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PubMed1996

Silencing of the constitutive activity of the dopamine D1B receptor. Reciprocal mutations between D1 receptor subtypes delineate residues underlying activation properties.

Recently, we have shown that the dopamine D1B/D5 receptor displays binding and coupling properties that are reminiscent of those of the constitutively activated G protein-coupled receptors when compared with the related D1A/D1 receptor subtype (Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925-27931). The carboxyl-terminal region of the third cytoplasmic loop of several G protein-coupled receptors has been demonstrated to be important for the regulation of the equilibrium between inactive and active receptor conformations. In this cytoplasmic region, the primary structure of dopamine D1A and D1B receptors differs by only two residues: Phe264/Arg266 are present in D1A receptor compared with Ile288/Lys290 in the D1B receptor. To investigate whether these structural differences could account for the distinct binding and coupling properties of these dopamine receptor subtypes, we swapped the variant residues located in the carboxyl-terminal region by site-directed mutagenesis. The exchange of the D1A receptor residue Phe264 by the D1B receptor counterpart isoleucine led to a D1A receptor mutant exhibiting D1B-like constitutive properties. In contrast, substitution of D1B receptor Ile288 by the D1A receptor counterpart phenylalanine resulted in a loss of constitutive activation of the D1B receptor with binding and coupling properties similar to the D1A receptor. The Arg/Lys substitution had no effect on the function of either receptor. These results demonstrate that the carboxyl-terminal region, and in particular residue Ile288, is a major determinant of the constitutive activity of the dopamine D1B receptor. Moreover, these results establish that not only can agonist-independent activity of a receptor be induced, but when given the appropriate mutation, it can be reversed or silenced.

Adenylyl Cyclases↗

PubMed1996

Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation.

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present studies, the constitutively active Akt and a nonmyristoylated control mutant were expressed in 3T3-L1 cells that can be induced to differentiate into adipocytes. The constitutively active Akt induced glucose uptake into adipocytes in the absence of insulin by stimulating translocation of the insulin-responsive glucose transporter 4 to the plasma membrane. The constitutively active Akt also increased the synthesis of the ubiquitously expressed glucose transporter 1. The increased glucose influx in the 3T3-L1 adipocytes directed lipid but not glycogen synthesis. These results indicate that Akt can regulate glucose uptake and metabolism.

3T3 Cells↗

PubMed1996

Differential storage of prolactin, granins (Chromogranin B and secretogranin II), and constitutive secretory markers in rat pituitary GH4C1 cells.

The rat pituitary cell line GH4C1 secretes granins (chromogranin B and secretogranin II) and prolactin by the regulated secretory pathway. The intracellular storage of prolactin is preferentially induced by hormone treatment with estradiol, insulin, and epidermal growth factor. The goal of this study was to determine the effect of hormone treatment on storage of granins and constitutive secretory markers. The granins were efficiently stored in both hormone-treated and -untreated cells (17% of total secreted in 4 h). Secreted alkaline phosphatase (SEAP), a truncated membrane protein that would not be expected to enter secretory granules, and glycosaminoglycan, a marker for the constitutive secretory pathway, exhibited 70 80% secretion under both conditions. In comparison, the relative prolactin secretion was 31 and 68% from hormone-treated and -untreated cells, respectively. Phorbol ester and KCl stimulated prolactin secretion 2.3-fold from untreated cells and 5. 5-fold from hormone-treated cells. In contrast, SEAP secretion was stimulated 1.5-fold from both treated and untreated cells, consistent with secretion by the constitutive secretory pathway. Stimulated secretion of granins was detected from both hormone-treated and -untreated cells. These results suggest that granin and prolactin storage are differentially regulated and that the constitutive secretory pathway is not affected by hormone treatment.

Alkaline Phosphatase↗

PubMed1997

Constitutive activation of the beta2 adrenergic receptor alters the orientation of its sixth membrane-spanning segment.

