Involvement of Actin in Agonist-induced Endocytosis of the G Protein-coupled Receptor for Thromboxane A₂

The role of actin in endocytosis of G protein-coupled receptors is poorly defined. In the present study, we demonstrate that agents that depolymerize (latrunculin B and cytochalasin D) or stabilize (jasplakinolide) the actin cytoskeleton blocked agonist-induced endocytosis of the β isoform of the thromboxane A₂ receptor (TPβ) in HEK293 cells. This suggests that endocytosis of TPβ requires active remodeling of the actin cytoskeleton. On the other hand, disruption of microtubules with colchicine did not affect endocytosis of the receptor. Expression of wild-type and mutant forms of the small GTPases RhoA and Cdc42 potently inhibited endocytosis of TPβ, further indicating a role for the dynamic regulation of the actin cytoskeleton in this pathway. Agonist treatment of TPβ in HEK293 cells resulted in the formation of actin stress fibers through Gα_q/11 signaling. Because we previously showed that endocytosis of TPβ is dependent on arrestins, we decided to explore the relation between arrestin-2 and -3 and actin in endocytosis of this receptor. Interestingly, we show that the inhibition of TPβ endocytosis by the actin toxins in HEK293 cells was overcome by the overexpression of arrestin-3, but not of arrestin-2. These results indicate that the actin cytoskeleton is not essential in arrestin-3-mediated endocytosis of TPβ. However, arrestin-3 could not promote endocytosis of the TPβY339A and TPβI343A carboxyl-terminal mutants when the actin cytoskeleton was disrupted. Our data provide new evidence that the actin cytoskeleton plays an essential role in TPβ endocytosis. Furthermore, our work suggests the existence of actin-dependent and -independent arrestin-mediated pathways of endocytosis.