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The A
Volume 270,
Number 23,
Issue of June 9, pp. 13987-13997, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Adenosine Receptor
adenosine receptor is a member of the
G-protein coupled receptor family, and its activation stimulates cyclic
AMP production. To determine the residues which are involved in ligand
binding, several residues in transmembrane domains 5-7 were
individually replaced with alanine and other amino acids. The binding
properties of the resultant mutant receptors were determined in
transfected COS-7 cells. To study the expression levels in COS-7 cells,
mutant receptors were tagged at their amino terminus with a
hemagglutinin epitope, which allowed their immunological detection in
the plasma membrane by the monoclonal antibody 12CA5. The functional
properties of mutant receptors were determined by measuring stimulation
of adenylate cyclase. Specific binding of [
H]CGS
21680 (15 nM) and [H]XAC (4
nM), an A agonist and antagonist, respectively,
was absent in the following Ala mutants: F182A, H250A, N253A, I274A,
H278A, and S281A, although they were well expressed in the plasma
membrane. The hydroxy group of Ser-277 is required for high affinity
binding of agonists, but not antagonists. An N181S mutant lost affinity
for adenosine agonists substituted at N
or C-2, but not at
C-5`. The mutant receptors I274A, S277A, and H278A showed full
stimulation of adenylate cyclase at high concentrations of CGS 21680.
The functional agonist potencies at mutant receptors that lacked
radioligand binding were >30-fold less than those at the wild type
receptor. His-250 appears to be a required component of a hydrophobic
pocket, and H-bonding to this residue is not essential. On the other
hand, replacement of His-278 with other aromatic residues was not
tolerated in ligand binding. Thus, some of the residues targeted in
this study may be involved in the direct interaction with ligands in
the human A adenosine receptor. A molecular model based on
the structure of rhodopsin, in which the 5`-NH in NECA is hydrogen
bonded to Ser-277 and His-278, was developed in order to visualize the
environment of the ligand binding site.
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