The binding site of the beta2 adrenergic receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Methanethiosulfonate ethylammonium (MTSEA), a charged, hydrophilic, lipophobic, sulfhydryl-specific reagent, had no effect on the binding of agonist or antagonist to wild-type beta2 receptor expressed in HEK 293 cells. This suggested that no endogenous cysteines are accessible in the binding site crevice. In contrast, in a constitutively active beta2 receptor, MTSEA significantly inhibited antagonist binding, and isoproterenol slowed the rate of reaction of MTSEA. This implies that at least one endogenous cysteine becomes accessible in the binding site crevice of the constitutively active beta2 receptor. Cys-285, in the sixth membrane-spanning segment, is responsible for the inhibitory effect of MTSEA on ligand binding to the constitutively active mutant. The acquired accessibility of Cys-285 in the constitutively active mutant may result from a rotation and/or tilting of the sixth membrane-spanning segment associated with activation of the receptor. This rearrangement could bring Cys-285 to the margin of the binding site crevice where it becomes accessible to MTSEA.

Adrenergic beta-Agonists↗

PubMed1997

Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.

Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.

Adrenergic beta-Agonists↗

PubMed1997

Constitutive activation of JAK1 in Src-transformed cells.

We have previously found that the signal transducer and activator of transcription (Stat) 3 is constitutively activated in cells stably transformed by the v-Src oncoprotein. While activation of Stat proteins has also been observed following epidermal growth factor or platelet-derived growth factor stimulation, Stat3 activation is more commonly associated with signaling through cytokine receptors and activation of the Janus family tyrosine kinases JAK1 or JAK2. We therefore investigated whether JAK1 or JAK2 were activated in Src-transformed cells. In three v-Src-transformed fibroblast cell lines (NIH3T3, Balb/c, and 3Y1), JAK1 displayed increased tyrosyl phosphorylation compared to non-transformed cells. The level of tyrosyl phosphorylation of JAK1 was significantly greater in NIH3T3 cells transformed by expression of v-Src or high levels of a constitutively active mutant of c-Src (Y527F) than in cells overexpressing the less transforming normal c-Src. Enzymatic activity of JAK1 was assessed using autophosphorylation assays. In anti-JAK1 immunoprecipitates from v-Src-transformed NIH3T3 cells, a protein with the same migration as JAK1 showed substantially increased levels of 32P incorporation compared to immunoprecipitates from non-transformed cells. Similar results were obtained using anti-JAK2 immunoprecipitates; however, the level of JAK2 tyrosyl phosphorylation and 32P incorporation in anti-JAK2 immunoprecipitates were markedly lower than in anti-JAK1 immunoprecipitates. We conclude that JAK1, and possibly JAK2, are constitutively activated in Src-transformed cells, raising the possibility that Janus family kinases contribute to the constitutive activation of Stat3 previously observed in these cells and/or other properties of Src-transformed cells.

3T3 Cells↗

PubMed1997

Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

A new and essential cis-element AEE (aldose reductase enhancer element), necessary for the constitutive activity and the osmotic stress response of rat aldose reductase transcription in a rat liver cell line, has been identified. In transient transfection assays, an increase in promoter activity, up to 3.8-fold, was observed with osmotic stress (600 mosm/kg H2O) using a luciferase reporter gene construct containing aldose reductase promoter sequence from -1,094 base pair (bp) to +23 bp. A deletion between -1,071 and -895 bp reduced the constitutive activity and abolished the osmotic response of the promoter. Exonuclease III mediated in vivo DNA footprinting and dimethyl sulfate in vivo footprinting revealed DNA protection of a 32-bp region and two guanosines (G) within this region protected from methylation, respectively. Electrophoretic gel mobility shift assays using whole liver cell extracts showed protein binding, under both normal and stressed conditions. Deletion of the sequence between the two guanosines protected by in vivo dimethyl sulfate DNA footprinting (GAAGAGTG) in a luciferase construct (-1,094 bp to +23 bp) abolished the constitutive promoter activity. One copy of AEE fused to the thymidine kinase promoter gave a maximum constitutive activity of 7.7-fold and a maximum osmotic response activity of 6. 7-fold.

Aldehyde Reductase↗

PubMed1998

Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation.

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.

