A role for amino acid residues in the third cytoplasmic loop in defining the ligand binding characteristics of the α(2D)-adrenergic receptor

In this report, 5 amino acid residues (aa) in the third cytoplasmic loop of the α(2D)-adrenergic receptor are identified which (individually or together) alter its ligand-binding characteristics. An important structural discrepancy exists in the third cytoplasmic loop of the α(2D)-ARs encoded by the rat cDNA and the rat gene - five aa are different. The newly identified bovine receptor as well as the mouse receptor contained the 5 aa identical to that encoded by the rat cDNA. Site-directed mutation of these residues to those of the rat gene encoded receptor resulted in alteration of binding characteristics: significant changes in the ability of the mutant receptor to bind to a number of agonists and antagonists were observed - ranging from a decrease by half in the case of oxymetazoline, to near total loss of binding in the case of prasozin. Thus, the mutant receptor was no longer pure α(2D)-AR. This indicated a hitherto unrealized role of the third cytoplasmic loop in defining the ligand-binding characteristics of the receptor, and also suggested that the rat gene sequence was most probably in error.