Functional and molecular evidence for heteromeric association of P2Y(1) receptor with P2Y(2) and P2Y(4) receptors in mouse granulocytes

Abstract

Background: All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. Methods: Pharmacological characteristics of P2 receptors were evaluated by Ca2+ measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. Results: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca2+ increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y(1) receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y(1) receptor and the P2Y(2) and P2Y(4) receptors was shown by immunoprecipitation and FRET analysis. Conclusion: Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.