G protein coupling to M1 and M3 muscarinic receptors in sublingual glands

Rat sublingual glandM₁ and M₃ muscarinic receptors each directlyactivate exocrine secretion. To investigate the functional role ofcoreceptor expression, we determined receptor-G protein coupling.Although membrane proteins of 40 and 41 kDa are ADP-ribosylated bypertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), bothcarbachol-stimulated high-affinity GTPase activity and the GTP-inducedshift in agonist binding are insensitive to CTX or PTX. Carbacholenhances photoaffinity labeling([-³²P]GTP-azidoaniline) of only 42-kDa proteins thatare subsequently tractable to immunoprecipitation by antibodiesspecific for G_q or G₁₁ but notG₁₂ or G₁₃. Carbachol-stimulatedphotoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate(PIP₂) hydrolysis is reduced 55% and 60%, respectively,by M₁ receptor blockade with m1-toxin.G_q/11-specific antibody blocks carbachol-stimulated PIP₂ hydrolysis. We also provide estimates of the molarratios of receptors to G_q and G₁₁.Although simultaneous activation of M₁ and M₃receptors is required for a maximal response, both receptor subtypesare coupled to G_q and G₁₁ to stimulateexocrine secretion via redundant mechanisms.

     

The internalization of the M2 and M4 muscarinic acetylcholine receptors involves distinct subsets of small G-proteins

Multiple mechanisms exist for the endocytosis of receptors from the cell surface. While the M1, M3, and M4 subtypes of muscarinic acetylcholine receptor and M4 receptors transduce their signals through the same second messengers but internalize though different pathways, we tested the ability of several small G-proteins to regulate the agonist-induced endocytosis of M2 and M4 in JEG-3 human choriocarcinoma cells. Dominant-negative Rab5 as well as both wild-type and dominant-negative Rab11 inhibited M4 but not M2 endocytosis. In contrast, a dominant-negative Arf6 as well as wild-type Rab22 increased M2 but not M4 endocytosis. We used immunocytochemistry to show that in unstimulated cells, the M2 and M4 receptors co-localize on the cell surface, whereas after stimulation M2 and M4 are in distinct vesicular compartments. In this study, we demonstrate that agonist-induced internalization of the M2 receptor utilizes an Arf6, Rab22 dependent pathway, while the M4 receptor undergoes agonist-induced internalization through a Rab5, Rab11 dependent pathway. Additionally, we show that Rab15 and RhoA are not involved in either pathway in JEG-3 cells.     

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.     

Pronounced conformational changes following agonist activation of the M(3) muscarinic acetylcholine receptor

The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M(3) receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M(3) receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M(3) receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.     

Transient hypoxia induces sequestration of M1 and M2 muscarinic acetylcholine receptors

Oxidative stress has been implicated in impairing muscarinic acetylcholine receptor (mAChR) signaling activity. It remains unclear, however, whether alterations in the cell surface distribution of mAChRs following oxidative stress contribute to the diminished mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient hypoxia was associated with an increase in phosphorylation of these receptors. Over-expression of a catalytically inactive G protein-coupled receptor kinase 2 (GRK2 K220R) blocked the increased phosphorylation and sequestration of the M2, but not M1, mAChRs following transient hypoxia. Hypoxia induced phosphorylation and sequestration of the M1 mAChR was, however, blocked by over-expression of a catalytically inactive casein kinase 1 alpha (CK1alpha K46R). These results are the first demonstration that M1 and M2 mAChRs undergo sequestration following transient hypoxia. The data suggest that increased phosphorylation of M1 and M2 mAChRs underlies the mechanism responsible for sequestration of these receptors following transient hypoxia. We report here that distinct pathways involving CK1alpha and GRK2 mediated sequestration of M1 and M2 mAChRs following transient hypoxic-induced oxidative stress.     

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists

Background and Objective

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M₁ mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).

Methods

Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M₁ mAChR, respectively. The optimized FRET receptor construct (M₁-cam5) was expressed stably in HEK293 cells.

Results

The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα_q/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M₁ FRET (F_EYFP/F_ECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.

Conclusion

The M₁ FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M₁ mAChR activation.     

M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways

When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades.     

Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice

Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M?, M?, and M? muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R(-/-), M3R(-/-), or M5R(-/-) mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R(-/-), M3R(-/-), M5R(-/-), and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M(3) receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R(-/-), M5R(-/-), and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R(-/-) mice. Deletion of either M?, M?, or M? receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M? receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M? nor M? receptors appear to be involved in cholinergic responses of the three vascular preparations tested.     

