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Molecular Recognition Section, Laboratory of Bioorganic Chemistry,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892 (Q.J., D.G.,
B.X.L., A.M.v. R., Y.-C.K., K.A.J.), and
Department of Pharmacology,
University of North Carolina School of Medicine, Chapel Hill, North
Carolina 27599 (R.A.N., J.R.S., T.K.H.)
We conducted a mutational analysis of residues potentially involved in
the adenine nucleotide binding pocket of the human P2Y1
receptor. Mutated receptors were expressed in COS-7 cells with an
epitope tag that permitted confirmation of expression in the plasma
membrane, and agonist-promoted inositol phosphate accumulation was
assessed as a measure of receptor activity. Residues in transmembrane
helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to
be involved in ligand recognition were replaced with alanine and, in
some cases, by other amino acids. The potent P2Y1 receptor
agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing
the R128A, R310A, and S314A mutant receptors, and a markedly reduced
potency of 2-MeSATP was observed with the K280A and Q307A mutants.
These results suggest that residues on the exofacial side of TM3 and
TM7 are critical determinants of the ATP binding pocket. In contrast,
there was no change in the potency or maximal effect of 2-MeSATP with
the S317A mutant receptor. Alanine replacement of F131, H132, Y136,
F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold
reduction in potency compared with that observed with the wild-type
receptor. These residues thus seem to subserve a less important
modulatory role in ligand binding to the P2Y1 receptor.
Because changes in the potency of 2-methylthio-ADP and
2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at
these mutant receptors, the 
- and 
-phosphates of the adenine
nucleotides seem to be less important than the 
-phosphate in
ligand/P2Y1 receptor interactions. However, T221A and T222A
mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in
diphosphate or monophosphate potency. This result may be indicative of
a greater role of these TM5 residues in -phosphate recognition.
Taken together, the results suggest that the adenosine and
-phosphate moieties of ATP bind to critical residues in TM3 and TM7
on the exofacial side of the human P2Y1 receptor.
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