRACT: The nucleic acid and deduced amino acid sequence of the Drosophila insulin receptor homologue (dir) has been determined. The coding sequence of dir is contained within 10 exons spanning less than 8 kilobase pairs of genomic DNA. The deduced amino acid sequence of the dir encodes a protein of 2148 amino acids, larger than the human insulin receptor due to amino- and carboxyl-terminal extensions.
The overall level of amino acid identity between the DIR and human insulin and insulin-like growth factor-I receptors is 32.5
and 33.3%, respectively. Higher levels of identity are found in exon 2 (45 and 43%, respectively) and in the β subunit (50
and 48%, respectively), and the positions of most cysteine residues in the α subunit cysteine-rich domain are conserved. A
novel, 400-amino acid, carboxyl-terminal extension contains 9 tyrosine residues, four of which are present in YXXM or YXXL motifs, suggesting that they function as binding sites for SH2 domain-containing signaling proteins. The presence of multiple
putative SH2 domain binding sites in the DIR represents a significant difference from its mammalian homologues and suggests
that, unlike the human insulin and insulin-like growth factor-I receptors, the DIR forms stable complexes with signaling molecules
as part of its signal transduction mechanism.
[Show abstract][Hide abstract] ABSTRACT: Ligand-dependent autophosphorylation and immunoprecipitation have been used to distinguish insulin and insulin-like growth factor-I (IGF-I) receptor beta-subunits in the permissive and inducible subclones of the C2 myoblast cell line. Permissive myoblasts differentiate spontaneously, whereas myoblasts of the inducible subclone require exogenous IGFs to undergo terminal differentiation. Permissive myoblasts contain beta-subunits of 95 and 101 kilodalton (kDa) mol wt. The 95-kDa subunits are immunoprecipitated with antipeptide antibodies directed against tyrosine kinase (AbP2), juxtamembrane (AbP4), and carboxy-terminal (AbP5) domains of the insulin receptor and insulin receptor monoclonal antibody 29B4. The tryptic phosphopeptide map of the 95-kDa band suggests that it contains both insulin and IGF-I receptor beta-subunits. The 101-kDa subunit is immunoprecipitated by AbP2, AbP4, and AbP5, because it forms a hybrid complex with the 95-kDa protein, but it does not react directly with AbP4, AbP5, or antibody 29B4. Phosphorylation of the 101-kDa subunit is more responsive to IGF-I than to IGF-II or insulin, indicating that it is a second IGF-I receptor beta-subunit. Inducible myoblasts exhibit a single major beta-subunit of 106 kDa mol wt. Its immunoreactivity and phosphopeptide map are virtually identical to those of the 101-kDa IGF-I receptor beta-subunit from permissive cells. However, unlike the 101-kDa beta-subunit, phosphorylation of the 106-kDa protein appears to be more responsive to IGF-II than to either IGF-I or insulin. It is lost upon differentiation of myoblasts into myotubes concomittant with the appearance of 95- and 101-kDa beta-subunits. These data demonstrate 1) an alpha 2 beta 2 IGF receptor that has high sensitivity for IGF-II in inducible, but not in permissive, myoblasts; 2) the beta-subunit of this receptor exhibits different migration in sodium dodecyl sulfate-polyacrylamide gels from either of those found in permissive cells; and 3) expression of this beta-subunit is developmentally regulated. This suggests that the inducible cell beta-subunit is a component of a stage-specific alpha 2 beta 2 IGF receptor subtype that functions as an IGF-II receptor.
[Show abstract][Hide abstract] ABSTRACT: Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.
