Phenylalanine in the Second Membrane-Spanning Domain of α_1A-Adrenergic Receptor Determines Subtype Selectivity of Dihydropyridine Antagonists

Abstract

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.

Phe-308 and Phe-312 in Transmembrane Domain 7 Are Major Sites of α1-Adrenergic Receptor Antagonist Binding

“…These consecutive residues (Gln-177, Ile-178, and Asn-179) are involved only in ␣ 1a -AR versus ␣ 1b -AR selectivity issues and then for only three ligands, phentolamine, WB4101, and partial effects on 5-methylurapidil (7). Another group has shown that mutagenesis of a phenylalanine residue (F86M) at the surface of TM2 in the ␣ 1a -AR accounts for the ␣ 1a versus ␣ 1d selectivity of dihydropyridine antagonists such as niguldipine (12). This aromatic residue is also modeled to be in the first helical turn of TM2.…”

“…A single phenylalanine residue about two turns into the TM7 domain of the ␣ 2 -AR (Phe-412) was shown to promote high-affinity binding of yohimbine (11). Mutagenesis of a phenylalanine residue (Phe-86) at the surface of TM2 in the ␣ 1a -AR accounts for the ␣ 1a versus ␣ 1d selectivity of dihydropyridine antagonists such as niguldipine (12). Furthermore, a phenylalanine residue (Phe-310 of the ␣ 1b -AR) in TM6 has been identified as being important not only for agonist binding but also for the binding of certain ␣ 1 -antagonists (13).…”

Mutational Analysis of the .ALPHA.1a-Adrenergic Receptor Binding Pocket of Antagonists by Radioligand Binding Assay

“…8,9,19) To verify the modeling data and the involvement of corresponding amino acids (Asp106 and Gln167) in the human a 1a -AR, these amino acids were mutated to alanine and phenylalanine. (Table 2).…”

Structure-Activity Relationships of Receptor Binding of 1,4-Dihydropyridine Derivatives

“…[31][32][33] SNAP 5540 is selective at the a 1a -adrenoceptor over a 1b and a 1d receptors and other ion channels and receptors. [34][35][36] In the preset study, compound 58 exhibited 64% inhibition of specific [ 3 H]glibenclamide binding. Thus, the binding activity of compounds 81, 84, 108 and 109 for K ATP channel was shown to be 1.6-3.8 times greater than that for 1,4-DHP receptors.…”