MUSCARINIC RECEPTOR SEQUESTRATION IN SH-SY5Y NEUROBLASTOMA-CELLS IS INHIBITED WHEN CLATHRIN DISTRIBUTION IS PERTURBED

The possibility that clathrin plays a role in the agonist-mediated seq uestration of muscarinic cholinergic receptors in human SH-SY5Y neurob lastoma 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 hyperto nic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70-80% at 550 mOsm), which was reve rsed when cells were returned to isotonic medium. Depletion of intrace llular K+ or acidification of the cytosol also resulted in >80% inhibi tion of muscarinic receptor sequestration. Under conditions of hyperto nicity, depletion of intracellular K+, or acidification of cytosol, mu scarinic receptor-stimulated phosphoinositide hydrolysis and Ca2+ sign aling events were either unaffected or markedly less inhibited than re ceptor sequestration. That these same experimental conditions did pert urb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K+ resulted in a pronounc ed accumulation of clathrin in the perinuclear region, whereas acidifi cation of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The res ults are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.