[Show abstract][Hide abstract] ABSTRACT: Insulin and insulinlike growth factor 1 (IGF-1) receptors are present in brain, yet their function remains obscure. Expression of these tyrosine kinase-bearing growth factor receptors during rat brain development was examined by using three antipeptide antibodies directed against epitopes in the beta subunits (AbP2, AbP4, and AbP5). All three antibodies recognized both insulin and IGF-1 receptors. Membranes were prepared from fetal brains (14 to 21 days of gestation), neonatal brain (postnatal day 1), and adult brain. Immunoblot analyses using AbP4 and AbP5 revealed a 92-kilodalton (kDa) protein that corresponded to the beta subunit of the insulin and IGF-1 receptors. Densitometric scanning of immunoblots indicated that receptor proteins were 4- to 10-fold more abundant in fetal brain membranes than in membranes from adult brain. Expression was highest during 16 to 18 days of gestation and declined thereafter to the relatively low level found in adult brain. Immunoblot analyses with AbP2 as well as ligand-activated receptor autophosphorylation revealed an additional protein of 97 kDa. This protein was phosphorylated in response to IGF-1 and was not directly recognized by AbP4 or AbP5. The covalent association of the 97-kDa protein with the 92-kDa beta subunit was indicated by the ability of AbP4 and AbP5 to immunoprecipitate both proteins under nonreducing conditions but only the 92-kDa protein after reduction. In contrast, AbP2 immunoprecipitated both proteins regardless of their association. This immunospecificity remained unchanged after deglycosylation of the isolated proteins. Two-dimensional tryptic phosphopeptide analysis showed that the 92- and 97-kDa subunits of the IGF-1 receptor are related but distinct proteins. Taken together, the data suggest that the 92- and 97-kDa subunits differ in primary amino acid sequence. Thus, two distinct beta subunits may be present in a single IGF-1 receptor in brain. These subunits have in common an epitope recognized by an antibody to the tyrosine kinase domain (AbP2) but differ in regions thought to be important in receptor kinase regulation and signal transduction.
Preview · Article · Aug 1989 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: The Drosophila melanogaster insulin receptor (Drosophila insulin receptor homolog [dIRH]) is similar to its mammalian counterpart in deduced amino acid sequence, subunit structure, and ligand-stimulated protein tyrosine kinase activity. The function of this receptor in D. melanogaster is not yet known. However, a role in development is suggested by the observations that levels of insulin-stimulated kinase activity and expression of dIRH mRNA are maximal during Drosophila midembryogenesis. In this study, a 2.9-kilobase (kb) cDNA clone corresponding to both the dIRH tyrosine kinase domain and some of the 3' untranslated sequence was used to determine the tissue distribution of dIRH mRNA during development. Two principal mRNAs of 11 and 8.6 kb hybridized with the dIRH cDNA in Northern (RNA) blot analysis. The abundance of the 8.6-kb mRNA increased transiently in early embryos, whereas the 11-kb species was most abundant during midembryogenesis. A similar pattern of expression was previously determined by Northern analysis, using a dIRH genomic clone (L. Petruzzelli, R. Herrera, R. Arenas-Garcia, R. Fernandez, M. J. Birnbaum, and O. M. Rosen, Proc. Natl. Acad. Sci. USA 83:4710-4714, 1986). In situ hybridization revealed dIRH transcripts in the ovaries of adult flies, in which the transcripts appeared to be synthesized by nurse cells for eventual storage as maternal RNA in the mature oocyte. Throughout embryogenesis, dIRH transcripts were ubiquitously expressed, although after midembryogenesis, higher levels were detected in the developing nervous system. Nervous system expression remained elevated throughout the larval stages and persisted in the adult, in which the cortex of the brain and ganglion cells were among the most prominently labeled tissues. In larvae, the imaginal disk cells exhibited comparatively high levels of dIRH mRNA expression. The broad distribution of dIRH mRNA in embryos and imaginal disks is compatible with a role for dIRH in anabolic processes required for cell growth. The apparently elevated expression of dIRH mRNA in nervous tissue during mid- and late embryogenesis coincides with a period of active neurite outgrowth and suggests that dIRH may be involved in this process.
Preview · Article · May 1988 · Molecular and Cellular Biology