Associate editor: J. WessRegulation of muscarinic acetylcholine receptor signaling
Introduction
The muscarinic acetylcholine (ACh) receptors (mAChRs) mediate most of the actions of the neurotransmitter ACh in the CNS and peripheral nervous system, as well as in the end organs of parasympathetic nerves. In mammals, 5 distinct mAChR subtypes (M1–M5) have been identified, with each receptor subtype being the product of a different gene. The mAChR belongs to the superfamily of seven-transmembrane receptors, which activate signal transduction pathways through their interaction with heterotrimeric guanine nucleotide binding regulatory proteins (G proteins). The M1, M3, and M5 receptor subtypes are efficiently coupled to the pertussis toxin-insensitive Gαq/11 and Gα13 subtype G proteins, leading to, for example, activation of phospholipase C (PLC) and phospholipase D (PLD), while M2 and M4 receptors preferentially couple to pertussis toxin-sensitive Gi and Go proteins, leading to the inhibition of adenylyl cyclase Caulfield, 1993, Rümenapp et al., 2001.
The different mAChR subtypes display unique, but not exclusive, expression patterns in the CNS and peripheral organs, such as the heart, exocrine glands, and smooth muscle tissues Caulfield, 1993, Levey et al., 1991. Recent studies in genetically modified mice lacking one of the five mAChR subtypes have identified important, but hitherto unknown, functions of the various receptor subtypes in the CNS and peripheral nervous system. For example, M1 mAChRs in the CNS are the main mAChRs that mediate ACh-induced MAP kinase activation, a process essential for memory Hamilton & Nathanson, 2001, Hamilton et al., 1997. M1 mAChR deficiency also leads to significantly elevated dopaminergic transmission in the striatum and strongly increased locomotor activity Gerber et al., 2001, Miyakawa et al., 2001. In addition, M1 mAChR-deficient mice have an increased response to the stimulatory effects of amphetamine (Gerber et al., 2001). The brain M2 mAChRs contribute to the centrally mediated antinociception, and M2 mAChR knockout mice display disrupted agonist-induced tremor and attenuated agonist-induced hypothermia (Gomeza et al., 1999a). In addition, M2 mAChRs are essential for mAChR-dependent bradycardia and contribute (although only to a small extent) to agonist-induced contraction of stomach fundus, urinary bladder, and trachea (Stengel et al., 2000). The M3 mAChRs play a key role in salivary secretion, pupillary constriction, and bladder detrusor contraction in mice (Matsui et al., 2000). Importantly, M3 mAChRs are involved in the regulation of food intake and appetite: mice deficient in M3 mAChRs display a significant decrease in food intake, reduced body weight, and peripheral fat deposits (Yamada et al., 2001). Like the M1 mAChRs, brain M4 mAChRs are involved in the modulation of central dopaminergic responses: M4 mAChR-deficient mice show an increase in basal locomotor activity and greatly enhanced locomotor responses after activation of D1 dopamine receptors (Gomeza et al., 1999b). M4 mAChRs, however, appear to play a negligible role in regulating peripheral smooth muscle tone (Stengel et al., 2000). Finally, M5 mAChRs facilitate mAChR-induced dopamine release in the striatum and modulate both morphine reward and withdrawal processes (Basile et al., 2002). In addition, M5 mAChRs are required for cholinergic dilation of cerebral blood arteries and arterioles (Yamada et al., 2001).
A general feature of mAChRs and other G protein-coupled receptors (GPCRs) is that they are highly regulated. Impressive advances have been made in understanding the molecular mechanisms that regulate mAChR signaling. As the mAChRs are widely regarded as paradigms for other GPCRs, especially those that efficiently couple to Gαi and Gαq/11 proteins, this knowledge applies to a large number of other GPCRs as well. In the present review, we have summarized our current understanding of the regulation of the various mAChR subtypes.
