Role of the M1 receptor in regulating circadian rhythms.

Cholinergic stimuli are potent regulators of the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). Using a brain slice model, we have found that the SCN clock is subject to muscarinic regulation, a sensitivity expressed only during the night of the clock's 24-h cycle. Pharmacological and signal transduction characteristics are compatible with a response mediated by an M1-like receptor. Molecular manipulation of muscarinic receptors will provide important insights as to the receptor subtype(s) regulating circadian rhythms.

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons.

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.

Alteration of cardiovascular and neuronal function in M1 knockout mice.

We used gene targeting to generate mice lacking the M1 muscarinic acetylcholine receptor. These mice exhibit a decreased susceptibility to pilocarpine-induced seizures, loss of regulation of M-current potassium channel activity and of a specific calcium channel pathway in sympathetic neurons, a loss of the positive chronotropic and inotropic responses to the novel muscarinic agonist McN-A-343, and impaired learning in a hippocampal-dependent test of spatial memory.

Elucidating the role of muscarinic receptors in psychosis.

Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.

The M1 receptor is required for muscarinic activation of mitogen-activated protein (MAP) kinase in murine cerebral cortical neurons.

Muscarinic acetylcholine receptors (mAChR) in the central nervous system are involved in learning and memory, epileptic seizures, and processing the amyloid precursor protein. The M(1) receptor is the predominant mAChR subtype in the cortex and hippocampus. Although the five mAChR fall into two broad functional groups, all five subtypes, when expressed in recombinant systems, can activate the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway has been implicated in learning and memory, amyloid protein processing, and neuronal plasticity. We used M(1) knock-out mice to determine the role of this receptor subtype in signal transduction in the mouse forebrain. In primary cortical cultures from mice lacking the M(1) mAChR, agonist-stimulated phosphoinositide hydrolysis was reduced by more than 60% compared with cultures from wild type mice. Although muscarinic agonists induced robust activation of MAPK in cortical cultures from wild type mice, mAChR-mediated activation of MAPK was virtually absent in cultures from M(1)-deficient mice. These results indicate that the M(1) mAChR is the major subtype that mediates activation of phospholipase C and MAPK in mouse forebrain.

Identification of a basolateral sorting signal for the M3 muscarinic acetylcholine receptor in Madin-Darby canine kidney cells.

Muscarinic acetylcholine receptors (mAChRs) can be differentially localized in polarized cells. To identify potential sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M(2) subtype is sorted apically, whereas M(3) is targeted basolaterally. Expression of M(2)/M(3) receptor chimeras revealed that a 21-residue sequence, Ser(271)-Ser(291), from the M(3) third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M(3) sorting signal into the homologous regions of M(2) was sufficient to confer basolateral localization to this apical receptor. Sequences containing the M(3) sorting signal also conferred basolateral targeting to M(2) when added to either the third intracellular loop or the C-terminal cytoplasmic tail. Furthermore, addition of a sequence containing the M(3) basolateral sorting signal to the cytoplasmic tail of the interleukin-2 receptor alpha-chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M(3) basolateral sorting signal is dominant over apical signals in M(2) and acts in a position-independent manner. The M(3) sorting signal represents a novel basolateral targeting motif for G protein-coupled receptors.

M1 muscarinic acetylcholine receptors activate extracellular signal-regulated kinase in CA1 pyramidal neurons in mouse hippocampal slices.

Activation of extracellular signal-regulated kinases (ERK) is crucial for many neural functions, including learning, memory, and synaptic plasticity. As muscarinic acetylcholine receptors (mAChR) modulate many of the same higher brain functions as ERK, we examined mAChR-mediated ERK activation in mouse hippocampal slices. The cholinergic agonist carbachol caused an atropine-sensitive ERK activation in the dendrites and somata CA1 pyramidal neurons. To determine the responsible mAChR subtype, we combined pharmacologic and genetic approaches. Pretreatment with M1 antagonists inhibited ERK activation. Furthermore, mAChR-induced ERK activation was absent in slices from M1 knockout mice. ERK activation was normal in slices derived from other mAChR subtype knockouts (M2, M3, and M4), although these other subtypes are expressed in many of the same neurons. Thus, we demonstrate divergent functions for the different mAChR subtypes. We conclude that M1 is responsible for mAChR-mediated ERK activation, providing a mechanism by which M1 may modulate learning and memory.

Developmental expression of muscarinic acetylcholine receptors in chick retina: selective induction of M2 muscarinic receptor expression in ovo by a factor secreted by muller glial cells.

