Comparison of the kinetics and extent of muscarinic M₁–M₅ receptor internalization, recycling and downregulation in Chinese hamster ovary cells

Abstract

We characterized agonist-induced internalization, recycling and downregulation of each muscarinic receptor subtype (M₁–M₅) stably expressed in Chinese hamster ovary (CHO) cells. The radioligands [³H]QNB and [³H]NMS were used to measure the total and plasma membrane populations of muscarinic receptors, respectively. Following carbachol treatment (1 mM), the rank orders for the rate of carbachol-induced internalization of the muscarinic subtypes were M₂ > M₄ = M₅ > M₃ = M₁, respectively. Unlike the M₂ receptor, M₁, M₃, M₄ and M₅ receptors recycled back to the plasma membrane after 1 h carbachol treatment. The receptor downregulation elicited to 24 h carbachol treatment was similar for M₂, M₃, M₄ and M₅ receptors, whereas that for the M₁ receptor was greater. Our results indicate that there are subtype-specific differences in the rate and extent of agonist-induced muscarinic receptor internalization, recycling and downregulation in CHO cells.

Introduction

Muscarinic acetylcholine receptors are G protein-coupled receptors and five subtypes (M₁–M₅) have been cloned (see review, Hulme et al., 1990). When expressed in cells lacking endogenous muscarinic receptors, M₁, M₃, and M₅ receptors mediate stimulation of phosphoinositide hydrolysis, whereas M₂ and M₄ receptors mediate inhibition of adenylyl cyclase activity (Hammer, 1980, Kashihara et al., 1992). Consistent with the second messenger signaling, M₁, M₃, and M₅ receptors couple with G_q/11 proteins and M₂ and M₄ receptors to couple with G_i/o proteins (DeLapp et al., 1999, Offermanns et al., 1994, Parker et al., 1991).

Typically, prolonged agonist exposure activates mechanisms that regulate G protein-coupled receptor activity and there are three major mechanisms: desensitization, internalization, and downregulation. Homologous receptor desensitization is thought to be initiated by phosphorylation of G protein-coupled receptors by G protein-coupled receptor kinases (GRKs), which causes the recruitment of β-arrestin and a subsequent reduction in second messenger signaling by uncoupling the receptor from associated G proteins (see review, Shenoy and Lefkowitz, 2003). β-arrestin binding also leads to receptor internalization via clathrin coated vesicles and β-arrestin has binding sites for the heavy chain of clathrin and the clathrin adaptor AP2 (Goodman et al., 1996, Laporte et al., 2000). Once internalized, G protein-coupled receptors may be recycled back to the plasma membrane or degraded in lysosomes in a process referred to as downregulation.

Agonist binding to muscarinic receptors, like other G protein-coupled receptors, leads to receptor desensitization, internalization, and downregulation depending upon the concentration and duration of agonist exposure. Several studies have characterized these agonist-dependent processes and subtype- and cell-specific differences have been observed. For instance, when exposed to the muscarinic agonist carbachol, M₁ and M₃ receptors internalized to a lesser extent than M₂ and M₄ receptors in CHO and COS-7 cells (Koenig and Edwardson, 1996, Tsuga et al., 1998b). The rate of carbachol-dependent M₃ receptor internalization was approximately 10-fold greater in SH-SY5Y neuroblastoma cells compared to CHO cells transfected with a M₃ receptor construct (Koenig and Edwardson, 1996). These are a few of many examples of subtype- and cell-specific differences in the agonist-dependent regulation of muscarinic receptors.

To date, the agonist-dependent internalization, recycling and downregulation of all five subtypes of muscarinic receptor have not been characterized in one cell type. This comparison is important because differences in the kinetics or extent of internalization, recycling or downregulation would be an indication that distinct mechanisms regulate the activity of muscarinic receptors in a subtype-specific manner. In this investigation, we compared the carbachol-dependent internalization and downregulation of M₁–M₅ receptors in CHO cells. We also compared the recycling of M₁–M₅ receptors after a brief treatment with carbachol. In general, M₂ receptors internalized faster and more extensively than the other subtypes. M₃, M₄, and M₅ receptors recycled more extensively than M₁ and M₂ receptors. Lastly, the extent of receptor downregulation elicited to 24 h carbachol treatment was greater for the M₁ receptor than for the M₂, M₃, M₄ and M₅ receptors.