Figure 3.

M₃-muscarinic receptors expressed in Chinese hamster ovary (CHO) cells and cerebellar granule neurons are differentially phosphorylated. Cerebellar granule neurons were cultured from 6-day-old neonatal mice and maintained for 6 days before being labelled for phosphorylation studies. CHO cells expressing the recombinant mouse M₃-muscarinic receptor or cerebellar granule neurons were labelled with [³²P]orthophosphate for 1–2 h (100–200 μCi ml⁻¹). The cells were then stimulated for 5 min with the muscarinic agonist methacholine (100 μM). Cells were then solubilized and the M₃-muscarinic receptor immunoprecipitated using a receptor-specific antibody. The immunoprecipitate was resolved by 8% SDS-PAGE and the radioactive band associated with the receptor was excised. The receptor band was then digested with trypsin and the resulting peptides resolved in the first dimension by electrophoresis and in the second dimension by thin layer chromatography. The position of the radioactive phospho-peptides was then determined in a phospho-imager. The numbered peptides are those that run in the same position and represent phosphorylation sites that are the same in the two cell types. The arrows represent phosphorylation events that are specific to the cell in which the receptor is expressed. The experiment shown was adapted from Torrecilla et al. (2007) where details of the methods can be obtained.