Muscarinic acetylcholine receptor-interacting proteins (mAChRIPs): targeting the receptorsome

Current Drug Targets, 2011, 12, 00-00 1 Muscarinic Acetylcholine Receptors Interacting Proteins (mAChRIPs): Targeting the Receptorsome D. O. Borroto-Escuela1, Luigi F. Agnati2, Kjell Fuxe1 and F. Ciruela*,3 1 Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 17177 Stockholm, Sweden; 2IRCCS San Camillo, Lido Venezia, Italy; 3Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, Universitat de Barcelona, L’Hospitalet de Llobregat, 08907 Barcelona, Spain Abstract : Muscarinic acetylcholine receptors comprise a large family of G protein-coupled receptors that are involved in the regulation of many important functions of the central and peripheral nervous system. To achieve such a large range of physiological effects, these receptors interact, in addition to the canonical heterotrimeric G proteins, with a large array of accessory proteins including scaffold molecules, ion channels and enzymes which operate as molecular transducers of muscarinic function. Interestingly, as demonstrated for others G protein-coupled receptors this type of receptors are also able to oligomerize, a fact that has been shown to play a critical role in their subcellular distribution, trafficking, and finetuning modulation of cholinergic signalling. On the other hand, the specificity of these receptor interactions may be largely determined by the occurrence of precise protein-interacting motifs, posttranslational modifications, and the differential tissue distribution and stoichiometry of the receptor-interacting proteins. Thus, the exhaustive cataloguing and documentation of muscarinic acetylcholine receptors interacting proteins and the grasp of their specific function will explain some key physiological differences in muscarinic-mediated cholinergic transmission. Overall, a better comprehension of the muscarinic receptor interactome will have by sure a great impact on the cholinergic pharmacology and thus providing previously unrealized opportunities to achieve greater specificity in muscarinic-related drug discovery and diagnostic. Keywords: G protein-coupled receptors, muscarinic acetylcholine receptors, interacting proteins, dimerization, interactome, signal transduction, intracellular loops, scaffold proteins. INTRODUCTION Acetylcholine, by activating muscarinic acetylcholine receptors (mAChRs), has been shown to mediate various functions in the central and peripheral nervous systems (CNS and PNS, respectively) [1]. mAChRs belong to the class I of heptahelical, transmembrane G protein-coupled receptors (GPCRs) [2], which are activated by both endogenously produced acetylcholine and exogenously administered muscarinic compounds. Pharmacological, anatomical and molecular studies have demonstrated the existence of five mAChRs subtypes, namely M1R, M2R, M3R, M4 R and M5R. Uncovering the physiological roles of the distinct mAChRs subtypes has been possible thanks to the gradual development of more selective receptor agonists and antagonists, and also by the detailed phenotypic characterization of knockout mice [3]. Thus, different experimental approaches, including immunohistochemical and mRNA hybridization studies, showed that mAChRs are present in virtually all organs, tissues, or cell types (Table 1) [4-6]. In the CNS and PNS mAChRs have discrete distribution and they are expressed pre- and postsynaptically [2]. On the other hand, peripheral mAChRs mediate the classical muscarinic actions of acetylcholine on organs or tissues that are innervated by parasympathetic nerves (Table 1). Consequently, mAChRs are found in visceral smooth muscle, in cardiac muscle, in *Address correspondence to this author at the Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina-Bellvitge, Pavelló de Govern, Av. Feixa Llarga, s/n, 08907 L'Hospitalet del Llobregat, Barcelona, Spain; Tel: +34-934024280/+34934035820; Fax: +34-934029082; E-mail: fciruela@ub.edu 1389-4501/11 $58.00+.00 secretory glands, and in the endothelial cells of the vasculature [7, 8]. Except for endothelial cells, all the sites mentioned above receive cholinergic innervations. Thus, the most common responses mediated by these peripheral mAChRs include reduction of heart rate, stimulation of exocrine glandular secretion, vasodilatation and smooth muscle contraction (Table 1). On the other hand, central mAChRs are involved in the regulation of an extraordinarily large number of cognitive, behavioural, sensory, motor, and autonomic functions [3]. Finally, it is important to mention here that there are also evidences