184 Biochimica et Biophysica Acta. 1012 (1989) 184-190 Elsevter BBAMCR 12477 Photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor Dale E. Mais 1, Christiane Yoakim 3, Yvan Guindon 3, John W. Gillard 3, Joshua Rokach 3 and Perry V. Halushka 1,2 I Departments of Cell and Molecular Pharmacologyand Experimental Therapeutics and " Medicine. Medical University of South Carolina. Charleston. SC (U.S,A.) and ~ Merck Frosst Canada Inc.. Dorval, Quebec (Canada) (Received 27 December 1988) Key words: Receptor; Thromboxane A 2/prostaglandin H 2 receptor: Photoaffinity labeling; 13-Azapinane; Thromboxane A 2 (Human platelet) The synthesis, binding and photoincorporation of a thromboxane Az/prostaglandin H 2 (TXA2/PGH 2) analog (9,11-dimethylmethano-I 1,12-methano-16-(3-112s lliodo-4-azidophenyl)-13,14-dihydro-13-aza- 15afl-0J-tetranor-TXA 2) ([ ns llPTA-Azido) to washed human platelets was characterized. Kinetic analysis of the binding of [t~ IIPTA.Azido at 300C yielded a kt of 1.83 • 107 M -t. min-i and k -t of 0.195 rain-l, K d - k_dkt = 11 nM. Incubation of washed human platelets with ll2SllPTA-Azido followed by photolysis resulted in the radiolabelling of a number of platelet proteins as assessed by SDS-PAGE autoradiography. The radiolabelling of three ,of these protein bands could be either uniformly blocked or reduced with a series of structurally dissimilar TXA 2/PGH 2 receptor antagonists or agonists and corresponded to proteins with a molecular mass of 43, 39 and 27 kDa. In addition, the incorporation of [~2sllPTA-Azido into the three proteins was stereoselectively blocked by a pair of optically active stereoisomers that are TXA,/PGH 2 receptor antagonists. Two-dimensional gel electrophoresis indicated that the 43 kDa protein possessed a pl value of 5.6 and that the 27 kDa protein exists in at least three isoforms with pl values of 4.9, 5.1 and 5.3. The labelling pattern was not altered by a mixture of proteinase inhibitors. The data suggest that one or more of these specifically radiolabelled proteins may represent the human platelet TXA2/PGH 2 receptor. Introduction Since 1975 when the structure of unstable thrombox- ane A 2 (TXA2) was suggested [1], a variety of stable analogs, both mimetics and antagonists, have been pre- Abbreviations: [12s llPTA-Azido. 9,11-dimethylmethano-I 1,12- methano-16-(3-[ las l]iodo,4.azidophenyl)-I3,14-dihydro-I 3-aza-15af- ~-tetranorthromboxane A 2; I-PTA.OH, 9,1 l-dimethylmethano-I 1,12- methanol- 16-(3-iodo-4-hydroxyphenyl)- 13,14-dihydro-I 3-aza-I 5aft- ~-tetranorthromboxane A2; SQ26655, [IS-(la,2f(5Z),3a(IE,3S), 4 a )1-7-[3-( 3-hydroxy- 1-octenyl)-7-oxbicyclo-[2.2,1 ]hept-2-yl]-5-hepten- oic acid: SQ29548, [1S-(la,2f(5Z),3f,4a)].7-{3.[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl}-5-heptenoic acid; U46619, 15-(S)-hydro×y-lla,9a-(epoxymethano)prosta.(SZ, 13E)dienoic acid; L-657925( - ) or L657926( + ), ( - )- or ( + )-9-p- chlorobenzyl-6-fluoro-l,2,3A-tetrahydrocarbazol-l-yl acetic acid: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophore- sis: TXA2/PGH2, thromboxane A2/prostaglandin H2; PMSF, phenylmethylsulfonyl fluoride. Correspondence: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, U.S.A. pared and their pharmacology examined [2]. One of the pharmacologic effects of TXA 2, prostaglandin H 2 (PGH2) and their stable agonists is activation of plate- lets which is mediated through specific receptors [2]. Recently, radioligand binding studies have begun to characterize a putative TXA 2/PGH 2 receptor in washed human platelets and membranes prepared from human platelets [5-9]. Furthermore, this putative receptor has been solubilized from human platelet membranes in an active form [10] and a molecular mass of 140 kDa was determined based on its hydrodynamic properties [11]. Photoaffinity ligands have been used in a variety of ways to characterize a broad spectrum of receptors. Included in these uses in combination with SDS-PAGE is determination of the molecular mass of the receptors. Two previous reports have described photoaffinity ligands for the human platelet TXA2/PGH 2 receptor [12,13]. In the first of these reports, an aryl diazonium salt was photolyzed in the presence of human platelet membranes and irreversible incorporation of the ligand into the TXA2/PGH 2 receptor was shown [12]. This incorporation could be inhibited by co-incubation with 0167-4781/89/$03,50 .~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)