Activation of Protein Kinase C Induces Rapid Internalization and Subsequent Degradation of Muscarinic Acetylcholine Receptors in Neuroblastoma Cells

摘要

The tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C, acted synergistically with A23187 to decrease muscarinic acetylcholine receptor (mAChR) number in neuroblastoma cells (clone N1E-115) as determined by a filter binding assay using 3Hquinuclidinyl benzilate in membrane homogenates. After a 6-hincubation, .0000001m PMA and .0000003 m A23187 reduced mAChR NUMBER 30-40%, compared to the 40-50% reduction observed after treatment with .001 carbachol, a muiscarinic agonist. Incubation with 3 x .0000001 m A23187 and .0000003 m 4 alpha-phorbol ester, did not alter mAChR number. The addition of PMA and A23187 to cultures incubated with .001 m carbachol caused only a modest 6% further reduction in mACHr number as compared to incubated with carbachol alone. The kinetics of the decrease in mAChR number produced by PMA/A23187 was blocked by treatment with the protein synthesis inhibitor cycloheximide. intact cell binding studies employing 3Hn-methylscopolamine showed that treatment with either PMA/A23187 or carbacnol caused a rapid (within 15 min) loss of receptors from the cell surface prior to the decrease in total mAChR number. PMA (.0000001 m), but not 4 alpha-phorbol 12,13-didecanoate, promoted the translocation of protein kinase C activity from the cytosol to the membrane.