Phenoxybenzamine Binding Reveals the Helical Orientation of the Third Transmembrane Domain of Adrenergic Receptors

Abstract

Phenoxybenzamine (PB), a classical α-adrenergic antagonist, binds irreversibly to the α-adrenergic receptors (ARs). Amino acid sequence alignments and the predicted helical arrangement of the seven transmembrane (TM) domains suggested an accessible cysteine residue in transmembrane 3 of the α2-ARs, in position C3.36 (in subtypes A, B, and C corresponding to amino acid residue numbers 117/96/135, respectively), as a possible site for the PB interaction. Irreversible binding of PB to recombinant human α2-ARs (90 nM, 30 min) reduced the ligand binding capacity of α2-, α2B-, and α 2C-AR by 81, 96, and 77%. When the TM3 cysteine, Cys117, of α2A-AR was mutated to valine (α2A-C117V), the receptor became resistant to PB (inactivation, 10%). The β2-AR contains a valine in this position (V3.36; position number 117) and a cysteine in the preceding position (Cys116) and was not inactivated by PB (10 μM, 30 min) (inactivation 26%). The helical orientation of TM3 was tested by exchanging the amino acids at positions 116 and 117 of the α2A-AR and β2-AR. The α2A-F116C/C117V mutant was resistant to PB (inactivation, 7%), whereas β2-V117C was irreversibly inactivated (inactivation, 93%), confirming that position 3.36 is exposed to receptor ligands, and position 3.35 is not exposed in the binding pocket.