The Timing of Endocytosis after Activation of a G-Protein-Coupled Receptor in a Sensory Neuron

Endocytosis is a fundamental cellular event in membrane retrieval after exocytosis and in the regulation of receptor-mediated signal transduction. In contrast to the well-studied depolarization-induced membrane recycling, little is known about the kinetics of ligand-induced endocytosis of G-protein-coupled receptors in neurons. Here we investigated the kinetics of ligand-receptor binding-induced endocytosis in rat sensory neurons using a membrane capacitance assay. The time constant of ADP-induced endocytosis of P2Y-receptors was determined as 1.7 s. The ADP-induced endocytosis was blocked by antagonists against P2Y, phosphorylation, and clathrin. However, block of dynamin was without effect. The ADP-induced endocytosis was confirmed independently by a single vesicle image technique using a styryl FM2-10. Finally, the receptors were internalized in response to ADP, as determined by GFP-labeled P2Y. We conclude that ligand-receptor binding leads to rapid endocytosis in the cytoplasm of rat dorsal root ganglion neurons.