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Published online 30 April 2001. doi:10.1083/jcb.153.3.491
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© The Rockefeller University Press, 0021-9525/2001//491 $5.00
The Journal of Cell Biology, Volume 153, Number 3, , 2001 491-502

Original Article

An Rgd Sequence in the P2y2 Receptor Interacts with {alpha}Vβ3 Integrins and Is Required for Go-Mediated Signal Transduction



Laurie Erba, Jun Liua, Jonathan Ockerhausenb, Qiongman Konga, Richard C. Garrada, Korey Griffina, Chris Neala, Brent Krugha, Laura I. Santiago-Pérezc, Fernando A. Gonzálezc, Hattie D. Greshamd, John T. Turnerb, and Gary A. Weismana

a Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65212
b Department of Pharmacology, University of Missouri-Columbia, Columbia, Missouri 65212
c Department of Chemistry, University of Puerto Rico, Rio Piedras, Puerto Rico 00931
d Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico 87131
Department of Biochemistry, M121 Medical Science Building, University of Missouri-Columbia, Columbia, MO 65212.(573) 884-4597(573) 882-1708

erbl{at}missouri.edu

The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against {alpha}Vβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that {alpha}V integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-{alpha}V integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go.

Key Words: purinergic receptors • cell surface receptors • integrins • GTP-binding proteins • signal transduction

© 2001 The Rockefeller University Press

Abbreviations used in this paper: AI, adhesion index; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; ERK1/2, extracellular regulated kinase; FAK, focal adhesion kinase; GPCR, G protein–coupled receptor; HSA, human serum albumin; IP, inositol phosphate; HA, hemagglutinin; MAP, mitogen-activated protein; pAb, polyclonal Ab; P2YR, P2Y nucleotide receptor; RGD, arginine-glycine-aspartic acid.


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