Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases.
Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.
Field
K A
KA
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Apgar
J R
JR
Hong-Geller
E
E
Siraganian
R P
RP
Baird
B
B
Holowka
D
D
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000235:genetics / Q000648:ultrastructure; D004789:Enzyme Activation; D015151:Immunoblotting; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D050356:Lipid Metabolism; D008407:Mast Cells / Q000166:cytology / Q000502:physiology*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000502:physiology*; D015398:Signal Transduction; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D017455:Receptors, IgE; D019000:Phosphotyrosine; D020741:rho GTP-Binding Proteins; D002118:Calcium