Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receptors.
Phenoxybenzamine (PB), a classical alpha-adrenergic antagonist, binds irreversibly to the alpha-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 alpha(2)-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 microm, 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.
Frang
H
H
Department of Pharmacology, University of Turku, FIN-20520 Turku and Orion Pharma, FIN-20101 Turku, Finland.
Cockcroft
V
V
Karskela
T
T
Scheinin
M
M
Marjamäki
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010643:Phenoxybenzamine / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011941:Receptors, Adrenergic / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014162:Transfection
D011941:Receptors, Adrenergic; D010643:Phenoxybenzamine