Downregulation of M1 and M2 Muscarinic Receptor Subtypes in Y1 Mouse Adrenocarcinoma Cells

Abstract

Muscarinic acetylcholine receptors are important in mediating neurotransmission in the central and peripheral nervous systems and regulate a broad spectrum of physiologic responses. Chronic exposure of cells to muscarinic agonists results in a decrease in receptor number and diminished coupling to effectors. We examined this phenomenon in 2 of the 5 described receptor subtypes: ml, one of the predominant subtypes expressed in brain, and m2, the major cardiac subtype. DNA clones for these receptors were transfected into the Y1 mouse adrenal cell line and a variant Y1 clone. Kin 8, in which cAMP-dependent protein kinase activity is greatly reduced. Transfected receptors could be expressed at high levels and were functional as determined by their ability to bind muscarinic ligands and stimulate phosphoinositide turnover (ml) or inhibit adenylyl cyclase (m2). We determined the susceptibility of the receptor subtypes to internalization after chronic exposure to the muscarinic agonist, carbachol, and as a consequence of activation of cAMP-dependent protein kinase and protein kinase C. These experiments suggest that the mechanisms for internalization of the m1 and m2 receptor subtypes differ. The ml, but not the m2, receptor was internalized in response to activation of protein kinase C and this internalization was dependent on the presence of a functional cAMP-dependent protein kinase. Activation of either protein kinase C or cAMP-dependent protein kinase did not mimic agonist-induced internalization of either m1 or m2. There was a large surplus of receptors for coupling to the effector enzymes in cells expressing several hundred fmol receptor per mg membrane protein. At low receptor numbers, agonist-induced desensitization was correlated with internalization of receptors.