Why are A(2B) receptors low-affinity adenosine receptors? Mutation of Asn273 to Tyr increases affinity of human A2B receptor for 2-(1-hexynyl) adenosine

Adenosine A(2B) receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despit e numerous synthetic efforts, 5'-N-ethylcarboxamidoadenosine (NECA) still i s the reference agonist, albeit nonselective for this receptor. In our sear ch for higher affinity agonists, we developed decision schemes to select am ino acids for mutation to the corresponding residues in the most homologous , higher affinity, human A(2A) receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second sch eme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the aff inity for NECA, (R)-N-6(phenylisopropyl) adenosine (R-PIA), and 2Cl Ado. Th e affinity of the N273Y mutant for NECA and R-PIA and for the antagonists x anthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. Ho wever, this mutant had a slightly increased affinity for a 2-substituted ad enosine derivative, CGS21680. This prompted us to investigate other 2-subst ituted adenosines, with selectivity and high affinity for A(2A) receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl) adenosine had submicromolar affinity for the N273Y rec eptor, 0.18 +/- 0.10 muM, with a 61-fold affinity gain over the wt receptor . In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 +/- 0.5 muM for the wt receptor. The high affinity of (S)-PHP adenosine fo r the wt receptor suggests that further modifications at the 2- position ma y yield agonists with even higher affinity for A(2B) receptors.