Muscarinic acetylcholine receptors (mAChRs) play an important role in signal processing in the retina. We have used subtype-specific antibodies to identify the changes in the localization of mAChR expression during embryonic development of the retina in vivo and their relationship to the changes in mAChRs in retinal cells in culture. We have demonstrated previously that treatment of fresh retinal cultures with conditioned media from mature retinal cultures specifically induces expression of the M(2) mAChR (McKinnon et al., 1998). We show that the M(2)-inducing activity, which we tentatively have called MARIA (muscarinic acetylcholine receptor-inducing activity) is produced by Müller glial cells in culture, because significant activity can be found in media conditioned by essentially neuron-free cultures of Müller glia, as well as by a Müller glial cell line but not several neuroblastoma cell lines. We also demonstrate that the appearance of the M(2) receptor in vivo occurs concomitantly with the appearance of significant numbers of Müller glial cells in the developing retina. Furthermore, the administration of crude or partially purified preparations of MARIA to developing chick embryos in ovo induces precocious expression of M(2) mAChRs in the appropriate cell types in the retina. These results show that a factor secreted by cultured retinal Müller glia can regulate M(2) mAChR expression in vivo and in vitro and suggest that the secretion of MARIA by Müller glia in vivo may be responsible for the normal induction of M(2) mAChR expression during embryonic development.

Multiple topological domains mediate subtype-specific internalization of the M2 muscarinic acetylcholine receptor.

Endocytosis of agonist-activated G protein-coupled receptors (GPCRs) is required for both resensitization and recycling to the cell surface as well as lysosomal degradation. Thus, this process is crucial for regulation of receptor signaling and cellular responsiveness. Although many GPCRs internalize into clathrin-coated vesicles in a dynamin-dependent manner, some receptors, including the M(2) muscarinic acetylcholine receptor (mAChR), can also exhibit dynamin-independent internalization. We have identified five amino acids, located in the sixth and seventh transmembrane domains and the third intracellular loop, that are essential for agonist-induced M(2) mAChR internalization via a dynamin-independent mechanism in JEG-3 choriocarcinoma cells. Substitution of these residues into the M(1) mAChR, which does not internalize in these cells, is sufficient for conversion to the internalization-competent M(2) mAChR phenotype, whereas removal of these residues from the M(2) mAChR blocks internalization. Cotransfection of a dominant-negative isoform of dynamin has no effect on M(2) mAChR internalization. An internalization-incompetent M(2) mutant that lacks a subset of the necessary residues can still internalize via a G protein-coupled receptor kinase-2 and beta-arrestin-dependent pathway. Furthermore, internalization is independent of the signal transduction pathway that is activated. These results identify a novel motif that specifies structural requirements for subtype-specific dynamin-independent internalization of a GPCR.

Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling.

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF- and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately N-terminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide.

Differential regulation of leukemia inhibitory factor-stimulated neuronal gene expression by protein phosphatases SHP-1 and SHP-2 through mitogen-activated protein kinase-dependent and -independent pathways.

The neurally active cytokine leukemia inhibitory factor (LIF) signals through a bipartite receptor complex composed of LIF receptor alpha (LIFR) and gp130. gp130 and LIFR contain consensus binding motifs for the protein tyrosine phosphatase SHP-2 surrounding tyrosines 118 and 115 (Y118 and Y115) of their cytoplasmic domains, respectively. These sites are necessary for maximal activation of mitogen-activated protein kinase (MAPK). Coexpression of catalytically inactive, but not wild-type, SHP-2 reduced LIFR- and gp130-mediated activation of MAPK up to 75%. Conversely, coexpression of the wild-type, but not catalytically inactive, SHP-1, a related phosphatase, reduced activity up to 80%, demonstrating that SHP-2 and SHP-1 have opposing effects on the MAPK pathway. Mutation of Y115 of the cytoplasmic domain of LIFR eliminates receptor-mediated tyrosine phosphorylation of SHP-2. In contrast, SHP-1 association with gp130 and LIFR is constitutive and independent of Y118 and Y115, respectively. SHP-1 has a positive regulatory role on LIF-stimulated vasoactive intestinal peptide (VIP) reporter gene expression in neuronal cells, whereas the effect of SHP-2 is negative. Furthermore, LIF-stimulated MAPK activation negatively regulates this VIP reporter gene induction. SHP-2 also negatively regulates LIF-dependent expression of choline acetyltransferase, but this regulation could be dissociated from its effects on MAPK activation. These data indicate that SHP-1 and SHP-2 are important regulators of LIF-dependent neuronal gene expression via both MAPK-dependent and -independent pathways.

Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene.

The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.

Muscarinic-induced modulation of potassium conductances is unchanged in mouse hippocampal pyramidal cells that lack functional M1 receptors.

