PHENYLALANINE IN THE 2ND-MEMBRANE-SPANNING DOMAIN OF ALPHA(1A)-ADRENERGIC RECEPTOR DETERMINES SUBTYPE SELECTIVITY OF DIHYDROPYRIDINE ANTAGONISTS

The alpha(1)-adrenergic receptors (alpha(1)-AR) belong to the G-protei n coupled seven-transmembrane biogenic amine receptor family, Three su btypes have been successfully cloned in the alpha(1)-adrenergic recept or family, and they share 50% identical amino acid sequences and 70% s imilarity. We have constructed seven chimeric receptors of the alpha(1 A)-AR Each of the chimeras contains alpha(1D)-subtype amino acid seque nces within the membrane-spanning domains, Comparisons of ligand affin ities with these chimeras has provided information on the importance o f certain amino acid residues in determining receptor subtype specific ity in the alpha(1A)- and alpha(1D)-ARs. With ligands in the dihydropy ridine series, the niguldipine analog 1 was found to have respective p K(i)'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 w as also exhibited by two other niguldipine analogs, 2 and 3, which had similar pK(i)'s toward alpha(1D)-AR and alpha(1A/D)(TM2) This subtype selectivity was also maintained in the piperdine derivative, 4, an al pha(1A)-AR selective ligand, which showed the same parallel trends in binding affinities with alpha(1A)-AR and the six chimeras as the nigul dipine analogs, Since in considering the second membrane-spanning doma in, 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 pheny lalanine 86 is solely responsible for the selectivity found in the chi meric receptor alpha(1A/D)(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the am ino acid at position 86 in the final turn toward the extracellular reg ion, This is four helical turns above aspartic acid-79, a conserved am ino acid in the second membrane-spanning domain, This is the first rep ort that suggests a significant involvement of the second membrane-spa nning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.