Abstract

In the present study, we investigated the desensitization and trafficking of the P2Y₁ and P2Y₁₂ receptors after agonist-induced stimulation of platelets or astrocytoma cells transfected with the P2Y₁ or P2Y₁₂ receptors fused to green fluorescent protein. In platelets and in transfected cells, exposure to 10 μM ADP caused desensitization of the P2Y₁ receptor-driven calcium signal, whereas the P2Y₁₂ receptor-mediated inhibition of cAMP formation was not affected. Plasma membranes from ADP-stimulated platelets also retained P2Y₁₂ activity. Agonist-induced P2Y₁ receptor desensitization was accompanied by its internalization in platelets and transfected cells. In contrast, although a substantial fraction of P2Y₁₂ receptors was rapidly and transiently internalized, most of the P2Y₁₂ receptors remained at the plasma membrane. Activated P2Y₁ receptors were internalized through a clathrin-dependent pathway in cells and platelets, whereas the P2Y₁₂ receptors seemed to use a distinct, clathrin-independent pathway. Together, these data indicate that the P2Y₁ and P2Y₁₂ receptors are differentially regulated upon activation. The absence of desensitization of the Gi protein-coupled P2Y₁₂ receptor-dependent responses could represent a mechanism to preserve the hemostatic properties of otherwise unresponsive platelets.