High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT.
In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.
Wilson
B S
BS
Department of Pathology and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. bwilson@salud.unm.edu
Pfeiffer
J R
JR
Surviladze
Z
Z
Gaudet
E A
EA
Oliver
J M
JM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000737:chemistry; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D015471:Leukemia, Basophilic, Acute; D008213:Lymphocyte Activation; D008407:Mast Cells / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000378:metabolism; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000032:analysis* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000032:analysis / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D010750:Phosphoproteins; D017455:Receptors, IgE; D010738:Type C Phospholipases; C490893:Gab2 protein, rat