that mAChRs are expressed in several non-innervated tissues and cells [9, 10] as well as in primary and metastatic tumour cells, where they act as inductors of transformation, growth and proliferation [11, 12]. Each mAChR subtype is characterized by its differential selectivity for heterotrimeric G protein coupling (Table 1). Thus, M1 R, M3 R and M5R are mostly coupled to Gαq/11 proteins which stimulate phospholipase C activity and results in the generation of inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG), thus ending in the mobilization of intracellular Ca2+ and in the activation of protein kinase C (PKC) [13]. On the other hand, M2 R and M4 R are coupled to Gαi/o proteins which inhibit adenylate cyclase. Also, the activation of these receptors prolongs the opening of potassium channel, non-selective cation channel, and transient receptor potential channel opening [14, 15]. Overall, by this differential G protein coupling mAChRs can trigger distinct, and eventually opposed, signalling pathways in response to the same stimuli (e.g. acetylcholine) when co-expressed in the same cells. As a consequence, by controlling related © 2011 Bentham Science Publishers 2 Current Drug Targets, 2011, Vol. 12, No. 13 Table 1. Borroto-Escuela et al. G Protein Association, Tissue Distribution and Physiology of Muscarinic Acetylcholine Receptor Subtypes in Neural and Non-Neuronal Systems Subtype G protein Organ and tissue Physiological effects M1R Gαq/11 Autonomic ganglion including myenteric plexus, cerebral cortex, canine saphenous vein, Lymphocyte and keratinocyte Depolarization, contraction, increased gastric acid secretion, IL-2 production, proliferation M2R Gαi/o Heart, atrium, ileum smooth muscle, sinu-atrial node Reduce Ach release, contractile force and rate M3R Gαq/11 Urinary bladder, iris circular muscle, blood vessels-endothelium, Smooth muscle and salivary glands, pancreatic β cells, Lymphocyte and keratinocyte, colon and thyroid cancer cells Contraction, vasodilatation via release of NO, inhibition of apoptosis, inhibition of cell migration, proliferation M4R Gαi/o Corpus striatum, Uterus (guinea pig), lung, muscle (rabbit), keratinocyte Contraction, NO-dependent relaxation, cell migration M5R Gαq/11 Substantia nigra pars compacta, ventral tegmental area, brain microvasculature Growth and proliferation second messengers mAChRs are able to regulate a large array of signalling processes [16-18]. Although the first proteins found to functionally interact with mAChRs were, of course, G proteins, an increasing amount of evidence are now suggesting that this simplistic stoichiometric model defined as “one receptor-one G protein-one effector” no longer exists. Interestingly, a large number of proteins have been shown to interact with GPCR in general and with mAChRs in particular. Thus, the interaction with these proteins (i.e. scaffolding or accessory proteins) and also the oligomerization with other GPCRs, determine in part the mAChRs signalling efficiency and specificity. As a consequence, receptors are now considered as complex signalling units (i.e. signallosomes or recaptorsomes) that dynamically couple to multiple G proteins and other molecules. Thus, the spatiotemporal regulation of these interactions will be responsible for the formation of receptor entities with specific pharmacological profile and functional characteristics [19]. In the present review, effort will be made to discuss all the mAChR interacting partners, and special attention will be paid to these that link the receptors to alternative signalling pathways beyond G proteins [20]. Also, in addition to emphasize on the role of these accessory proteins in mAChRs-mediated signal transduction we will discuss how receptor oligomerization might impinge in mAChR functioning. Overall, such knowledge will allow us to address some fundamental questions concerning the molecular mechanisms hidden behind the pharmacological properties of each particular mAChR subtype, and thus providing new insights into the large array of muscarinic-mediated physiological and pathophysiological processes. MOLECULAR ASPECTS INTERACTIONS BEHIND RECEPTOR A critical question in GPCRs signalling is to know what determines the specificity of a named signal transduction process. Thus, in contrast with the classical model “one receptor-one G protein-one effector” mentioned above [21], nowadays the trend when analyzing a GPCR signal transduction system is to knowhow receptor accessory proteins impinge in the transduction process. Extensive studies concerning GPCR signalling mechanisms have demonstrated