A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor.
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 beta- and gamma-phosphates of the adenine nucleotides seem to be less important than the alpha-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 gamma-phosphate recognition. Taken together, the results suggest that the adenosine and alpha-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor.
Jiang
Q
Q
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Guo
D
D
Lee
B X
BX
Van Rhee
A M
AM
Kim
Y C
YC
Nicholas
R A
RA
Schachter
J B
JB
Harden
T K
TK
Jacobson
K A
KA
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism / Q000502:physiology; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D008024:Ligands; C546382:P2RY1 protein, human; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D000249:Adenosine Monophosphate; D010738:Type C Phospholipases