A Mutational Analysis of Residues Essential for Ligand Recognition at the Human P2Y₁Receptor

Abstract

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.

ADP Is the Cognate Ligand for the Orphan G Protein-coupled Receptor SP1999

“…Identification of the Ligand for SP1999 -To understand the function of SP1999, we set out to identify its endogenous ligand; SP1999 was cotransfected with a mixture of chimeric G protein plasmids encoding G␣ q/12 , G␣ 16 , G␣ q/i , G␣ q/z , G␣ q/i3 , FIG. 1.…”

“…Amino acids Phe-105, Tyr-106, Tyr-110, Phe-198, His-253, Arg-256, and Ser-288 are identical to residues highly conserved among the P2Y1, P2Y2, P2Y4, and P2Y6 receptors (16,17). Through the use of molecular modeling and site-directed mutagenesis, these residues have been shown to be involved in the binding of purinergic ligands to the P2Y1 receptor.…”

“…Through the use of molecular modeling and site-directed mutagenesis, these residues have been shown to be involved in the binding of purinergic ligands to the P2Y1 receptor. Although several residues found to be absolutely required for ligand interaction at the P2Y1 receptor (Arg-128 and Arg-310 in P2Y1) are not strictly conserved in SP1999, it is highly possible that ligands bind to SP1999 in an orientation different from P2Y1, allowing other residues present in SP1999 to become involved in the binding site (16,17). Further studies involving molecular modeling and site-directed mutagenesis of SP1999 will be required to specifically define the ligand binding pocket of this receptor.…”

A two‐entropies analysis to identify functional positions in the transmembrane region of class A G protein‐coupled receptors

“…14,17 The second assumption is that endogenous binding sites are located in a region embedded between TM helices. This has been shown experimentally for a large number of receptors, including those for biogenic amines, 49 nucleotides, 50 melatonin, 51 and prostacyclin. 52 In the entropy-variability plot, both entropy and variability are measures of conservation of each position of class A GPCRs.…”

Prediction of the 3-D structure of rat MrgA G protein-coupled receptor and identification of its binding site

“…The resulting TM sequence alignment is shown in Fig. 9 where the key residues in adenosine receptors and P2Y 1 receptor identified from the binding or functional assay experiments are bolded and underlined [42,43].…”