Activation of M3 muscarinic acetylcholine receptors inhibits voltage-dependent calcium influx in small cell lung carcinoma

Small cell carcinoma of the lung (SCC) expresses several characteristics of neuronal cells, including voltage-gated Ca2+ channels (VGCC), and also expresses muscarinic acetylcholine receptors (mAChR). In testing the possibility that VGCC may be functionally coupled to mAChR in SCC cell lines, we found that depolarization-dependent Ca2+ influx was inhibited by carbachol (IC50 = 0.78 microM) and oxotremorine (IC50 = 0.69 microM). Equilibrium dissociation constants for several mAChR antagonists indicated that a mAChR of M3 subtype was involved. Exposure of SCC to carbachol induced the hydrolysis of phosphoinositides and increased the cytosolic free Ca2+ concentration ([Ca2+]i). The carbachol-mediated inhibition of depolarization-dependent Ca2+ influx did not directly correlate with increased [Ca2+]i but did correlate with inositol poly-phosphate generation. The protein kinase C activators phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetyl-sn-glycerol neither mimicked nor amplified the inhibitory effect of carbachol on Ca2+ influx. However, phorbol 12-myristate 13-acetate suppressed the carbachol-induced inositol polyphosphate generation and inhibition of depolarization-dependent Ca2+ influx. The inactive compound 4 alpha-phorbol had no effect. These data suggest that the inhibition of VGCC caused by carbachol is not due to protein kinase C activation, but rather is due to events mediated by inositol polyphosphates. This is the first documentation of a role for phosphoinositide hydrolysis in the functional coupling of mAChR and VGCC. The expression of M3 mAChR functionally coupled to VGCC could have therapeutic implications for SCC, in light of recent demonstrations that cell proliferation can be influenced by activation of neurotransmitter receptors.     

A CalDAG-GEFI/Rap1/B-Raf cassette couples M(1) muscarinic acetylcholine receptors to the activation of ERK1/2

In this study we examine signaling pathways linking the M(1) subtype of muscarinic acetylcholine receptor (M(1) mAChR) to activation of extracellular signal-regulated kinases (ERK) 1 and 2 in neuronal PC12D cells. We first show that activation of ERK1/2 by the M(1) mAChR agonist carbachol takes place primarily via a Ras-independent pathway that depends largely upon Rap1, another small GTP-binding protein in the Ras family. Rap1 in turn activates B-Raf, an upstream activator of ERK1/2. Consistent with these results, carbachol was found to activate Rap1 more potently than Ras. Similar to other small GTP-binding proteins, activation of Rap1 requires a guanine nucleotide exchange factor (GEF) to promote its conversion from the GDP- to GTP-bound form. Using specific antibodies, we show that a recently identified Rap1 GEF, calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is expressed in PC12D cells and that carbachol stimulates the formation of a complex containing CalDAG-GEFI, Rap1, and activated B-Raf. Finally, we show that expression of CalDAG-GEFI antisense RNA largely blocks carbachol-stimulated activation of hemagglutinin (HA)1-tagged B-Raf and formation of the CalDAG-GEFI/Rap1/HA1-tagged B-Raf complex. Together, these data define a novel signaling pathway for M(1) mAChR, where increases in Ca(2+) and diacylglycerol stimulate the sequential activation of CalDAG-GEFI, Rap1, and B-Raf, resulting in the activation of MEK and ERK1/2.     

Evidence for the simulaneous translocation of muscarinic acetylcholine receptor and G protein by carbachol

Interactions between guanine nucleotide regulatory proteins (G proteins) and muscarinic acetylcholine receptors (mAChRs) were studied in vivo following carbachol treatment. Rat brain homogenates were separated by high speed ultracentrigation into heavy and light membrane and 300,000 g supernate franctions. The G proteins were partially purified by Sephadex-G200 and heptylamine-Sepharose and the mAChRs by (3,2′-aminobenzhydryloxy)-tropane-(ABT)-affinity chromatographies. Radioligand binding assays showed that acute carbachol induced a biphasic translocation of the mAChRs and G proteins into the light membrane fraction with an initial release at 5–10 min and a second phase at 60 min. Portions of the released mAChRs and the G proteins, were found in the 300,000 g supernates and light membranes and were eluted in the same peak fractions from a Sephadex G-200 column. This dually labelled peak dissociated in the presence of digitonin, suggesting close association between the mAChR and G protein. ABT-affinity chromatography yielded dually labelled mAChR-G protein fractions which eluated as a single radioactive peak on a second ABT column. the partially purified G proteins from these fractions were photoaffinity labelled with 8-azidoguanosine-5′-triphosphate, [γ-³²P]. SDS-PAGE autoradiography revealed the presence of G_0ξ and G_i which may be released simultaneously with the mAChRs from the plasma membrane. In addition, a 110,000 molecular weight polypeptide was dually labelled by [³H]-PrBCM and [γ-³²P]-8-azido-GTP suggesting the presence of a “mAChR-G protein complex.” These findings provide direct evidence for the release of mAChRs and G proteins and a mAChR-G protein complex by agonist occupation of the mAChRs.     

