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PDBsum entry 1mmh
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G protein-coupled receptor
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PDB id
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1mmh
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Contents |
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32 a.a.
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29 a.a.
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26 a.a.
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28 a.a.
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30 a.a.
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29 a.a.
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34 a.a.
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References listed in PDB file
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Key reference
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Title
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Site-Directed mutagenesis identifies residues involved in ligand recognition in the human a2a adenosine receptor.
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Authors
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J.Kim,
J.Wess,
A.M.Van rhee,
T.Schöneberg,
K.A.Jacobson.
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Ref.
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J Biol Chem, 1995,
270,
13987-13997.
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PubMed id
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Abstract
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The A2a 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 [3H]CGS 21680
(15 nM) and [3H]XAC (4 nM), an A2a 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
N6 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 A2a 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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Secondary reference #1
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Title
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Modelling the p2y purinoceptor using rhodopsin as template.
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Authors
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A.M.Van rhee,
B.Fischer,
P.J.Van galen,
K.A.Jacobson.
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Ref.
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Drug Des Discov, 1995,
13,
133-154.
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PubMed id
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