Stable expression of truncated inositol 1,4,5-trisphosphate receptor subunits in 3T3 fibroblasts. Coordinate signaling changes and differential suppression of cell growth and transformation.
Stable clones of NIH 3T3 fibroblasts transfected with the cDNA of either the wild-type or deletion forms of the rat type I (or cerebellar) inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) were investigated. The delta form, missing the NH2-terminal sequence that includes the IP3-binding site, is expected to be still assembled with wild-type subunits to yield a tetrameric Ca2+ channel across the endoplasmic reticulum membrane; the s form, missing the membrane-spanning sequences, is expected to remain as a soluble monomer in the cytosol. With respect to control clones transfected with the vector only, the synthesis fo IP3Rs was markedly stimulated in the receptor-transfected clones. The mass accumulation, however, was increased only moderately (deletion forms = 15-30% of the endogenous IP3R), apparently because of a compensatory increase in receptor turnover. Coordinate changes in IP3 generation and Ca2+ release were revealed in the delta clones by experiments in both intact and permeabilized cells. In these clones, the IP3R was more sensitive to IP3, and IP3 generation at the ATP P2u surface receptor was decreased. This latter effect was due neither to a defect in G protein coupling nor to changes in phospholipase C expression, but to down-regulation of the P2u receptor. In the cells expressing the s- and delta-IP3R subunits, no differences with respect to the controls were observed in epidermal growth factor-induced DNA synthesis, whereas long-term growth stimulated by serum was reduced. Even more marked, especially in the delta clones (-90%), was the inhibition of cell transformation induced by autocrine stimulation with transforming growth factor alpha of the overexpressed epidermal growth factor receptors or by other growth factor receptors and oncogenes (platelet-derived growth factor/platelet-derived growth factor receptor beta, HER2/neu, and v-erbB). These effects appear not to be connected to the signaling processes mediated by tyrosine phosphorylation since the latter was unchanged in the delta clones. These results demonstrate for the first time (a) that the changes in cellular homeostasis directly induced by deleted IP3R subunits (increased receptor synthesis and increased IP3R sensitivity) are largely compensated by indirect coordinate changes apparently aimed to keep near normal the signaling properties of the cells; (b) that modulation of intracellular Ca2+ channels induces profound consequences that differentially affect growth and oncogenesis; and (c) that IP3Rs and the Ca2+ stores are important cross-roads of intracellular signaling pathways.
Department of Molecular Biology, Max-Planck-Institut fur Biochemie, Martinsried, Federal Republic of Germany.
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GA
Fischer
E
E
Clementi
M
M
Raichman
T
T
Südhof
A
A
Ullrich
J
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Meldolesi
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division; D002531:Cerebellum / Q000737:chemistry; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D051379:Mice; D010766:Phosphorylation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D016211:Transforming Growth Factor alpha / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D066246:ErbB Receptors; D002118:Calcium