Section snippets
Classical orthosteric muscarinic acetylcholine receptor ligands
As indicated above, mAChRs can interact with different G proteins to influence multiple effector activities. The final outcome of receptor stimulation with respect to the different effector systems is not only dependent on the concentration and intrinsic efficacy of the agonist. Over the last decade, substantive evidence has accumulated that GPCRs, including mAChR, can adopt multiple receptor conformations. These different receptor conformations are either generated spontaneously or
Regulation of muscarinic acetylcholine receptor oligomerization
The pioneering studies by Wess and colleagues have demonstrated that in a heterologous expression system of COS-7 cells, M3 mAChRs are able to form functional dimers Maggio et al., 1993a, Maggio et al., 1993b, Schöneberg et al., 1995. As different subtypes of muscarinic receptors are coexpressed in the same cell type in several tissues, for example, M1 and M2 mAChRs in neurons and M2 and M3 mAChRs in smooth muscle cells, it has been proposed that mAChR subtypes may heterodimerize. However,
Protein kinase C and G protein-coupled receptor kinases
Although ACh is rapidly hydrolyzed after release, desensitization of mAChRs occurs under physiological conditions, as the vagal nerves are tonically active in the intact animal. Furthermore, in vivo administration of muscarinic receptor antagonists blocking the action of released ACh or inducing the inactive state of the mAChR can evoke mAChR up-regulation Wall et al., 1992, Witt-Enderby et al., 1995. As with a large number of GPCRs, agonist-induced desensitization of mAChRs usually involves
Heterotrimeric G proteins, receptor kinases, β-arrestin, and clathrin
Prolonged agonist activation of mAChRs does not only facilitate uncoupling of the receptor from the G protein, but can also induce mAChR internalization in a large number of cell types (Koenig & Edwardson, 1996). An important question has been whether mAChR internalization requires activation of the receptors by agonists and activation of the heterotrimeric G proteins. This does not seem to be the case. First, incubation of HEK293 cells expressing M1 mAChRs with the muscarinic antagonist N
Role of receptor internalization and recycling in muscarinic acetylcholine receptor desensitization and resensitization
It is now well established that internalization and recycling of most GPCRs is required for resensitization of the desensitized receptors Pippig et al., 1995, Ferguson, 2001. It is envisaged that receptor activation stimulates receptor phosphorylation and the binding of β-arrestin to the phosphorylated receptor, which, in turn, causes uncoupling of the receptor from its G protein and internalization into the cell interior. In an endocytic compartment, a GPCR phosphatase, designated as GRP,
Role of receptor phosphorylation and internalization in muscarinic acetylcholine receptor-mediated activation of ERK1/2
The traditional model of GPCR signaling has been that receptor signaling arises from the ability of the activated receptor to catalyze the exchange of GDP for GTP on the heterotrimeric G proteins, whose dissociated subunits interact with effector molecules. Over the last several years, it has become clear that receptor phosphorylation and β-arrestin binding to the phosphorylated receptors may not only uncouple the receptors from the heterotrimeric G proteins, but can also initiate other
Homologous and heterologous down-regulation of muscarinic acetylcholine receptors
In many cells, exposure to muscarinic agonists induces rapid desensitization and internalization of the cell surface mAChRs that is followed by a loss of total number of cellular receptors in the following hours. This process, also termed down-regulation, can only be overcome by de novo receptor synthesis. Based on studies using cell permeable inhibitors of de novo protein synthesis and lysosomal proteolysis, increased receptor degradation is thought to be an important underlying mechanism for
Heterologous regulation of M2 muscarinic acetylcholine receptor gene expression
A large number of groups have been investigating whether expression of the PLC-coupled M1 and M3 mAChRs is subject to negative feedback inhibition via activation of the 1,2-diacylglycerol-PKC pathway. Indeed, treatment of cells with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) reduces the number of mAChR binding sites in the human embryonic pituitary cell line Flow 9000 (Lo & Hughes, 1988), mouse neuroblastoma N1E-115 cells (Liles et al., 1986), and human embryonic lung HEL299
Is muscarinic acetylcholine receptor internalization required for muscarinic acetylcholine receptor down-regulation?
Although it seems plausible to assume that receptor down-regulation requires prior internalization of the receptors into the cell interior, several reports suggest that mAChR down-regulation proceeds independent of receptor internalization. First, deletion of a large portion of the third cytoplasmic loop of the mouse M1 mAChR strongly reduces agonist-induced M1 mAChR down-regulation without affecting short-term receptor internalization in Y1 adrenal carcinoma cells (Shapiro & Nathanson, 1989).
