Restoration of Ca2+ influx and degranulation capacity of variant RBL-2H3 cells upon implantation of isolated cromolyn binding protein.
Recently, variants of the rat basophilic leukemia cells (RBL-2H3), deficient in their binding capacity for the antiallergic drug cromolyn but displaying unimpaired ability to bind IgE, were selected and cloned [Mazurek, N., Bashkin, P., Petrank, A. & Pecht, I. (1983) Nature (London) 303, 528-530]. Although the histamine content and the number of IgE receptors in these variants are similar to those of the parental cells, they cannot be stimulated immunologically to allow Ca(2+) influx and to degranulate. In contrast, the Ca(2+) ionophore A23187 causes these variants to degranulate, indicating that the mechanism distal to the Ca(2+) gating is intact in the variants. The cromolyn binding protein (CBP), present in the membranes of RBL-2H3 cells, has recently been isolated by affinity chromatography under nondenaturing conditions. In the current study we have used Sendaivirus envelopes as fusogenic carriers to implant the purified CBP into the membrane of variant basophils that were defective in it. This fusion leads to the restoration of Ca(2+) uptake and degranulation capacity of the variants after IgE-mediated stimulation. These restored activities seem to show a sigmoidal dependence on the amount of incorporated CBP. Saturation values comparable to those of the parental line are reached when the level of implanted CBP approaches its density on the latter line. The restored capacity is due to the implanted CBP, because the reinstated immunological response can be blocked by the inhibitory drug cromolyn and by monoclonal antibodies specific to CBP, both shown to prevent Ca(2+) uptake and degranulation in mast cells and parental RBL-2H3 cells. These results point out that CBP plays an important role in the Ca(2+) gating process resulting in degranulation.
D000818:Animals; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000276:immunology*; D002459:Cell Fusion; D002460:Cell Line; D014644:Genetic Variation; D007942:Leukemia, Experimental / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins; D051381:Rats; D011919:Rats, Inbred Strains
D002352:Carrier Proteins; D008565:Membrane Proteins; C039229:cromolyn binding protein, rat; D002118:Calcium