Recruitment of the cross-linked opsonic receptor CD32A (FcgammaRIIA) to high-density detergent-resistant membrane domains in human neutrophils.
We have previously shown that CD32A (or FcgammaRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-beta-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-beta-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.
Rollet-Labelle
Emmanuelle
E
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL and Department of Medicine, Faculty of Medicine, Laval University, Sainte-Foy, QC, Canada G1V 4G2. emmanuelle.rollet@crchul.ulaval.ca
Marois
Sébastien
S
Barbeau
Kathy
K
Malawista
Stephen E
SE
Naccache
Paul H
PH
D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry; D003594:Cytoplasmic Granules / Q000737:chemistry; D003902:Detergents / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D011743:Pyrimidines / Q000494:pharmacology; D017452:Receptors, IgG / Q000737:chemistry* / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D000072377:Syk Kinase; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019061:src-Family Kinases / Q000737:chemistry
C412373:AG 1879; D015703:Antigens, CD; D003432:Cross-Linking Reagents; D003902:Detergents; D004792:Enzyme Precursors; C080568:Fc gamma receptor IIA; D047908:Intracellular Signaling Peptides and Proteins; D011743:Pyrimidines; D017452:Receptors, IgG; D011971:Receptors, Immunologic; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C091196:opsonin receptor; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D019061:src-Family Kinases