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The alpha 1-adrenergic receptors (alpha 1-AR) belong to the G-protein coupled seven-transmembrane biogenic amine receptor family. Three subtypes have been successfully cloned in the alpha 1-adrenergic receptor family, and they share 50% identical amino acid sequences and 70% similarity. We have constructed seven chimeric receptors of the alpha 1A-AR. Each of the chimeras contains alpha 1D-subtype amino acid sequences within the membrane-spanning domains. Comparisons of ligand affinities with these chimeras has provided information on the importance of certain amino acid residues in determining receptor subtype specificity in the alpha 1A- and alpha 1D-ARs. With ligands in the dihydropyridine series, the niguldipine analog 1 was found to have respective pKi's of 9.32 +/- 0.17 for alpha 1A-AR; 6.84 +/- 0.24 for alpha 1D-AR; and 6.76 +/- 0.28 for alpha 1A/D(TM2), respectively. This trend was also exhibited by two other niguldipine analogs, 2 and 3, which had similar pKi's toward alpha 1D-AR and alpha 1A/D(TM2). This subtype selectivity was also maintained in the piperdine derivative, 4, and alpha 1A-AR selective ligand, which showed the same parallel trends in binding affinities with alpha 1A-AR and the six chimeras as the niguldipine analogs. Since in considering the second membrane-spanning domain, the alpha 1A- and alpha 1D-ARs only differ at positions 76, 77, 85, and 86, we were able to show through mutational studies that phenylalanine 86 is solely responsible for the selectivity found in the chimeric receptor alpha 1A/D(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the amino acid at position 86 in the final turn toward the extracellular region. This is four helical turns above aspartic acid-79, a conserved amino acid in the second membrane-spanning domain. This is the first report that suggests a significant involvement of the second membrane-spanning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.

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9,1 1-Dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15a/3-w-tetranor-TXA2 (I-PTA-OH), a recently synthesized thromboxane (TX) A2/prostaglandin (PG) H2 receptor antagonist, was shown to be a competitive antagonist of human platelet aggregation induced by the stable endoperoxide analog U46619. This antagonism was due to competitive blockade of the platelet TXA2/PGH2 receptor since I-PTA-OH did not antagonize the first phase of ADP-induced aggregation which is TXA2/PGH2 independent, nor did it inhibit TXA2 synthesis. In addition, analysis of dose-response curves to U46619 (0.1 to 40 ,M) in the presence of increasing concentrations of I-PTA-OH (0.5 to 10 ,M) showed that I-PTA-OH produced a parallel rightward shift of the dose-response curve. Further analysis of the data in the form of a Schild plot yielded a straight line with a slope (m = 1.03) not significantly different from -1. These results are consistent with the notion that I-PTA-OH acts as a competitive antagonist of the TXA2/PGH2 receptor. Circulation 72, No. 6, 1202No. 6, -1207No. 6, , 1985 SINCE THE DISCOVERY of the prostaglandin (PG) endoperoxides PGG2 and PGH2 -3 and thromboxane (TX) A2,4 their actions on platelets and vascular smooth muscle have been the subject of numerous investigations. Initial studies were descriptive in nature, characterizing PGG2, PGH2, and TXA2 as potent vasoconstrictors2-7 and stimulators of platelet aggregation.3' Since the development of TX synthetase inhibitors, the potential roles of the PG endoperoxides and TXA2 in platelet aggregation'0 and in cardiovascular diseases`' have been studied. It was anticipated that this group of compounds could provide new therapeutic approaches for the treatment of a variety of cardiovascular diseases."More recently, the development of stable analogs of the PG endoperoxides and TXA2, which act as either mimics or antagonists of the actions of the naturally occurring eicosanoids, has provided tools for fundamental investigations into the mechanisms of action of these compounds and the nature of their receptors. Studies with the receptor antagonists have provided

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