Molecular and Cellular PharmacologyComparison of the kinetics and extent of muscarinic M1–M5 receptor internalization, recycling and downregulation in Chinese hamster ovary 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 Gq/11 proteins and M2 and M4 receptors to couple with Gi/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.
Section snippets
Cell culture
CHO cells stably expressing each subtype of muscarinic receptor (M1–M5) were obtained from Dr. Tom I. Bonner at the National Institutes of Mental Health. The preparation of these cell lines was described by Buckley et al. (1989). Cell lines were stored in the vapor phase of liquid nitrogen and revived in growth medium (F-12K, supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin). Cells were maintained in growth medium containing geneticin (500 μg/ml) in a humidified incubator
Comparison of the kinetics and extent of muscarinic M1–M5 receptor internalization.
We investigated the kinetics of agonist-induced internalization of human muscarinic M1–M5 receptors stably expressed in CHO cells. CHO cells expressing muscarinic M1–M5 receptors were incubated with the muscarinic receptor agonist carbachol (1 mM) for various times up to 4 h and then receptor binding at the cell surface was measured using a single concentration of [3H]NMS (1.6 nM). As shown in Fig. 1, these data were consistent with a first-order decay process.
The estimates of the rate constant
Discussion
Each subtype of human muscarinic receptor was stably expressed in CHO cells using a viral promoter (i.e., CMV). Consequently, the wild-type transcriptional control of the receptor subtypes was lost. However, agonist-dependent internalization, recycling and downregulation of muscarinic receptors was observed and several proteins that play a role in these agonist-dependent processes (e.g., G protein-coupled receptor kinases, β-arrestin, clathrin, dynamin, etc.) are expressed in CHO cells (Gaborik
Acknowledgements
This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant 1R15-NS057742].
We would like to thank Crystal A. Shults for her excellent technical assistance and Drs. Fred Ehlert and Craig W. Stevens for reading and critiquing our manuscript.
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