Degradation of rat brain cholinergic muscarinic receptors in vitro: enhancement by agonists and inhibition by antagonists.

Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.