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

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Frang H, Cockcroft V, Karskela T, Scheinin M, Marjamaki A

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

J Biol Chem. 2001 Aug 17;276(33):31279-84. Epub 2001 Jun 6.

PubMed ID

11395517 [ View in PubMed

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Abstract

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.

DrugBank Data that Cites this Article

Drugs

Phenoxybenzamine

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Phenoxybenzamine	Alpha-2A adrenergic receptor	Protein	Humans	Yes	Antagonist	Details
Phenoxybenzamine	Alpha-2B adrenergic receptor	Protein	Humans	Unknown	Antagonist	Details
Phenoxybenzamine	Alpha-2C adrenergic receptor	Protein	Humans	Unknown	Antagonist	Details
Phenoxybenzamine	Beta-2 adrenergic receptor	Protein	Humans	Unknown	Not Available	Details