that the cytoplasmatic face of these receptors and, particularly, the third intracellular loop (3IL) and the Cterminal tail, play a critical role both in mediating the signal transfer to G proteins and in the direct interactions with accessory proteins [22]. On the other hand, the receptor transmembrane domains have been shown to participate in hydrophobic protein-protein interactions with plasma membrane proteins (e.g. GPCRs and ion channels). Interestingly, sequence analysis of the cytoplasmatic hydrophilic domains of the five muscarinic receptor subtypes reveals high sequence similarity (~90% of amino acid identity) in the case of the first and second intracellular loops. In contrast, a lower similarity is observed when the 3IL is analyzed (~40% of amino acid identity and high variability in length). In addition, a close analysis of the charge distribution (e.g. amino acids with potential positive and negative charge) showed a great number of clustered charges within the 3IL and the C-terminal tail, an issue that has been involved in coulombic epitope-epitope interactions [23]. The GPCR 3IL play a key role in the G protein activation and multiprotein complex formation, thus controlling the signal transduction mediated by the receptor. Indeed, sitedirected mutagenesis studies demonstrated that the Nterminal portion of the 3IL of some GPCRs in general and of the mAChRs in particular represents a fundamental structural determinant for direct G protein coupling (Fig. (1)) [24, 25]. Thus, insertion mutagenesis studies with the rat M3R suggested that this region forms an amphiphilic alpha helix and that the hydrophobic side of this helix represents an important G protein recognition surface (Fig. (1)). Interestingly, further analysis of this receptor segment showed that the Tyr-254 located at the N-terminus of the 3IL and the Ala-Ala-Gln-Thr-Leu (AAQTL) motif at the Cterminal of the 3IL of the M3 R (Fig. (1); blue encircled Y254 and AAQTL amino acids, respectively) play a key role in M3R-mediated Gαq activation [26]. In contrast, a short sequence motif (Val-Thr-Ile-Leu) located at the junction between the TM VI and the 3IL of the M2R is responsible for the recognition of the C-terminus of Gαi/0. On the other hand, expression of a peptide containing the entire 3IL of mAChRs abrogated the interaction of these receptors with their respective G proteins and also disrupted the signalling of other GPCRs that couple to the same G protein population mAChR Interacting Proteins Current Drug Targets, 2011, Vol. 12, No. 13 3 Fig. (1). Scheme showing the M3R sequence motifs associated with protein-protein interactions. A serpentine two-dimensional representation of the M3R sequence with the extracellular space at the top and the intracellular space at the bottom is shown. The residues within the intracellular domains of the receptor that are involved in protein-protein interactions are encircled. The most relevant binding motifs are depicted in filled circles (A: receptor-receptor interface interaction; B: G protein βγ subunit; C: GRK2; D: Gαq/11; E: oncogene SET protein; F: calmodulin; G: RhoA; H: CK1α). Note that in the N-terminal region of the 3IL some recognition sites overlap. [9, 27, 28]. Overall, the mAChRs 3IL, which also binds to Gβγ, calmodulin and RGS protein [29, 30], can be considered as a receptor intracellular antagonist that targets Gαq/11 proteins. The C-terminal domain of GPCRs is recognized as one of the most important domains for the regulation of GPCR function [22]. Thus, this region is implicated in posttranslation modifications and it is crucial for receptor phosphorylation, desensitization and trafficking in some GPCRs [31]. Interestingly, while a large array of interacting proteins has been described for many GPCRs (i.e. adenosine receptors [32]) in the case of mAChRs only a few as been reported. The sequence, the length, and the different recognition motifs involved in protein-protein interactions are specific for each GPCR C-terminus. The predicted Cterminal tail of the muscarinic receptors is a highly conserved domain both in sequence similarity and in length. This domain contains a conserved region following the seventh transmembrane domain composed of a cluster of basic amino acids that confer anti-apoptotic properties to the receptor, but the molecular mechanisms still remains known [33]. Overall, the ability of mAChRs to interact with different proteins probably begins at the sequence motif level of both the cytoplasmatic face and the transmembrane domains of the receptor. Indeed, in the cytoplasmatic domain of mAChRs