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis

Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes), scaffolded by hematopoietic protein 1 (Hem-1), that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE)2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference–mediated knockdown of Hem-1–containing complexes in neutrophil-like cells: (a) dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b) substantially weakens Rac activation and phosphatidylinositol-(3,4,5)-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5)-tris-phosphate)/Rac/F-actin–mediated feedback circuit that organizes the leading edge; and (c) prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1–containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.     

M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms.

Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (alpha), novel (delta), and atypical (iota) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 microM), calphostin C (IC50 = 2.4 microM), or chelerythrine (IC50 = 8.0 microM). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.     

Proliferative effect of acetylcholine on rat trachea epithelial cells is mediated by nicotinic receptors and muscarinic receptors of the M1-subtype

Acetylcholine (ACh), synthesized in mammalian non-neuronal cells such as epithelial cells of the airways, digestive tract and skin, is involved in the regulation of basic cell functions (so-called non-neuronal cholinergic system). In the present experiments rat trachea epithelial cells have been cultured to study the proliferative effect of applied ACh by [3H]thymidine incorporation. ACh (exposure time 24 h) caused a concentration-dependent increase in cell proliferation with a doubling of the [3H]thymidine incorporation at a concentration of 0.1 microM. This effect was partly reduced by 30 microM tubocurarine and completely abolished by the additional application of 1 microM atropine. The stimulatory effect of acetylcholine, remaining in the presence of tubocurarine, was prevented by 1 microM pirenzepine (preferentially acting at M1-receptors), but neither by 1 microM AFDX 116 (preferentially acting at M2-receptors) nor by 1 microM hexahydrosiladifenidol (preferentially acting at M3-receptors). The combination of tubocurarine and pirenzepine halved the basal [3H]thymidine incorporation. In conclusion, ACh produces a proliferative effect in rat trachea epithelial cells, the effect being mediated by both nicotinic receptors and muscarinic receptors of the M1-subtype.     

Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1-receptor selective antagonists pirenzepine (10 microM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 microM) and AF-DX 116 (10 microM). The M4-receptor selective antagonists tropicamide (1 microM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 microM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals.     

Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy.

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.     

Salmonella enterica serotype Typhimurium usurps the scaffold protein IQGAP1 to manipulate Rac1 and MAPK signalling

Salmonella enterica serotype Typhimurium invades eukaryotic cells by re-arranging the host-cell cytoskeleton. However, the precise mechanisms by which Salmonella induces cytoskeletal changes remain undefined. IQGAP1 (IQ motif-containing GTPase-activating protein 1) is a scaffold protein that binds multiple proteins including actin, the Rho GTPases Rac1 and Cdc42 (cell division cycle 42), and components of the MAPK (mitogen-activated protein kinase) pathway. We have shown previously that optimal invasion of Salmonella into HeLa cells requires IQGAP1. In the present paper, we use IQGAP1-null MEFs (mouse embryonic fibroblasts) and selected well-characterized IQGAP1 mutant constructs to dissect the molecular determinants of Salmonella invasion. Knockout of IQGAP1 expression reduced Salmonella invasion into MEFs by 75%. Reconstituting IQGAP1-null MEFs with wild-type IQGAP1 completely rescued invasion. By contrast, reconstituting IQGAP1-null cells with mutant IQGAP1 constructs that specifically lack binding to either Cdc42 and Rac1 (termed IQGAP1ΔMK24), actin, MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase] or ERK only partially restored Salmonella entry. Cell-permeant inhibitors of Rac1 activation or MAPK signalling reduced Salmonella invasion into control cells by 50%, but had no effect on bacterial entry into IQGAP1-null MEFs. Importantly, the ability of IQGAP1ΔMK24 to promote Salmonella invasion into IQGAP1-null cells was abrogated by chemical inhibition of MAPK signalling. Collectively, these results imply that the scaffolding function of IQGAP1, which integrates Rac1 and MAPK signalling, is usurped by Salmonella to invade fibroblasts and suggest that IQGAP1 may be a potential therapeutic target for Salmonella pathogenesis.