Dianne Alewood

Subtype-selective noncompetitive or competitive inhibition of human α1-Adrenergic receptors by ρ-TIA

The 19-amino acid conopeptide (ρ-TIA) was shown previously to antagonize noncompetitively α1B-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human α1-AR subtypes (α1A, α1B, and α1D). Radioligand binding assays showed that ρ-TIA was 10-fold selective for human α1B-over α1A- and α1D-ARs. As observed with hamster α1B-ARs, ρ-TIA decreased the number of binding sites (Bmax) for human α1B-ARs without changing affinity (KD), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, ρ-TIA had opposite effects at human α1A-ARs and α1D-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. ρ-TIA reduced maximal NE-stimulated [3H]inositol phosphate formation in HEK293 cells expressing human α1B-ARs but competitively inhibited responses in cells expressing α1A- or α1D-ARs. Truncation mutants showed that the amino-terminal domains of α1B- or α1D-ARs are not involved in interaction with ρ-TIA. Alanine-scanning mutagenesis of ρ-TIA showed F18A had an increased selectivity for α1B-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at α1B-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus ρ-TIA noncompetitively inhibits α1B-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at α1B-ARs.