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A mutant fibroblast, 2A4b, was isolated from the Chinese hamster lung cell line CCL39 by a previously described selection (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390) for cells deficient in thrombin-induced signaling. Although the antiporter activation by thrombin in 2A4b is only approximately 60% that in CCL39, the stimulation by serum is not significantly impaired, indicating that the defect in 2A4b lies upstream of the antiporter in the signaling pathway. The addition of thrombin to serum-starved 2A4b cells causes blunted responses both in production of inositol phosphates and in the cytosolic [Ca2+] transient, particularly when no Ca2+ is added to the external medium. The in vitro inositol phospholipid-specific phospholipase C (PLC) activity of 2A4b cytosol plus membrane extracts exceeds that in CCL39. However, immunoblots with antibodies to PLC isozymes show that although the levels of PLC-delta 1, PLC-gamma 1, and PLC-beta 3 are at least as great as those in CCL39, the amount of PLC-beta 1 in 2A4b is markedly deficient (< or = 10%). PLC-beta 1 is found primarily in the nucleus and in non-nuclear membranes of CCL39 and is proportionately low in these subcellular locations of 2A4b. Thrombin activation of phospholipases D and A2 is impaired in 2A4b. We postulate that the deficiency in PLC-beta 1 causes defective targeting of protein kinase C-alpha to specific membrane sites, which may be required for activation of these downstream phospholipases.

We report here further characterization of the Chinese hamster lung fibroblast mutant D1-9b displaying elevated agonist-induced phosphatidylinositol (PI) turnover responses relative to CCL39, the parental cell line (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390). These differences in PI turnover responses are further enhanced by long term (24 h) pretreatment of cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Short term pretreatment (15 min) with PMA attenuates the agonist-induced PI turnover response of both wild type and mutant D1-9b cell lines to that of unstimulated controls, suggesting that PMA-induced desensitization responses in D1-9b are intact. Both cytosolic and membrane/particulate fractions prepared from mutant D1-9b have decreased phospholipase C (PLC) activities relative to wild type when assayed with either of four different PI/phosphatidylinositol 4,5-bisphosphate-mixed micelle/vesicle substrate combinations. Thermolability studies, Mono Q anion-exchange chromatography, and Western blot studies identified the source of cytosolic PLC deficiency in D1-9b as being due to the absence of PLC delta 1, one of at least two PLC isozymes previously shown to be in wild type CCL39 cytosol (Rath, H. M., Fee, J. A., Rhee, S. G., and Silbert, D. F. (1990) J. Biol. Chem. 265, 3080-3087). We also report here the presence of two peaks of PLC activities (i.e. peaks mA and mB) following Mono Q chromatography of wild type CCL39 membrane/particulate extracts; membrane/particulate extracts prepared from mutant D1-9b are missing the first of these two peaks of activities (peak mA). Immunoblot analysis confirms the presence of PLC beta 1, but not PLC gamma 1 or PLC delta 1, in peak mB. Comparisons are made between D1-9b and the previously characterized mutant D1-6b, which displays diminished agonist-induced PI turnover responses yet in vitro biochemical deficiencies similar to those of mutant D1-9b. Somatic cell hybridization experiments suggest that the defects found in mutant D1-9b are codominant relative to wild type phenotype and that mutant D1-9b belongs to the same genetic complementation group as mutant D1-6b. Possible explanations to explain these different agonist-induced responses in light of the two mutants similar in vitro biochemical deficiencies are addressed.