TY - JOUR
T1 - Ligand binding and subtype selectivity of the human A2A adenosine receptor: Identification and characterization of essential amino acid residues
AU - Jaakola, Veli-Pekka
AU - Lane, Jonathan
AU - Lin, Judy
AU - Katritch, Vsevolod
AU - IJzerman, Adriaan P
AU - Stevens, Raymond
PY - 2010
Y1 - 2010
N2 - The crystal structure of the human A2A adenosine receptor
bound to the A2A receptor-specific antagonist, ZM241385, was
recently determined at 2.6-AEs resolution. Surprisingly, the
antagonist binds in an extended conformation, perpendicular to
the plane of the membrane, and indicates a number of interactions
unidentified before in ZM241385 recognition. To further
understand the selectivity of ZM241385 for the human A2A
adenosine receptor, we examined the effect of mutating amino
acid residues within the binding cavity likely to have key interactions
and that have not been previously examined. Mutation
of Phe-168 to Ala abolishes both agonist and antagonist binding
as well as receptor activity, whereas mutation of this residue to
Trp or Tyr had only moderate effects. The Met-1773Ala mutation
impeded antagonist but not agonist binding. Finally, the
Leu-249 3 Ala mutant showed neither agonist nor antagonist
binding affinity. From our results and previously published
mutagenesis data, we conclude that conserved residues Phe-
168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a
central role in coordinating the bicyclic core present in both agonistsandantagonists.
Bycombiningthe analysis of the mutagenesis
data with a comparison of the sequences of different adenosine
receptor subtypes from different species, we predict that the interactions
that determine subtype selectivity reside in the more divergent
a??uppera?? region of the binding cavity while the a??lowera?? part of
the binding cavity is conserved across adenosine receptor subtypes.
AB - The crystal structure of the human A2A adenosine receptor
bound to the A2A receptor-specific antagonist, ZM241385, was
recently determined at 2.6-AEs resolution. Surprisingly, the
antagonist binds in an extended conformation, perpendicular to
the plane of the membrane, and indicates a number of interactions
unidentified before in ZM241385 recognition. To further
understand the selectivity of ZM241385 for the human A2A
adenosine receptor, we examined the effect of mutating amino
acid residues within the binding cavity likely to have key interactions
and that have not been previously examined. Mutation
of Phe-168 to Ala abolishes both agonist and antagonist binding
as well as receptor activity, whereas mutation of this residue to
Trp or Tyr had only moderate effects. The Met-1773Ala mutation
impeded antagonist but not agonist binding. Finally, the
Leu-249 3 Ala mutant showed neither agonist nor antagonist
binding affinity. From our results and previously published
mutagenesis data, we conclude that conserved residues Phe-
168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a
central role in coordinating the bicyclic core present in both agonistsandantagonists.
Bycombiningthe analysis of the mutagenesis
data with a comparison of the sequences of different adenosine
receptor subtypes from different species, we predict that the interactions
that determine subtype selectivity reside in the more divergent
a??uppera?? region of the binding cavity while the a??lowera?? part of
the binding cavity is conserved across adenosine receptor subtypes.
U2 - 10.1074/jbc.M109.096974
DO - 10.1074/jbc.M109.096974
M3 - Article
SN - 0021-9258
VL - 285
SP - 13032
EP - 13044
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -