Hetero-oligomerization and Functional Interaction between Purinergic Receptors Expressed in Platelets to Regulate Platelet Shape Change

Abstract

  Adenosine and its precursors, ATP and ADP, exert various physiological effects via binding to purinergic receptors. We previously used co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and immunoelectron microscopy to demonstrate the hetero-oligomerization of purinergic receptor subtypes. Furthermore, pharmacological studies found significant changes in receptor-mediated signaling in human embryonic kidney (HEK) 293T cells co-transfected with these receptors. These findings suggest that heterodimers of purinergic receptors may have distinct pharmacological profiles, possibly due to dimerization-induced conformational changes, further suggesting that hetero-dimerization may be employed to “fine-tune” purinergic receptor signaling. Adenosine A_2A receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. ADP activates human platelets by stimulating both P2Y₁R and P2Y₁₂R, which act sequentially and in concert to achieve complete platelet aggregation. In contrast, adenosine stimulates Gs-coupled A_2AR, followed by activativation of adenylate cyclase, leading to an increase in intracellular cAMP levels, which potently inhibits platelet activation. We examined the hetero-oligomerization and functional interactions of A_2AR, P2Y₁R, and P2Y₁₂R. In HEK293T cells triply expressing all three receptors, hetero-oligomerization was observed among the three receptors. Additionally, P2Y₁R agonist-evoked Ca²⁺ signaling was significantly inhibited by co-treatment with an A_2AR antagonist in HEK293T cells. In human platelets, we identified endogenous A_2AR/P2Y₁R and A_2AR/P2Y₁₂R heterodimers. We also observed functional Ca²⁺-signaling-related cross-talk similar to those found in HEK293T cells, and found that they appeared to affect platelet shape. These results collectively suggest that intermolecular signal transduction and specific conformational changes occur among components of the hetero-oligomers formed by these three receptors.