A Mutational Analysis of Residues Essential for Ligand Recognition at the Human P2Y1 Receptor

  1. Qiaoling Jiang1,
  2. Danping Guo1,
  3. Brian X. Lee1,
  4. A. Michiel Van Rhee1,
  5. Yong-Chul Kim1,
  6. Robert A. Nicholas2,
  7. Joel B. Schachter2,
  8. T. Kendall Harden2 and
  9. Kenneth A. Jacobson1
  1. 1Molecular 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 2Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 (R.A.N., J.R.S., T.K.H.)

    Abstract

    We conducted a mutational analysis of residues potentially involved in the adenine nucleotide binding pocket of the human P2Y1receptor. 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 β- and γ-phosphates of the adenine nucleotides seem to be less important than the α-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 γ-phosphate recognition. Taken together, the results suggest that the adenosine and α-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor.

    Footnotes

    • Send reprint requests to: Dr. K. A. Jacobson, Chief, Molecular Recognition Section, Bldg. 8A, Room B1A-17, NIDDK, NIH, Bethesda, MD 20892. E-mail:kajacobs{at}helix.nih.gov

    • ↵2 The coordinates of the P2Y1 receptor model are available from the protein database (Brookhaven National Laboratory) using the URLhttp://www.pdb. bnl.gov/cgi-bin/browse under the ID code 1ddd.

    • ↵3 Residue identifiers as described in van Rhee and Jacobson (4) specify the helix (X) and the position relative to a key conserved residue in that helix, which is designated (X0.50). For example, for the helices mutated in the present study, the key conserved residues are R149(3.50), P229(5.50), P275(6.50), and P321(7.50).

    • ↵1 P2Y1-P2Y7 sequences have been cloned and named in the literature (3), however, several of these may be either receptors of a different family (e.g., P2Y5and possibly P2Y7) or possibly species homologues (e.g., P2Y3 and P2Y6). The identity and relationship among these clones require further study.

    • Abbreviations:
      2-MeSATP
      2-methylthio-ATP
      2-MeSADP
      2-methylthio-ADP
      DMEM
      Dulbecco’s modified Eagle’s medium
      ELISA
      enzyme-linked immunosorbent assay
      FBS
      fetal bovine serum
      HA
      hemagglutinin
      HT-AMP
      2-(hexylthio)adenosine-5′-monophosphate
      PBS
      phosphate-buffered saline
      PCR
      polymerase chain reaction
      TM
      (helical) transmembrane domain
      PPADS
      pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid
      • Received February 26, 1997.
      • Accepted May 27, 1997.
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    1. Molecular Pharmacology September 1, 1997 vol. 52 no. 3 499-507
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