Amino Acid Sequence↗

PubMed1998

Constitutive activation of NF-kappaB causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells. Autocrine role of tumor necrosis factor and reactive oxygen intermediates.

How tumor cells develop resistance to apoptosis induced by cytokines and chemotherapeutic agents is incompletely understood. In the present report, we investigated apoptosis induction by tumor necrosis factor (TNF) in two human T cell lines, Jurkat and HuT-78. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells. It was further found that HuT-78 cells constitutively expressed the nuclear transcription factor NF-kappaB. TNF activated NF-kappaB in Jurkat cells but not in HuT-78 cells. HuT-78 cells were also resistant to NF-kappaB activation induced by phorbol ester, H2O2, ceramide, endotoxin, and interleukin-1. Despite the presence of preactivated NF-kappaB, HuT-78 cells also expressed high levels of IkappaB-alpha, the inhibitory subunit of NF-kappaB and, unlike Jurkat cells, were resistant to TNF-induced degradation of IkappaB-alpha. Its half-life in HuT-78 cells was 12 h as opposed to 45 min in Jurkat cells. Antibodies against TNF blocked the constitutive activation of NF-kappaB and proliferation of HuT-78 cells but had no significant effect on Jurkat cells, suggesting an autocrine role for TNF. The antioxidant pyrrolidine dithiocarbamate also suppressed constitutive NF-kappaB activation and it reversed the cell's sensitivity to TNF-induced cytotoxicity and activation of caspase-3. Overall, these results suggest that constitutive activation of NF-kappaB, TNF, and prooxidant pathway in certain T cell lymphomas causes resistance to apoptosis, and this can be reversed by antioxidants.

Apoptosis↗

PubMed1998

Identification of a conserved switch residue responsible for selective constitutive activation of the beta2-adrenergic receptor.

A cysteine-to-phenylalanine mutation of residue 116 in the third transmembrane domain of the beta2-adrenergic receptor caused selective constitutive activation of Na+/H+ exchange through a pathway not involving cAMP. This selectivity was identified by comparing binding and signaling characteristics of wild-type (WT) versus C116F mutant receptors transiently transfected into COS-1 cells. Indicating constitutive activity, ligand binding to the C116F mutant showed a 78-fold higher than WT affinity for isoproterenol and a 40-fold lower than WT affinity for ICI 118551. Although agonist-independent activation of cAMP production was not exhibited by the C116F mutant, a constitutive stimulation of the Na+/H+ exchanger (NHE1) was observed. This was identified by measuring either basal intracellular pH (pHi) or rate of pHi recovery from cellular acid load. Due to a higher rate of H+ efflux through NHE1, C116F transfectants exhibited a significantly higher pHi (7.42) than did WT transfectants (7.1). Furthermore, the rate of pHi recovery from acid load facilitated by NHE1 was 2.1-fold faster in mutant transfectants than in WT transfectants. The lower rate seen in the WT case was stimulated by epinephrine, and the higher rate seen in the mutant case was inhibited by ICI 118551. These findings, which show that a C116F mutation of the beta2-adrenergic receptor evokes selective constitutive coupling to NHE1 over cAMP, form the basis of our prediction that multiple and distinct activation states can exist in G protein-coupled receptors.

Adrenergic beta-Agonists↗

PubMed1999

Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1.

The transcriptional regulation of the Fas ligand (FasL) gene in Sertoli cells was investigated, as these cells are known to have constitutive expression of FasL and hence maintain an "immune privileged" environment within the testicle. Using the Sertoli cell line TM4, it was demonstrated that a gene segment of the 5'-untranslated region located between -318 and -237 relative to the translation start site is required for constitutive FasL transcription. Deletion and mutation analysis demonstrate that an Sp1 rather than an NFAT or NFKB-like DNA binding motif present within this region is necessary and sufficient for constitutive FasL gene transcription. Nuclear extracts of Sertoli cells contain Sp1 and Sp3 that specifically binds to the Sp1 motif present in the FasL gene, and overexpression of Sp1 but not Sp3 leads to a further increase of transcription from the FasL promoter-enhancer region. The data presented demonstrates that constitutive FasL gene transcription in Sertoli cells is regulated by Sp1. In addition, it is shown that basal FasL expression in Jurkat T cells is also controlled by Sp1 and this is in contrast to induced FasL expression, which is NFAT-dependent.

