title: Probing the binding site of the A1 adenosine receptor reengineered for orthogonal recognition by tailored nucleosides.
abstract: 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'-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5' position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, USA.
authors.forename: Krishnan K
authors.initials: KK
authors.lastname: Palaniappan
authors.forename: Zhan-Guo
authors.initials: ZG
authors.lastname: Gao
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Ivanov
authors.forename: Rebecca
authors.initials: R
authors.lastname: Greaves
authors.forename: Hayamitsu
authors.initials: H
authors.lastname: Adachi
authors.forename: Pedro
authors.initials: P
authors.lastname: Besada
authors.forename: Hea Ok
authors.initials: HO
authors.lastname: Kim
authors.forename: Ae Yil
authors.initials: AY
authors.lastname: Kim
authors.forename: Seung Ah
authors.initials: SA
authors.lastname: Choe
authors.forename: Lak Shin
authors.initials: LS
authors.lastname: Jeong
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
mesh_terms: D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D005818:Genetic Engineering; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008564:Membrane Potentials; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009705:Nucleosides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D043682:Receptor, Adenosine A1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D009705:Nucleosides; D043682:Receptor, Adenosine A1; D000242:Cyclic AMP; D002118:Calcium