Figure 4.

Stimulation of m1 muscarinic acetylcholine receptors results in the phosphorylation of the CTF of PS-1 by PKC. U373 cells stably transfected with the m1 receptor cDNA were labeled with [³⁵S]methionine (³⁵S-Met; Upper) for 14 h and then incubated for 2 h in the absence or presence of carbachol alone or in combination with either atropine or a PKC inhibitor. To activate PKC directly, cells were treated with PDBu for 2 h. The CTFs of PS-1 were immunoprecipitated with antibody 3027. The positions of unphosphorylated CTF (CTF) and phosphorylated CTF [CTF(P)] are marked by arrowheads. To analyze phosphate incorporation, cells were incubated in the presence of [³²P]orthophosphate during drug treatments. The CTFs of PS-1 were precipitated from cell lysates with antibody 3027. The ³²P-labeled ≈23-kDa and ≈24-kDa polypeptides are marked by arrowheads [CTF(P)], and the position of unphosphorylated CTF (not detectable by ³²P labeling) is marked by an arrow. Note that stimulation of m1 receptors with carbachol results in a marked increase in phosphate incorporation as well as a molecular mass shift, similar to that observed after PDBu-mediated PKC stimulation.