Activation of protein kinase C induces rapid internalization and subsequent degradation of muscarinic acetylcholine receptors in neuroblastoma cells

Abstract

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 [3H]quinuclidinyl benzilate in membrane homogenates. After a 6-h incubation, 10(-7) M PMA and 3 X 10(-7) M A23187 reduced mAChR number 30-40%, compared to the 40-50% reduction observed after treatment with 10(-3) M carbachol, a muscarinic agonist. Incubation with 3 X 10(-7) M A23187 and 10(-7) M 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, did not alter mAChR number. The addition of PMA and A23187 to cultures incubated with 10(-3) M carbachol caused only a modest 6% further reduction in mAChR number as compared to incubation with carbachol alone. The kinetics of the decrease in mAChR number produced by PMA/A23187 were similar to those seen after carbachol treatment. Recovery of mAChR number after treatment with either carbachol or PMA/A23187 was blocked by treatment with the protein synthesis inhibitor cycloheximide. Intact cell binding studies employing [3H]N-methylscopolamine showed that treatment with either PMA/A23187 or carbachol caused a rapid (within 15 min) loss of receptors from the cell surface prior to the decrease in total mAChR number. PMA (10(-7) M), but not 4 alpha-phorbol 12,13-didecanoate, promoted the translocation of protein kinase C activity from the cytosol to the membrane. Incubation with carbachol increased membrane-associated protein kinase C activity within 5 min with an EC50 of 3 X 10(-6) M. This increase persisted for at least 60 min in the continued presence of carbachol and was blocked by simultaneous incubation with atropine. These results suggest that activation of protein kinase C may be involved in the regulation of mAChR number in response to agonist.