title: Constitutive activation of a phosphoinositidase C-linked G protein in murine fibroblasts decreases agonist-stimulated Ca2+ mobilization.
abstract: We compared Ca2+ signaling and inositol polyphosphate metabolism in NIH-3T3 cells stably transfected with cDNA encoding either the wild-type G protein G16 alpha subunit or a GTPase-deficient alpha 16 subunit (Q212L-alpha 16). Constitutive activation of phosphoinositidase C (PIC) in cells expressing Q212L-alpha 16 was demonstrated by 1) an increased basal level of [3H]inositol polyphosphates, 2) an enhanced rate of [3H]inositol polyphosphate accumulation in cells treated with 10 mM LiCl, and 3) an increased rate of incorporation of [3H]inositol into cell lipids. Q212L-alpha 16 cells had a diminished cell growth rate. Basal intracellular Ca2+ concentration was equivalent in Fura-2 acetoxymethyl ester-loaded Q212L-alpha 16 cells compared with controls; however, calcium release in Q212L-alpha 16 cells exposed to ionomycin, ATP (a G protein-linked agonist), or platelet-derived growth factor (a tyrosine kinase-linked agonist) was decreased. Permeabilized, 45Ca-loaded Q212L-alpha 16 cells released less 45Ca at each concentration of inositol-1,4,5-trisphosphate than did control cells. Accordingly, the total amount of inositol trisphosphate (IP3) receptor protein was decreased in Q212L-alpha 16 cells relative to controls. These data demonstrate that Q212L-alpha 16 cells maintain physiological levels of cytoplasmic calcium and partially loaded Ca2+ stores in the face of constitutively active PIC. This is accomplished, at least in part, by down-regulation of IP3 receptor number. Thus, diminution in cell growth rate in Q212L-alpha 16 cells seems to be attributable to a combination of at least two effects: a direct effect of PIC activation leading to partial depletion of Ca2+ stores and an indirect, adaptive response resulting in a decreased IP3 receptor number.
authors.affiliation: Calcium Regulation Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Lobaugh
authors.forename: B
authors.initials: B
authors.lastname: Eisfelder
authors.forename: K
authors.initials: K
authors.lastname: Gibson
authors.forename: G L
authors.initials: GL
authors.lastname: Johnson
authors.forename: J W
authors.initials: JW
authors.lastname: Putney
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D018021:Lithium Chloride / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D007294:Inositol; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000252:Calcium-Transporting ATPases; D018021:Lithium Chloride; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium