The second extracellular loop of [alpha]2A-adrenoceptors contributes to the binding of yohimbine analogues

BACKGROUND AND PURPOSE: 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. 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 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. KEY 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 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 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.