Muscarinic Receptor Sequestration in SH-SY5Y Neuroblastoma Cells Is Inhibited when Clathrin Distribution Is Perturbed

Abstract: The possibility that clathrin plays a role in the agonist-mediated sequestration of muscarinic cholinergic receptors in human SH-SY5Y neuroblastoma cells has been investigated by the application of experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH-SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70–80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K⁺ or acidification of the cytosol also resulted in >80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K⁺, or acidification of cytosol, muscarinic receptor-stimulated phosphoinositide hydrolysis and Ca²⁺ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K⁺ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.