some clusters of charged amino acid have been identified and several post-translational modifications have been shown to occur (Fig. (1)). Thus, it is believed that these features are directly involved in the modulation of the docking site for most of the protein-protein interactions occurring in the muscarinic receptor family. TRANSMEMBRANE MUSCARINIC INTERACTING PROTEINS RECEPTOR mAChRs Oligomerization It is well accepted that GPCRs interact with each other to form functional oligomers [34-36]. Although the existence of 4 Current Drug Targets, 2011, Vol. 12, No. 13 Borroto-Escuela et al. Fig. (2). Muscarinic receptor family homo and heterodimerization. (A) Dimerization of muscarinic acetylcholine receptor family can result from either covalent (disulfide crosslink) and non-covalent unions (electrostatic and hydrophobic union) between receptor protomers. (B) Pharmacological evidences of the formation of muscarinic M2 and M3 heterodimer. Displacement of specific [3H]-NM binding to cotransfected M2/M3 wild-type and M2/M3-tail fragment receptors by pirenzepine in COS-7 cells. (C) BRET experiments showing an agonisticindependent dimerization of M2 and M3 muscarinic receptor subtypes. Table 2. mAChRs Oligomerization Overview. The Ability of Homo- and Heterodimerize of mAChRs is Shown. Specificity of Hetero-Oligomers is Depicted by Listing Receptors that have been Reported not being able to form Hetero-Oligomers with Relevant Receptors Receptor Homo- Heterodimerization Specificity M1R [38] M2R [38] α1d-AR [207], Smoothened [38] M2R [41, 208-210] M1R [38], M3R [38, 42, 59, 211] δOR [210], κOR [210], µOR [210], α1d-AR [207], Smoothened [38] M3R [38, 211, 212] α2c-AR [40, 211, 212], M1R [38], M2R [38, 42, 211], M5R [9] M1R [213], M2R [214], V2 R [215], α1d-AR [207], Smoothened [38] M4R [216] 5-HT2CR [217] α1d-AR [207], D2LR [216] M5R [9] M3R [9] mAChR dimers was predicted from early pharmacological analysis, during the last decade the acceptance of mAChRs oligomerization has become more evident due it to the robust experimental data obtained through different experimental approaches such as co-immunoprecipitation experiments using differentially tagged receptors, resonance energy mAChR Interacting Proteins transfer (RET) approaches -i.e. fluorescence-RET (FRET) and bioluminescence-RET (BRET)- and cooperative ligand binding experiments [37, 38] (Fig. (2B/C)). Pioneering studies by the group of Wess have demonstrated that in a heterologous expression system (i.e. COS-7 cells) the M3R is able to form functional homodimers [39, 40]. Also, co-purification and co-immunoprecipitation experiments in Sf9 cells showed the formation of M2 R oligomers [41]. Therefore, as different subtypes of mAChRs are co-expressed in the same cell type -i.e. M1R and M2 R in neurons; M2R and M3 R in smooth muscle cells; and M3 R and M5 R in lymphocytic Tand B-cells- it has been speculated that these receptors might form functional oligomers in native tissue [37]. Indeed, Maggio and Wess showed that M2R/M3R oligomerization is receptor subtype specific and occurs in both transfected cells and native tissues [42]. Also, by means of a BRET approach it has been demonstrated that M1R, M2 R, M3R and M5R can form constitutive homo- and heterodimers in living HEK 293 cells [9, 38, 43]. Interestingly, the analysis of the BRET saturation experiments reveals the existence of high affinity M1R/M2R, M2R/M3R, and M1R/M3R oligomers but with relative affinity values slightly lower than those observed for mAChRs homodimers (Table 2) [38]. A relatively controversial issue concerning GPCR oligomerization is the effect of ligand challenge on receptor oligomerization. Thus, as demonstrated for other GPCRs the agonists challenge or M3R did not affect the receptor oligomerization (Fig. (2C)) [9]. Interestingly, a similar behaviour has been described for the M2 R where receptor oligomerization was independent of either carbachol or atropine treatment [44]. However, by means of ratiometric FRET was demonstrated that the organization of human M3R oligomer can be modified by ligand binding. Thus, carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Hence, by means of cell surface homogeneous time-resolved FRET and the SNAP-tag technology (a 20 kDa mutant of the DNA repair protein O6-alkylguanineDNA alkyltransferase that reacts specifically and rapidly with benzylguanine derivatives) [45] it has been identified that human M3R oligomers are selectively enhanced by agonists incubation. Thus, it is concluded that depending on the method used to study receptor oligomerization it is possible to