A1 Refereed original research article in a scientific journal

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




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

PublisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Publication year2001

JournalJournal of Biological Chemistry

Journal name in sourceJOURNAL OF BIOLOGICAL CHEMISTRY

Journal acronymJ BIOL CHEM

Volume276

Issue33

First page 31279

Last page31284

Number of pages6

ISSN0021-9258

DOIhttps://doi.org/10.1074/jbc.M104167200(external)

Web address http://www.jbc.org/content/276/33/31279.full(external)


Abstract
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.



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