SITE-DIRECTED MUTAGENESIS OF THE HUMAN A(1) ADENOSINE RECEPTOR - INFLUENCES OF ACIDIC AND HYDROXY RESIDUES IN THE FIRST 4 TRANSMEMBRANE DOMAINS ON LIGAND-BINDING

To provide new insights into ligand/A(1) adenosine receptor (A(1) AR) interactions, site-directed mutagenesis was used to test the role of s everal residues in the first four transmembrane (TM) domains of the hu man A(1) AR. Based on multiple sequence analysis of all known ARs, bot h acidic (glutamic acid and aspartic acid) and polar hydroxy (serine a nd threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, an d Ser6 and Ser23 (TM1) were identified in all A(1) ARs. To test the ro le of these residues, each was individually mutated to alanine. When A la6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, a ffinities for [H-3]2-chloro-N-6-cyclopentyladenosine (CCPA) and [H-3]1 ,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen f or the wild-type receptor. After conversion of Glu16 to Ala16, the aff inity for [H-3]CCPA and other agonists fell 10-100-fold, whereas the a ffinity for [H-3]DPCPX and other antagonists was not affected. After c onversion of Asp55 to Ala55, the affinity for [H-3]CCPA and other agon ists increased less than or equal to 100-fold, whereas the affinity fo r [H-3]D-PCPX and other antagonists was not affected. Studies of the A la55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [H-3]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A(1) AR immunoreactivity was present on stable ce ll lines; however, functional binding sites could not be detected. Whe n Ser94 was converted to Thr94, the affinity for some xanthine antagon ists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play imp ortant roles in agonist/A(1) AR interactions and show that Asp55 is re sponsible for allosteric regulation of ligand/A(1) AR binding by sodiu m. We also identify Ser94 as an important site for ligand binding.