determine more or less efficiently the effect of ligands on such receptor complexes [46], a fact that might be related to a fast transient association of GPCRs within the oligomer [47]. Nevertheless, the M2 R expressed in JEG-3 cells showed a higher carbachol-mediated receptor-downregulation profile than the M3R. Therefore, co-expression of M3R with increasing amounts of the M2 R in these cells resulted in an increased agonist-induced down-regulation of M3R, thus suggesting that the formation of the M2 R/M3 R oligomer constitute a new mechanism of mAChRs long-term regulation [38, 48]. In addition, and based on the high selectivity displayed by muscarinic toxins, Marquer et al. studied the effect of the MT7 toxin in the human M1 R oligomerization state in both membrane preparation and in living cells. Therefore, their results suggest that MT7 binds to a dimeric form of human M1R, thus favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the preexisting muscarinic receptor homodimers [49]. Somewhat in line with this results, using fluorescence correlation spectro- Current Drug Targets, 2011, Vol. 12, No. 13 5 scopy was demonstrated that M1 R predominate as monomers in the absence of ligands and dimerized upon pirenzepine binding, suggesting that fast ligand binding to a peripheral receptor site indicates a sequence of conformational changes that allows the ligands to access to inner regions of the protein and drive ligand-receptor complexes toward a high affinity dimeric state [50]. Not without some controversy [51], it is generally accepted that there are some classes of GPCRs where oligomerization already occurs [52]. From the evidence gathered so far, it appears that class A GPCRs oligomers are formed before or during translocation from endoplasmic reticulum/ Golgi to the plasma membrane [53]. However, measuring BRET or FRET signals in cell fractions collected from a sucrose gradient, used to separate cell surface proteins from intracellular material, revealed that oligomers could already be detected in light weight fractions -i.e. the β2-AR [54] and the α1A-AR [55] homodimers and the D1 R/D2 Rheterodimer [56]-, thus suggesting their existence at the cell surface level. Indeed, it has been postulated that in order to modulate GPCR function, the GPCR homo- and heterodimers may be interconnected at the plasma membrane, forming higherorder oligomers, which are also termed receptor mosaics (RMs) [57]. In principle, dimerization/oligomerization of mAChRs can result from either noncovalent (ionic or hydrophobic) or covalent association of the receptor protomers (Fig. (2A)). In the M3 R, a cysteine pair (Cys140 and Cys220) is thought to be critically involved in the formation of covalently linked M3R (Fig. (2A)) [58]. These two cysteine residues are highly conserved within GPCRs of the rhodopsin family, and are able to form an intramolecular disulphide bond, thus covalently linking the first and second extracellular loops. However, they may also participate in the formation of intermolecular disulfide bonds. Apart from covalent interactions mediated by cysteine residues, it has been demonstrated that the mAChR 3IL actively participates in the heterodimerization of M2R and M3 R by means of coulombic interactions (Fig. (2A)). Thus, deleting 196 amino acids from the 3IL of short chimeric α2-AR/M3 R resulted in the loss of intermolecular interaction compared with receptors that kept the entire loop [59]. Despite the extensive literature on dimerization in mAChRs, there has been little discussion on the nature and molecular mechanism of dimerization. Accordingly, two basic hypotheses have been proposed, contact dimers and domain-swapped dimers. Thus, the domain swapping dimerization mechanism was suggested by Gouldson et al., based on their bioinformatics approach and biochemical experiments by other groups. In this mechanism, a monomer is divided into two substructures or two domains, and a substructure of a monomer is exchanged with the corresponding substructure of the other monomer. Thus, a large conformational change of a monomer structure is required. Truncation mutation experiments by Maggio et al. in muscarinic receptors are often regarded as evidence for domain swapping model. On the other hand, in contact dimerization, a dimer is formed between 2 different TM bundles (monomers) with separate binding sites by packing at the interface that would face the lipid environment in the monomeric receptor. However, for mAChRs experimental and in silico 6 Current Drug Targets, 2011, Vol. 12, No. 13 effort to identify a possible dimer interface in