Original Article

An RGD Sequence in the P2Y₂ Receptor Interacts with _Vß₃ Integrins and Is Required for G_o-mediated Signal Transduction

Correspondence to: Laurie Erb, Department of Biochemistry, M121 Medical Science Building, University of Missouri-Columbia, Columbia, MO 65212. Tel:(573) 882-1708 Fax:(573) 884-4597 E-mail: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 _Vß₃/ß₅ integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y₂Rs indicated that _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-_V integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y₂R. Pertussis toxin, an inhibitor of G_i/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 G_o, but not G_q. Since CD47 has been shown to associate directly with G_i/o family proteins, these results suggest that interactions between P2Y₂Rs, integrins, and CD47 may be important for coupling the P2Y₂R to G_o.

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

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