Activation of muscarinic acetylcholine (ACh) receptors (mAChRs) increases excitability of pyramidal cells by inhibiting several K+ conductances, including the after-hyperpolarization current (Iahp), the M-current (Im), and a leak K+ conductance (Ileak). Based on pharmacological evidence and the abundant localization of M1 receptors in pyramidal cells, it has been assumed that the M1 receptor is responsible for mediating these effects. However, given the poor selectivity of the pharmacological agents used to characterize these mAChR responses, rigorous characterization of the receptor subtypes that mediate these actions has not been possible. Surprisingly, patch clamp recording from CA1 pyramidal cells in M1 knockout mice revealed no significant difference in the degree of inhibition of Iahp, Im, or Ileak by the mAChR agonist, carbachol (CCh), as compared with wildtype controls. In addition, the M1-toxin was not able to block CCh's inhibition of the Iahp, Im, or Ileak These data demonstrate that the M1 receptor is not involved in increasing CA1 pyramidal cell excitability by mediating ACh effects on these K+ conductances.

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells.

We have characterized the muscarinic ACh receptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK) strain II epithelial cells. Binding studies with the membrane-impermeable antagonist N-[(3)H]methylscopolamine demonstrated that mAChRs are approximately 2.5 times more abundant on the basolateral than on the apical surface. Apical, but not basolateral, mAChRs inhibited forskolin-stimulated adenylyl cyclase activity in response to the agonist carbachol. Neither apical nor basolateral mAChRs exhibited detectable carbachol-stimulated phospholipase C activity. Carbachol application to the apical or the basolateral membrane resulted in a threefold increase in intracellular Ca(2+) concentration, which was completely inhibited by pertussis toxin on the apical side and partially inhibited on the basolateral side. RT-PCR analysis showed that MDCK cells express the M(4) and M(5) receptor mRNAs. These data suggest that M(4) receptors reside on the apical and basolateral membranes of polarized MDCK strain II cells and that the M(5) receptor may reside in the basolateral membrane of a subset of cells.

Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca(2+) channels by using knockout mice.

There are five known subtypes of muscarinic receptors (M(1)-M(5)). We have used knockout mice lacking the M(1), M(2), or M(4) receptors to determine which subtypes mediate modulation of voltage-gated Ca(2+) channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca(2+) channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca(2+) channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M(2) receptor knockout mice; the slow and voltage-independent pathway is absent from the M(1) receptor knockout mice; and neither pathway is affected in the M(4) receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.

Molecular analysis of the regulation of muscarinic receptor expression and function.

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

Tyrosines 905 and 915 of gp130 are required for maximum induction of m2 muscarinic acetylcholine receptor and VIP gene transcription by cytokines in neuronal cells.

1. Leukemia inhibitory factor action is mediated by a heterodimeric receptor consisting of two subunits, gp130 and the low-affinity leukemia inhibitory factor receptor (LIFR). 2. We used chimeric receptors containing the intracellular domain of either the LIFR or gp130 to identify regions of the receptors required for induction of the m2 muscarinic acetylcholine receptor gene in IMR-32 and SN56 neuronal cells. 3. While chimeric receptors containing the intracellular domain of gp130 were able to induce transcription from both the m2 and the vasoactive intestinal peptide (VIP) gene promoters, chimeric receptors containing the intracellular domain of the LIFR were incapable of mediating induction of the m2 gene despite being able to induce VIP transcription. 4. Deletion and mutagenesis studies identified two tyrosines, Y905 and Y915, which were required for maximal induction of the m2 and VIP genes. 5. Because Y905 and Y915 are reported to be the only tyrosine residues in gp130 that bind Stat1, these results suggest that this transcription factor plays a key role in the induction of transcription of both the m2 and the VIP genes.

Molecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors.

The regulation of muscarinic acetylcholine receptor expression and function was investigated in cultured cells and in knockout mice. Muscarinic agonist exposure causes m2 receptor desensitization and sequestration and decreases the expression of cardiac potassium channels. The expression of m2 receptors in chick retina is regulated by a developmentally regulated secreted factor. Mice lacking the m1 receptor exhibit a loss of muscarinic regulation of M-current potassium channel activity and pilocarpine-induced seizures.

Raf-1 independent stimulation of mitogen-activated protein kinase by leukemia inhibitory factor in 3T3-L1 cells.

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulated Raf-1 activity in a time- and dose-dependent manner. Although phorbol ester failed to activate Raf-1 directly, a protein kinase C-stimulated signal was found to be necessary, but not sufficient, for LIF-mediated activation of Raf-1. Elevation of intracellular cAMP levels completely blocked Raf-1 activation by LIF, but was without effect on the magnitude of mitogen-activated protein kinase (MAPK) stimulation by the cytokine, suggesting the presence of a Raf-1-independent, cAMP-insensitive MAPK kinase kinase (MAPKKK) pathway in 3T3-L1 cells. Mono Q-fractionation of LIF-stimulated 3T3-L1 extracts identified a single peak of MAPKKK activity that was largely insensitive to elevated intracellular levels of cAMP, and that failed to correlate with stimulation of either Raf-1 or MEKK1 protein kinases. Our results demonstrate that LIF-mediated activation of the MAP kinase cascade in 3T3-L1 cells proceeds through both Raf-1-dependent and -independent pathways which differ in their sensitivity to inhibition by intracellular cAMP.