Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receptors

Phenoxybenzamine (PB), a classical a-adrenergic antagonist, binds irreversibly to the a-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 alpha (2)-ARs, in position C-3.36 (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 a2-ARs (90 nm, 30 min) reduced the ligand binding capacity of alpha (2A)-, alpha (2B)-, and alpha (2C)-AR by 81, 96, and 77%. When the TM3 cysteine, Cys(117), of alpha (2A)-AR was mutated to valine (alpha (2A)-C117V), the receptor became resistant to PB (inactivation, 10%). The beta (2)-AR contains a valine in this position (V-3.36; position number 117) and a cysteine in the preceding position (Cys(116)) and was not inactivated by PB (10 mum, 30 min) (inactivation 26%). The helical orientation of TM3 was tested by exchanging the amino acids at positions 116 and 117 of the alpha (2A)-AR and beta (2)-AR. The alpha (2A)-F116C/C117V mutant was resistant to PB (inactivation, 7%), whereas beta (2)-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.