Probing the binding site of the A1 adenosine receptor reengineered for orthogonal recognition by tailored nucleosides

His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3‘ position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3‘-ureido, but not 3‘-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2‘- and 3‘-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3‘-ureido-3‘-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3‘-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2‘- and 3‘-ureid...