The second extracellular loop of α2A-adrenoceptors contributes to the binding of yohimbine analogues

Background and purpose: Rodent α2A-adrenoceptors bind the classical α2-antagonists yohimbine and rauwolscine with lower affinity than the human α2A-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 α2A-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding. Experimental approach: 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 α2A-adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays. Key results: Reciprocal effects on the binding of yohimbine and rauwolscine in human and mouse α2A-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 and implications: Positions 5.43, xl2.49 and xl2.51 are major determinants of the species preference for yohimbine and rauwolscine of the human versus mouse α2A-adrenoceptors. Residues at positions xl2.49 and xl2.51 determine the binding preference of RS-79948-197 for the human α2A-adrenoceptor. Thus, XL2 is involved in determining the species preferences of α2A-adrenoceptors of human and mouse for some antagonists. British Journal of Pharmacology (2007) 151, 1293–1304; doi:10.1038/sj.bjp.0707330