different receptor subtypes are still inconclusive. Overall, it is considered that the domain contact mechanism is somehow necessary for nearly all GPCR oligomerization processes. At this point, the functional implications and biological significance of homo- and heterodimerization of mAChRs are still poorly understood. A large body of evidence has progressively substantiated the role of receptor oligomerization in different functional aspects of GPCRs [36, 48]. For instance, dimerization can be required for receptor maturation and correct transport from the endoplasmic reticulum to the plasma membrane. In addition, receptor dimerization can also influence ligand recognition by each of the constituent protomers [60]. Thus, ligand binding to one protomer within the oligomer can modify the pharmacological properties (i.e. increase or decrease its ligand binding affinities) of the counterpart protomer [61]. Interestingly, it is believed that these ligand-mediated allosteric receptor-receptor modulations are based in trans-conformational rearrangements occurring within the oligomer interface (for review see [47]). Thus, as ligand-binding sites for muscarinic receptors are buried in a hydrophobic pocket formed by transmembrane domains, it is feasible that interactions between protomers through such domains might rearrange the counterpart ligand-binding site and modify ligand selectivity and affinity [62, 63]. mAChRs/Ion Channels Oligomerization Interaction among members of divergent families of receptors represents an additional layer of complexity when considering the mechanisms of receptor signal transduction crosstalk in cells. Interestingly, some reports provide compelling biochemical and functional information about direct interaction between GPCRs and receptor ion channels [64, 65]. However, mAChR agonist-mediated modulation of ion channels appears to be complex. Thus, it was observed that potassium channels may be activated by M2R and M4R after agonist challenge, although in the rat superior cervical ganglion topographical constraint effects appear to limit the M2R effect [66]. While some decades ago it was demonstrated that the release of acetylcholine from the vagus nerve produced bradycardia it not was until the discovery and characterization of regulators of G protein signalling proteins (RGSs) that the kinetic mechanisms behind ACh-mediated heart deceleration was explained. Interestingly, the existence of G protein gated inwardly rectifying K+ (GIRK) channels and the activation by M2 R in atrial myocytes was also demonstrated [67]. Subsequently, it was shown that this M2R-mediated GIRK activation was membrane-delimited, mimicked by non-hydrolysable GTP analogues and also involved a direct interaction with the Gβγ dimer but not with the Gα subunit (Fig. (3)) [68, 69]. Also, by using the yeast two-hybrid method and the pull-down assay it was possible to demonstrate that the Gβγ directly interacts with the Cterminal tail of the GIRK1 protein [70] (Fig. (3)). In addition, it has been observed that RGSs accelerate the activation and deactivation kinetics of GIRK channels [71]. Thus, while the RGS4 accelerated all the PTX-insensitive Gαi/o-coupled GIRK currents the RGS7 accelerated the Gαo coupled GIRK currents. Also, the co-expression of Gβγ, in Borroto-Escuela et al. addition to enhancing the kinetic effects of RGS7, caused a significant reduction in the GIRK currents steady-state, thus indicating that RGS7- Gβγ complexes disrupt G αo coupling. Overall, these results provide further evidence for the existence of a GPCR/Gβγ/GIRK signalling complex that is targeted by the RGS proteins and that controls the GIRK channel gating. A further example of complex regulation between muscarinic receptors and receptor channels is the activation of endogenous transient receptor potential-canonical subtype 6 channels (TRPC6) in neuronal PC12D cells by the M1 R [72]. Thus, activation of TRPC6 channels correlates with the formation of a multiprotein complex containing M1R, TRPC6 channels and PKC. Interestingly, the M1 R/TRPC6/ PKC multiprotein complex formation is transient and reaches the highest levels after 2 min of M1R challenge. In addition, a PKC-mediated TRPC6 phosphorylation in a conserved serine residue within the carboxyl-terminal domain (S768 in the TRPC6A isoform and S714 in the TRPC6B isoform) plays a key role in this process. Also, the immunophilin FKBP12, the phosphatase calcineurin, and the Ca2+binding protein calmodulin are recruited by the M1 R/TRPC6/ PKC complex after M1 R challenge and these proteins remain stably associated with