Animals↗

PubMed1999

Prostaglandin-endoperoxide H synthase-2 expression in human thyroid epithelium. Evidence for constitutive expression in vivo and in cultured KAT-50 cells.

Prostaglandin-endoperoxide H synthase (PGHS) (EC 1.14.99.1) expression was examined in human thyroid tissue and in KAT-50, a well differentiated human thyroid epithelial cell line. PGHS-1 is found constitutively expressed in most healthy tissues, whereas PGHS-2 is highly inducible and currently thought to be expressed, with few exceptions, only in diseased tissues. Surprisingly, PGHS-2 mRNA and protein were easily detected in normal thyroid tissue. KAT-50 cells express high levels of constitutive PGHS-2 mRNA and protein under basal culture conditions. Compounds usually associated with PGHS-2 induction, including interleukin-1beta (IL-1beta), phorbol 12-myristate 13-acetate, and serum transiently down-regulated PGHS-2 expression. Human PGHS-2 promoter constructs (-1840/+123 and -831/+123) fused to a luciferase reporter and transfected into untreated KAT-50 cells exhibited substantial activity. NS-398, a highly selective inhibitor of PGHS-2 could inhibit substantial basal prostaglandin E2 production. Exogenous IL-1 receptor antagonist or IL-1alpha neutralizing antibodies could attenuate constitutive PGHS-2 expression in KAT-50 cells, suggesting that endogenous IL-1alpha synthesis was driving PGHS-2 expression. Our findings suggest that normal thyroid epithelium expresses high constitutive levels of PGHS-2 in situ and in vitro and this enzyme is active in the generation of prostaglandin E2. Thus, unprovoked PGHS-2 expression might be considerably more widespread in healthy tissues than is currently believed.

Cell Line↗

PubMed1999

The N terminus of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor is necessary for high affinity chemokine binding but not for constitutive activity.

Kaposi's sarcoma-associated herpesvirus (KSHV) contains a gene encoding a G protein-coupled receptor (KSHV-GPCR) that is homologous to mammalian chemokine receptors. KSHV-GPCR signals constitutively (in an agonist-independent manner) via the phosphoinositide-inositol 1,4,5-trisphosphate pathway. Because it has been proposed that the N terminus (N-TERM) of other GPCRs may act as tethered agonists, we determined whether the N-TERM of KSHV-GPCR is necessary for constitutive signaling activity or ligand binding, or both. We show that replacement of the entire N-TERM of KSHV-GPCR with those of two other GPCRs, deletion of residues within the N-TERM, and disruption of a putative disulfide bond that may hold the N-TERM in close proximity to extracellular loop 3 do not affect constitutive signaling activity but decrease chemokine binding. There were differences in the effects of mutation of the N-TERM on binding of the chemokines growth-related oncogene alpha, which is an agonist, and interferon-gamma-inducible protein-10, which is an inverse agonist. The effects on chemokine binding were accompanied by changes in chemokine regulation of KSHV-GPCR signaling. We conclude that the N-TERM is not necessary for constitutive KSHV-GPCR signaling, i.e. the N-TERM is not a tethered agonist, but plays a crucial role in binding of chemokine ligands and of chemokine regulation of KSHV-GPCR signaling.

Animals↗

PubMed2005

Essential role of IkappaB kinase alpha in the constitutive processing of NF-kappaB2 p100.

