P2Y1 Receptor Radioligand
1257
tion of a radiolabeled agonist with a phospholipase C-linked P -purinergic recep-
protein, which is similar to the density of many different neu-
rotransmitter receptors in the brain and is greater than 100-
2
tor. J Biol Chem 264:6202–6206.
Dubyak GR and El-Moatassim C (1993) Signal transduction via P -purinergic re-
2
35
fold lower than the density of deoxyadenosine 5Ј([␣- S]thio)t-
riphosphate binding sites (39 pmol/mg protein) reported for rat
brain membranes (Simon et al., 1995a). More recently, Baurand
ceptors for extracellular ATP and other nucleotides. Am J Physiol 265:C577–C606.
Harden TK, Barnard EA, Boeynaems JM, Burnstock G, Dubyak GR, Hourani SMO,
and Insel PA (1998a) P2Y receptors, in The IUPHAR Compendium of Receptor
Characterization and Classification (Girdlestone D ed) pp 209–217, IUPHAR
Media, London.
33
et al. (2001) have P-labeled a P2Y1-R antagonist molecule,
MRS2179, also previously developed by our group (Boyer et al.,
Harden TK, Boyer JL, and Nicholas RA (1995) P -purinergic receptors: subtype-
2
associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35:
5
41–579.
1998). This molecule was shown to bind to intact human plate-
Harden TK, Lazarowski ER, and Boucher RC (1997) Release, metabolism and
interconversion of adenine and uridine nucleotides: implications for G protein-
coupled P2 receptor agonist selectivity. Trends Pharmacol Sci 18:43–46.
lets with relatively high affinity (K ϭ 109 nM). Binding was
d
inhibited by nonradioactive MRS2179 and by adenosine 3Ј,5Ј-
Harden TK, Nicholas RA, Schachter JB, Lazarowski ER, and Boyer JL (1998b)
Pharmacological selectivities of molecularly defined subtypes of P2Y receptors, in
The P2 Nucleotide Receptors (Turner JT, Weisman GA, and Fedan JS eds) pp
bisphosphate with K values similar to those observed in studies
i
of these two compounds as competitive antagonists of P2Y1-R–
1
09–134, Humana Press, Totowa, NJ.
33
promoted responses. Application of [ P]MRS2179 to the study
of P2Y1-R in other tissues was not reported. The 5- to 10-fold
higher affinity of MRS2279 than MRS2179, as well as the fact
that MRS2179 is a true nucleotide analog whereas MRS2279 is
not, are two important advantages inherent in the structure of
MRS2279.
Hechler B, Vigne P, Leon C, Breittmayer JP, and Gachet C (1998) ATP derivatives
are antagonists of the P2Y1 receptor: similarities to the platelet ADP receptor. Mol
Pharmacol 53:727–733.
Huang P, Lazarowski ER, Tarran R, Milgram SL, Boucher RC, and Stutts MJ (2001)
Compartmentalized autocrine signaling to cystic fibrosis transmembrane conduc-
tance regulator at the apical membrane of airway epithelial cells. Proc Natl Acad
Sci USA 98:14120–14125.
Khakh BS, Barnard EA, Burnstock G, Kennedy C, King BF, North RA, Seguela P,
Voigt M, and Humphrey PPA (2000) P2X receptors, in The IUPHAR Compendium
of Receptor Characterization and Classification (Girdlestone D ed) pp 291–305,
IUPHAR Media, London.
In summary, we have developed a binding assay for
P2Y1-R using a radiolabeled antagonist that has evolved
from a series of studies designed to produce high-affinity
P2Y1-R selective antagonists. This molecule is applicable for
the study of recombinant P2Y1-R in membrane or soluble
forms and natively expressed P2Y1-R in human, rat, and
mouse tissues. This receptor assay should prove valuable in
furthering our understanding of the biological significance
and regulation of the P2Y1-R and for further drug develop-
ment for this therapeutically important target protein.
Kim HS, Barak D, Harden TK, Boyer JL, and Jacobson KA (2001) Acyclic and
cyclopropyl analogues of adenosine bisphosphate antagonists of the P2Y1 receptor:
structure-activity relationships and receptor docking. J Med Chem 44:3092–3108.
Kim Y-C, Gallo-Rodriguez C, Jang S-Y, Nandanan E, Adams M, Harden TK, Boyer
JL, and Jacobson KA (2000) Acyclic analogues of deoxyadenosine 3Ј, 5Ј-
bisphosphates as P2Y1 receptor antagonists. J Med Chem 42:746–755.
Motte S, Swillens S, and Boeynaems JM (1996) Evidence that most high-affinity ATP
binding sites on aortic endothelial cells and membranes do not correspond to P2
receptors. Eur J Pharmacol 307:201–209.
Nandanan E, Camaioni E, Jang SY, Kim YC, Cristalli G, Herdewijn P, Secrist JA
3
rd, Tiwari KN, Mohanram A, Harden TK, et al. (1999) Structure-activity rela-
tionships of bisphosphate nucleotide derivatives as P2Y1 receptor antagonists and
partial agonists. J Med Chem 42:1625–1638.
Nandanan E, Jang SY, Moro S, Kim HO, Siddiqui MA, Russ P, Marquez VE, Busson
R, Herdewijn P, Harden TK, et al. (2000) Synthesis, biological activity and molec-
ular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y1
receptor ligands. J Med Chem 43:829–842.
Palmer RK, Boyer JL, Schachter JB, Nicholas RA, and Harden TK (1998) Agonist
action of adenosine triphosphates at the human P2Y1 receptor. Mol Pharmacol
Acknowledgments
We thank Eduardo Lazarowski for assistance in quantitating ATP
and ADP metabolism, Linda Brady in the University of North Caro-
lina Blood Bank for providing out-dated platelets, and David Rinker
and Meredith Jones for help in preparation of the manuscript.
54:1118–1123.
Perich JW and Johns RB (1988) Di-tert-butyl N,N-niethylphosphoramidite. A new
phosphorylating agent for the efficient phosphorylation of alcohols. Synthesis
2:142–144.
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Address correspondence to: Dr. T. Kendall Harden, Dept. of Pharmacology,
University of North Carolina School of Medicine, CB #7365, Chapel Hill, NC
27599. E-mail: tkh@med.unc.edu
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