the TRPC6 channels after M1R and PKC have disassociated. Thus, the activated TRPC6 channels form the core of a dynamic multiprotein complex that includes PKC and calcineurin, which respectively phosphorylate and dephosphorylate the channels. Interestingly, phosphorylation of the TRPC6 channels by PKC is required for the binding of FKBP12, which in turn is required for the binding of calcineurin and calmodulin. Thus, subsequent dephosphorylation of the channels by calcineurin is required for the disassociation of M1 Rs [72, 73]. Overall, in the two examples mentioned above still are many mechanistic details (i.e. second messengers involved, etc..) that remain unclear. INTRACELLULAR PROTEINS ACCESSORY INTERACTING The Receptor 3IL: The Prime Target for mAChRs Interacting Proteins Nowadays, it is well accepted that the mAChR 3IL operates as a receptor hub sustaining multiple protein-protein interactions, therefore apart of being the main target domain of G proteins is also a binding site for different accessory proteins. Thus, the mAChR 3IL physically interacts with a large array of proteins, in some cases forming a multiprotein complex that bypass the need of coupling to the heterotrimeric G proteins for signalling. Interestingly, one of theses cases is the activation of small G proteins by a large number of the GPCR via the Gα subunit activation or by direct interaction with the receptor [74, 75]. The muscarinic-mediated small G proteins regulation has been typically viewed as a downstream consequence of heterotrimeric G protein activation, particularly G αq, Gα11 or Gα12 activation, as described for some GPCRs [76]. Gα12/13mediated RhoA activation involves direct interactions with RhoGEF proteins (RhoGEF, LARG and p115-RhoGEF) [7779]. Similarly, Gαq/11-coupled receptors can signal through Rho via direct coupling of Gαq/11 with Rho GEF [80, 81], albeit with less efficacy when compared with Gα12/13 [82]. mAChR Interacting Proteins Current Drug Targets, 2011, Vol. 12, No. 13 7 Fig. (3). Schematic of the role of RGS4 in KG channel activity. A, The muscarinic M2 GPCR (M2) is activated by the binding of ACh, which activates the associated G-proteins by catalyzing the exchange of GDP for GTP on the -subunit. This causes the dissociation of the ß dimer, which binds and activates the KG channel. RGS4 is inhibited by PIP3. B, As calcium (Ca2+) levels rise in the cell after depolarization, the Ca2+/calmodulin complex binds RGS4 to relieve the PIP3-mediated inhibition. This allows RGS4 to exert its GAP activity on the subunit, resulting in the hydrolysis of GTP. C, The GDP-bound -subunit promotes the reassociation of the heterotrimeric G-protein complex, leading to an inactivation of the KG channel. As intracellular Ca2+ levels decrease, Ca2+/calmodulin dissociates from RGS4, allowing PIP3 to bind and inhibit the GAP activity of RGS4. Interestingly, there are also evidences for a direct mAChR activation of small G proteins, for instance the activation of the phospholipase D (PLD) by the small G proteins (i.e. ARF and RhoA) via M2 R and M3R [16, 83, 84]. In addition, both ARF and RhoA can be co-immunoprecipitated with the 3IL of the M3 R (Borroto-Escuela, unpublished results). Also, dominant negative constructs of ARF1/6 and PLD1/2 proved that the characteristic brefeldin A (BFA)-sensitive PLD activation shown by the M3 R appears to involve ARF1mediated activation of PLD1, where additional ARF6mediated component may involve PLD1 or PLD2 [85, 86]. Mutation in the conserved NPxxY amino acid sequence in their transmembrane VII domain prevents the association of these receptors with ARF or Rho and also signalling to PLD [87] (Borroto-Escuela, unpublished results). Overall, these results point to the existence of a multiprotein M3 R containing complex that it is stabilized by the interaction of ARF and Rho with the carboxyl terminal part of the 3IL and the NPxxY conserved motif localized in the transmembrane domain VII of M3R. However, while the Gαq coupled-mAChRs activate RhoA it is proposed that Gαi coupled-mAChRs activate Rac1, a process that involves the Gβγ dimers and phosphatidylinositol 3-kinase (PI3K) [88]. Interestingly, it has been postulated that this RhoA-Rac1 selectivity can be tuned by RGS3 association, thus RGS3 can function as a molecular switch that changes the canonical Gαi coupled-mAChRs signalling from Rac1 to RhoA [89]. Similarly, using the 3IL as bait it has been examined the selective recruitment of RGS proteins (i.e. RGS1, RGS2, RGS4, and RGS16) to the different subtypes of mAChRs [90]. In brief, while the M2R do not binds RGS1, RGS2 