Muscarinic acetylcholine receptors can induce down-regulation of signal transduction elements
While it is well recognized that down-regulation of GPCRs is predominantly associated with receptor loss at the cell surface, reduced mAChR responses may also result from a loss of downstream signal transduction elements. For example, prolonged activation of human M1 or M3 mAChRs in CHO cells does not only induce mAChR down-regulation, but also accelerate degradation of Gαq/11 proteins Mitchell et al., 1993, Mullaney et al., 1993, Van de Westerlo et al., 1995. Down-regulation of Gαq/Gα11 in CHO
Regulation of G protein-independent muscarinic acetylcholine receptor signaling
Recently, it has been shown that GPCRs are able to signal independently of heterotrimeric G proteins (for a review, see Brzostowski & Kimmel, 2001). In the case of mAChRs, there have only been a few studies that provide evidence for interaction of mAChRs with proteins other than heterotrimeric G proteins, kinases, and β-arrestins. Ilouz et al. (1999) and Linial et al. (1997) have shown in rat brain synaptosomes that presynaptic M2 (and to a lesser extent M4) mAChRs are in a high-affinity state
Muscarinic acetylcholine receptor up-regulation by muscarinic acetylcholine receptor antagonists
Chronic treatment with muscarinic antagonists (inverse agonists) such as atropine for up to 14 days induces a dose-dependent increase (up ∼100%) in mAChR number in several brain regions, heart, and airways Takeyasu et al., 1979, Wall et al., 1992, Westlind et al., 1981, Wise et al., 1980, Witt-Enderby et al., 1995. This increase in receptor number may be due in part to the inverse agonist activity of the muscarinic antagonist, which increases the proportion of receptors in the “inactive”
Regulation of intracellular muscarinic acetylcholine receptor distribution
Several studies have provided evidence for asymmetric distribution of mAChRs in polarized cells. In pancreatic and lacrimal acinar cells, release of Ca2+ following mAChR stimulation is selectively initiated in the luminal domain Tan et al., 1992, Toescu et al., 1992. In the CNS neurons, M1 mAChRs are most commonly found postsynaptically on somata and dendrites, whereas M2 mAChRs are located largely presynaptic (Levey et al., 1991). In polarized Madin-Darby canine kidney cells, endogenously
Functional activity of newly synthesized muscarinic acetylcholine receptors
There have been only a few published studies on the capacity of newly synthesized mAChRs to respond to stimulation. These studies have shown that mAChRs can be synthesized in a form that has diminished functional activity. This form of the receptor is slowly converted to a more physiologically active form Hunter & Nathanson, 1986, Ikegaya & Nathanson, 1993, Taylor et al., 1979. Unfortunately, during the past years, the underlying mechanism for this delayed acquisition of function has remained
Sensitization of muscarinic acetylcholine receptor-mediated phospholipase C activation
In contrast to rapid desensitization of mAChR-stimulated PLD (see Section 4.4), short-term exposure of M2 or M3 mAChRs to carbachol for 2 min followed by agonist washout can lead to a long-lasting, ∼2- to 3-fold increased PLC response upon a second mAChR challenge in HEK293 cells Schmidt et al., 1995, Schmidt et al., 1996, Schmidt et al., 1998. This potentiation of the PLC response is not restricted to mAChRs, but is also observed following prior activation of endogenously expressed P2Y
Concluding remarks and future studies
In this review, we have summarized our knowledge about the regulation of mAChR function. Research has generated a wealth of information, particularly on mAChR desensitization and internalization. In the years to come, several topics on mAChR signaling may deserve more attention, including compartmentation of receptors in signaling complexes, long-term potentiation of PLC by receptor activation, non-clathrin-mediated receptor internalization, functional consequences of differential receptor
Acknowledgements
The authors are supported by grants from the Deutsche Forschungsgemeinschaft and the IFORES program of the Universitätsklinikum Essen, Germany. This article is dedicated to the memory of Prof. E.J. Ariens.
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