Effects of synaptotagmin reveal two distinct mechanisms of agonist-stimulated internalization of the M4 muscarinic acetylcholine receptor.

1. Synaptotagmin has been reported to function in clathrin-mediated endocytosis. Here, we investigated its involvement in agonist-stimulated internalization of M4 muscarinic acetylcholine receptors exogenously expressed in human embryonic kidney (HEK-293 tsA201) cells. 2. Synaptotagmin I was present at low levels in these cells, and when overexpressed resided at the plasma membrane. 3. Synaptotagmin overexpression alone did not affect receptor internalization, but 'rescued' internalization that had been inhibited by either dominant-negative dynamin-1 or dominant-negative arrestin-2. Both normal and 'rescued' internalization were sensitive to inhibitors of clathrin-mediated endocytosis, but not to inhibitors of the function of caveolae. 4. There was no increase in AP-2 recruitment to the plasma membrane in cells overexpressing synaptotagmin. However, a mutant form of the receptor lacking a potential AP-2 recruitment motif, while being internalized normally in response to agonist stimulation, was not rescued by synaptotagmin in cells expressing dominant-negative dynamin or arrestin. 5. A mutant form of synaptotagmin (K326,327A), which binds phosphatidylinositol-4,5-bisphosphate (PIP2) much more weakly than the wild-type protein, did not rescue internalization. Furthermore, internalization was inhibited by the PH domain of phospholipase C-delta1, which sequesters PIP2, and synaptotagmin was now unable to rescue. 6. We propose that AP-2 binding to the C-terminal tail of the receptor is not normally required for its endocytosis, but that the synaptotagmin-mediated rescue involves the formation of a ternary complex with the receptor and AP-2. PIP2 might play a role as an intermediary in the formation of this complex.