Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes

• Published in: BMC pharmacology & toxicology Vol. 17; no. 1; p. 29
• Main Authors: Ribeiro-Filho, Antonio Carlos, Buri, Marcus Vinicius, Barros, Carlos Castilho, Dreyfuss, Juliana Luporini, Nader, Helena Bonciani, Justo, Giselle Zenker, Craveiro, Rogério Bastos, Pesquero, João Bosco, Miranda, Antonio, Ferreira, Alice Teixeira, Paredes-Gamero, Edgar Julian
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 07-07-2016; BioMed Central
• Subjects: Adenosine; Agonists; Analysis; Animals; Antibodies; Bone marrow; Calcium; Calcium ions; Desensitization; Evaluation; Female; Flow Cytometry; Fluorescence resonance energy transfer; Fura-2; G proteins; Gene expression; Granulocytes; Granulocytes - drug effects; Granulocytes - physiology; Hemopoiesis; Immunoprecipitation; Influence; Laboratory animals; Leukocytes (granulocytic); Mice; Mice, Inbred C57BL; Pharmacology; Polymerase chain reaction; Protein Binding - physiology; Proteins; Purine P2 receptors; Purinergic Agonists - pharmacology; Receptors; Receptors, Purinergic P2 - chemistry; Receptors, Purinergic P2 - physiology; Receptors, Purinergic P2Y1 - chemistry; Receptors, Purinergic P2Y1 - physiology; Receptors, Purinergic P2Y2 - chemistry; Receptors, Purinergic P2Y2 - physiology; Stem Cells - drug effects; Stem Cells - physiology; Western blotting; Calcium signaling; P2Y receptors; Granulocytes; Heteromeric association
• ISSN: 2050-6511; 2050-6511
• DOI: 10.1186/s40360-016-0072-y

All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown.
Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) 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.
Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) 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 P2Y1 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 P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis.
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.