Evidence for a Na+/Ca2+ exchanger in neuroblastoma x glioma hybrid NG108-15 cells.
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ABSTRACT:
SUBMITTER: Hsu LS
PROVIDER: S-EPMC1135752 | biostudies-other | 1995 Jul
REPOSITORIES: biostudies-other
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Project description:Clones of neuroblastoma x glioma hybrid, NH108-15, cells expressing differing levels of the human beta 2 adrenoceptor were isolated. Two clones were examined in detail, beta N22 which expressed some 4000 fmol/mg of membrane protein and clone beta N17 which expressed approx. 300 fmol/mg of membrane protein of the receptor. In beta N22 cells 'basal' adenylate cyclase activity measured in the presence of Mg2+ was significantly greater than that in wild-type NG108-15 or beta N17 cells. Both isoprenaline and iloprost were able to stimulate adenylate cyclase activity in each of beta N22 and beta N17 membranes. However, the EC50 for isoprenaline stimulation of adenylate cyclase in membranes of beta N22 cells (6 nM) was significantly lower than that in membranes of beta N17 cells (80 nM), whereas the EC50 for iloprost stimulation of adenylate cyclase (approx. 25 nM) was the same in the two clones and in parental NG108-15 cells. The high basal adenylate cyclase activity of beta N22 cell membranes was not a reflection of higher levels of expression of the adenylate cyclase catalytic unit, as adenylate cyclase activity measured in the presence of Mn2+ was equivalent in membranes of each of wild-type NG108-15 cells and clones beta N22 and beta N17. Basal adenylate cyclase activity measured in the presence of Mg2+ in clone beta N22 was significantly reduced, however, by the beta-receptor antagonist propranolol, whereas this agent was without effect on basal adenylate cyclase activity in membranes of wild-type NG108-15 cells. These data indicate that the elevated basal adenylate cyclase cascade in NG108-15 cells expressing high levels of the beta 2 adrenoceptor represents empty receptor activation of the signalling cascade.
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Project description:Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.
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Project description:Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.
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Project description:Regulation of ion-transport in the Na(+)/Ca(2+) exchanger (NCX) occurs via its cytoplasmic Ca(2+)-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca(2+)-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca(2+)-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca(2+). Striking differences in the electrostatic potential between the Ca(2+)-bound and -free forms strongly suggest that Ca(2+)-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C(2)-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca(2+)-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca(2+) is influenced by the closely located cassette exons. We further propose that Ca(2+)-binding to CBD2 induces a second electrostatic switch, required to alleviate Na(+)-dependent inactivation of Na(+)/Ca(2+) exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.
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