Transmembrane signaling in P815 mastocytoma cells by transfected IgE receptors.
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.
Miller
L
L
Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin, Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Alber
G
G
Varin-Blank
N
N
Ludowyke
R
R
Metzger
H
H
D000818:Animals; D000943:Antigens, Differentiation / Q000502:physiology; D000944:Antigens, Differentiation, B-Lymphocyte / Q000502:physiology*; D015152:Blotting, Northern; D002118:Calcium / Q000502:physiology; D015550:Cell Degranulation; D002772:Cholera Toxin / Q000494:pharmacology; D007073:Immunoglobulin E / Q000378:metabolism*; D015471:Leukemia, Basophilic, Acute; D046911:Macromolecular Substances; D008407:Mast Cells / Q000502:physiology*; D008970:Molecular Weight; D009218:Myosins / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011940:Receptor Aggregation; D011961:Receptors, Fc / Q000502:physiology*; D017455:Receptors, IgE; D017452:Receptors, IgG; D011994:Recombinant Proteins / Q000648:ultrastructure; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000943:Antigens, Differentiation; D000944:Antigens, Differentiation, B-Lymphocyte; D046911:Macromolecular Substances; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011961:Receptors, Fc; D017455:Receptors, IgE; D017452:Receptors, IgG; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D007073:Immunoglobulin E; D002772:Cholera Toxin; D011493:Protein Kinase C; D009218:Myosins; D002118:Calcium