Photoaffinity labeling of the leukotriene D4 receptor in guinea pig lung.
The leukotriene (LT)D4 receptor has been defined as a G-protein-coupled receptor. In order to characterize this receptor, an iodinated, photoactivatable azido derivative of LTD4 (125I-azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of 125I-azido-LTD4 specific binding to guinea pig lung membranes were directly comparable to those of [3H]LTD4 specific binding to this tissue. 125I-Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. 125I-Azido-LTD4 specific binding was also strongly inhibited by the nonhydrolyzable GTP analog, GTP gamma S, with ATP gamma S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G-protein. The cysteinyl leukotrienes competed for 125I-azido-LTD4 specific binding to guinea pig lung membranes with the following rank order of potency: LTD4 > LTE4 > LTC4, while the non-cysteinyl LTB4 was virtually inactive. Two structurally different LTD4 receptor antagonists, MK-571 and ICI 204,219, also competed for 125I-azido-LTD4 specific binding with nanomolar potency, whereas the leukotriene synthesis inhibitor, MK-886, was 10,000-fold less active. These data are in agreement with 125I-azido-LTD4 binding specifically to a G-protein-coupled LTD4 receptor. Photolysis of 125I-azido-LTD4 under equilibrium binding conditions resulted in the selective radiolabeling of a 45-kDa guinea pig lung membrane protein. The photolabeling of this 45-kDa protein was saturable, modulated by cations and inhibited by nucleotide analogs in an analogous way to 125I-azido-LTD4 specific binding. In addition, the photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the 125I-azido-LTD4 binding assay. It is proposed, therefore, that this novel 45-kDa protein is the guinea pig lung LTD4 receptor.
Department of Pharmacology, Merck Frosst Center for Therapeutic Research, Pointe Claire, Dorval, Quebec, Canada.
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Zamboni
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D017998:Leukotriene D4 / Q000031:analogs & derivatives*; D008024:Ligands; D008168:Lung / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D011971:Receptors, Immunologic / Q000032:analysis* / Q000037:antagonists & inhibitors; D018077:Receptors, Leukotriene; D013189:SRS-A / Q000031:analogs & derivatives / Q000378:metabolism*
D000345:Affinity Labels; D008024:Ligands; D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; D013189:SRS-A; C080349:iodinated azido-leukotriene D4; D017998:Leukotriene D4