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-delta 1, 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.