Processing of NF-kappaB2 precursor protein p100 to generate p52 is tightly controlled, which is important for proper function of NF-kappaB. Accordingly, constitutive processing of p100, caused by the loss of its C-terminal processing inhibitory domain due to nfkappab2 gene rearrangements, is associated with the development of various lymphomas and leukemia. In contrast to the physiological processing of p100 triggered by NF-kappaB-inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha), which requires the E3 ligase, beta-transducin repeat-containing protein (beta-TrCP), and occurs only in the cytoplasm, the constitutive processing of p100 is independent of beta-TrCP but rather is regulated by the nuclear shuttling of p100. Here, we show that constitutive processing of p100 also requires IKKalpha, but not IKKbeta (IkappaB kinase beta) or IKKgamma (IkappaB kinase gamma). It seems that NIK is also dispensable for this pathogenic processing of p100. These results demonstrate a general role of IKKalpha in p100 processing under both physiological and pathogenic conditions. Additionally, we find that IKKalpha is not required for the nuclear translocation of p100. Thus, these results also indicate that p100 nuclear translocation is not sufficient for the constitutive processing of p100.

Active Transport, Cell Nucleus↗

PubMed2006

Hyaluronan constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells.

Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-beta, PDGFR-beta, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-beta and PDGFR-beta require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-beta and PDGFR-beta are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation.

Breast Neoplasms↗

PubMed2000

Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells.

Apoptosis of cardiac muscle cells contributes to the development of cardiomyopathy. Recent studies showed that insulin-like growth factor I (IGF-I) inhibits apoptosis of cardiac muscle cells and improves myocardial function in experimental heart failure. This study was carried out to elucidate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the anti-apoptotic actions of IGF-I in cardiomyocytes and to explore whether expression of constitutively active PI 3-kinase can inhibit apoptosis in cardiomyocytes. Apoptosis of primary cardiomyocytes was induced by doxorubicin treatment and serum withdrawal. Transduction of cardiomyocytes with constitutively active PI 3-kinase specifically lead to serine phosphorylation of Akt, whereas phosphorylation of IGF-I receptor, IRS1/2 and p44/42 mitogen-activated protein kinase were not increased. In the cardiomyocytes transduced with constitutively active PI 3-kinase, activation of the pro-apoptotic caspase 3 was attenuated and fragmentation of DNA was reduced. Preincubating cells with PI 3-kinase inhibitor LY294002 was associated with loss of anti-apoptotic actions of IGF-I and PI 3-kinase. Neither IGF-I nor constitutively active PI 3-kinase lead to serine phosphorylation of Bad, suggesting that the anti-apoptotic effects of PI 3-kinase are not mediated through Bad phosphorylation in cardiac muscle cells. To determine whether activation of caspase 3 is sufficient to induce apoptosis in cardiomyocytes, an engineered TAT-caspase 3 protein was introduced to cardiomyocytes. Significant reduction of cell viability occurred in the cardiomyocytes transduced with active caspase 3, indicating that activation of caspase 3 is sufficient to cause cardiomyocyte death. These findings indicate the existence of an IGF-I receptor-PI 3-kinase-caspase 3 pathway in cardiomyocytes that plays an important role in the anti-apoptotic actions of IGF-I in heart. Moreover, these data suggest that modulation of PI 3-kinase activities may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in cardiomyopathy.

Animals↗

PubMed2001

Molecular basis for the constitutive activity of estrogen-related receptor alpha-1.

Some orphan nuclear receptors, including estrogen-related receptor alpha-1 (ERRalpha-1), can activate gene transcription in a constitutive manner. Little is known about the molecular basis of the constitutive activity of these receptors. Our results from site-directed mutagenesis experiments have revealed that Phe-329 (analogous to Ala-350 in estrogen receptor alpha (ERalpha)) is responsible for the constitutive activity of ERRalpha-1. The ERRalpha-1 mutant F329A lost the transactivation activity and acted as a dominant negative mutant. The mammalian cell transfection experiments revealed that the ERRalpha-1 mutant F329A, like wild-type ERalpha, recognized toxaphene (an organochlorine pesticide) as an agonist. This compound was previously shown to be an antagonist of wild-type ERRalpha-1. On the other hand, like wild-type ERRalpha-1, the ERalpha mutant A350F was found to be constitutively active (as demonstrated by mammalian cell transfection and yeast two-hybrid assays). These results indicate that Phe-329 in ERRalpha-1 and Ala-350 in ERalpha play important roles in both ligand binding and transactivation function.

Alanine↗

PubMed2001

Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.

Animals↗