title: The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues.
abstract: OBJECTIVE
  Rodent alpha(2A)-adrenoceptors bind the classical alpha(2)-antagonists yohimbine and rauwolscine with lower affinity than the human alpha(2A)-adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of alpha(2A)-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding.
  
  
  METHODS
  Amino acids facing the binding cavity were identified using molecular models: side chains of residues 5.43 in TM5 and xl2.49 and xl2.51 in XL2 differ between the mouse and human receptors. Reciprocal mutations were made in mouse and human alpha(2A)-adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays.
  
  
  RESULTS
  Reciprocal effects on the binding of yohimbine and rauwolscine in human and mouse alpha(2A)-adrenoceptors were observed for mutations at 5.43, xl2.49 and xl2.51. The binding profile of RS-79948-197 was reversed only by the XL2 substitutions.
  
  
  CONCLUSIONS
  Positions 5.43, xl2.49 and xl2.51 are major determinants of the species preference for yohimbine and rauwolscine of the human versus mouse alpha(2A)-adrenoceptors. Residues at positions xl2.49 and xl2.51 determine the binding preference of RS-79948-197 for the human alpha(2A)-adrenoceptor. Thus, XL2 is involved in determining the species preferences of alpha(2A)-adrenoceptors of human and mouse for some antagonists.
authors.affiliation: Department of Pharmacology, Drug Development and Therapeutics, University of Turku, Itäinen Pitkäkatu 4B, FL-20520 Turku, Finland. jomila@utu.fi
authors.forename: J M M
authors.initials: JM
authors.lastname: Laurila
authors.forename: H
authors.initials: H
authors.lastname: Xhaard
authors.forename: J O
authors.initials: JO
authors.lastname: Ruuskanen
authors.forename: M J M
authors.initials: MJ
authors.lastname: Rantanen
authors.forename: H K
authors.initials: HK
authors.lastname: Karlsson
authors.forename: M S
authors.initials: MS
authors.lastname: Johnson
authors.forename: M
authors.initials: M
authors.lastname: Scheinin
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009287:Naphthyridines / Q000494:pharmacology; D011807:Quinolizines / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects* / Q000378:metabolism; D013045:Species Specificity; D015016:Yohimbine / Q000494:pharmacology*
chemical_list: C547768:ADRA2A protein, human; C547769:Adra2a protein, mouse; D000317:Adrenergic alpha-Antagonists; D007546:Isoquinolines; D008024:Ligands; D009287:Naphthyridines; D011807:Quinolizines; C104875:RS 79948-197; D018341:Receptors, Adrenergic, alpha-2; C054290:L 657743; D015016:Yohimbine