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'-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.
Palaniappan
Krishnan K
KK
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.
Gao
Zhan-Guo
ZG
Ivanov
Andrei A
AA
Greaves
Rebecca
R
Adachi
Hayamitsu
H
Besada
Pedro
P
Kim
Hea Ok
HO
Kim
Ae Yil
AY
Choe
Seung Ah
SA
Jeong
Lak Shin
LS
Jacobson
Kenneth A
KA
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
D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D009705:Nucleosides; D043682:Receptor, Adenosine A1; D000242:Cyclic AMP; D002118:Calcium