Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.
A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.
Goldman
D W
DW
Department of Medicine, University of California Medical Center, San Francisco 94143-0724.
Gifford
L A
LA
Young
R N
RN
Marotti
T
T
Cheung
M K
MK
Goetzl
E J
EJ
D000345:Affinity Labels; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003432:Cross-Linking Reagents; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007975:Leukotriene B4 / Q000378:metabolism*; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D018102:Receptors, Leukotriene B4
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4; D006160:Guanosine Triphosphate