Abstract
In order to delineate structural-functional relationships of the mast cell receptor for IgE (Fc epsilon RI) by molecular-genetic analysis, a transfectable cell must be identified which resembles mast cells except for being deficient in receptors. We have found that the well known murine mastocytoma P815 is suitable. These cells express no Fc epsilon RI, lack mRNA for the alpha and beta subunits of the receptor, but contain some mRNA for gamma chains. After transfection with the cDNA for each of the subunits, stable clones could be isolated which expressed several hundred thousand normal Fc epsilon RI and synthesized large amounts of mRNA for alpha, beta, and gamma, the last at 3-fold higher levels than in the untransfected cells. Aggregation of the transfected receptors led to opening of presumptive calcium channels and to activation of phospholipase C, phospholipase A2, and protein kinase C. The kinetics and other characteristics of the signals were similar to those observed after stimulation of the rat tumor mast cells from which the receptor genetic material had been derived but were smaller in magnitude. These weaker signals most likely result from an overall reduced reactivity exhibited by the P815 cells since stimulation by other ligands led to weaker or even no responses. The cells failed to degranulate after either receptor aggregation or reaction with ionophores with or without phorbol ester. Both the transfected and untransfected P815 cells express Fc receptors for IgG (Fc gamma RII) which, interestingly, independently triggered similar responses despite their apparently simpler subunit structure.
Highlights
National Institute National Heart, and of Arthritis and Lung, and Blood Institute, In order to delineate structural-functional relationships of the mast cell receptor for IgE (Fc.RI) by molecular-genetic analysis, a transfectable cell must be identified which resembles mast cells except for being deficient in receptors
We have introduced cDNAs coding for each of the subunits of the rat high affinity IgE receptor into P815 HTR cells, a P815 subline selected to be highly transfectable by calcium phosphate precipitation (6)
We tested the transfected P815 cells for evidence of degranulation initiated by aggregation of the receptor, but they failed to respond in this way. That this reflects a defect late in the pathway is suggested by the similar failure of ionophore plus. Upon aggregation of their surface-bound receptors for IgE, the transfected P815 cells responded with a variety of biochemical changes that resemble those observed when the endogenous receptors on rat basophilic leukemia (RBL) and related cells are stimulated (2, 3)
Summary
E; Fc,RII, low affinity receptor for immunoglobulin G: DMEM, Dulbecco’s modified eagle’s medium; DNP, 2,4-. Many early biochemical events have been described which follow aggregation of the receptors (2, 3), none of the molecules that are responsible for these phenomena has been unambiguously identified. We plan to investigate which of these parts of the receptor are necessary to initiate one or more of the early biochemical signals Such information could be used to identify critical “postreceptor” molecules (see “Discussion”). Upon aggregation, even the wild-type transfected receptors on such cells failed to initiate some of the principal early biochemical events, such as hydrolysis of phosphoinositides and a rise in intracellular Ca’+, which are induced by the endogenous receptors on mast cells.. Functional IgE receptor complexes can be formed after transfecting receptor cDNAs into the P815 cells This cell line appears to be a suitable host for future work employing mutant receptors
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