or RGS16 the M1R binds only RGS2. Therefore, it has been postulated that RGS2 selectively interacts with the M1R and it acts as an effectors´ antagonist of phosphoinositide hydrolysis signal. On the other hand, it has been demonstrated that the knockdown of RGS3 and RGS5 alters the M3R-mediated Gαq activation in a receptorspecific manner [91]. In addition, it has been also demonstrated that RGS8 binds directly to the 3IL of the M1 R through the “MPRR” sequence at the N-terminus of RGS8 and this specifically inhibits the M1R signal transduction [92]. Interestingly, spinophilin has been identified as an RGS8 interacting protein and the RGS8 spinophilin-binding site is the “MPRR” sequence, thus both M1 R and spinophilin compete for the recruitment of RGS8 [93]. Overall, a model 8 Current Drug Targets, 2011, Vol. 12, No. 13 has been proposed in which the 3IL of mAChRs selectively recruit specific RGS protein(s) via their N-terminal, thus participating in the G protein-coupling and the regulation of receptor activation/desensitization mechanisms [30]. Another example of the involvement of accessory interating proteins in muscarinic-mediated signalling is the interaction of the oncogenic SET protein with the 3IL of M2R and M3R. The SET protein, also called TAF-1 (template activating factor I), was first described as part of the SET-CAN fusion gene in patients with acute undifferentiated leukaemia [90]. While several functions have been described for SET the cellular role of this protein still unclear. Thus, it has been demonstrated that SET is involved in the control of gene transcription as a member of the INHAT (inhibitor of histone acetyltransferase) complex, and also it has been considered an inhibitor of PP2A, a protein phosphatase involved in cell cycle progression and GPCR trafficking and regulation [94, 95]. Interestingly, by means of co-immunoprecipitation experiments and mass spectrometry it has been demonstrated a direct interaction of SET with the 3IL of M2R and M3R [96]. In addition, by means of siRNA knockdown experiments it has been demonstrated that SET acts to provide a brake on M3 R signalling, thus it has been proposed that SET interaction could impede the G protein coupling to the receptor. Interestingly, the epitope domain within the Cterminus of the M3R 3IL (I474-Q490) found to interact with SET is also involved of the G protein-M3R coupling, and thus responsible for the receptor activation [24, 58]. Alternatively, SET, by its ability to inhibit PP2A, may regulate the magnitude/duration of agonist-mediated receptor phosphorylation and thus play an important role in the M3 R signalling and trafficking observed following receptor challenge [97, 98]. Overall, the isolation of SET as a recaptor’s binding partner, as well as the functional characterization of this interaction, represents an unforeseen mechanism of mAChR regulation that might play a key role in both normal and pathological conditions. Similarly, a startling interaction between the M4R and the elongation factor 1A2 (eEF1A2) has been found both in vitro and in vivo [99]. Interestingly, the eEF1A is a GTP-binding protein that is essential in protein synthesis as it mediates the binding of the aminoacyl-tRNA to the acceptor site of the ribosome [100, 101]. Also, it is expressed only in skeletal and heart muscle, and in brain of adult mammals [102, 103]. Therefore, the M4R, via its 3IL, can modulate the protein translation and thus the synthesis of proteins through its direct interaction with eEF1A2. Finally, it has been demonstrated that calmodulin (CaM) can interact with the M1R and M3 R trough a motif located in the C-termini juxtamembrane region of the receptor 3IL (Fig. (1) and (3)) [104]. Interestingly, the CaM binding was found to be opposed to the PKC-mediated receptor phosphorylation, thus suggesting a role in receptor desensitization [105]. Also, this interaction may also be critical for mAChR-mediated ERK signalling pathway activation, which is dependent on Ca2+/CaM and involves agonist-mediated receptor internalization [106]. Regarding this last issue, it has been demonstrated that all arrestin subtypes directly interact with Ca2+/ CaM [107] and that the CaM binding site on arrestins overlaps with the binding site for GPCRs, thus indicating that both receptors and CaM will compete for the same arrestin binding site. Overall, it is suggested that the Borroto-Escuela et al. arrestin/CaM interaction might regulate the availability of these proteins to interact with GPCRs in general, and with M1R and M3R in particular, and thus tuning the receptor internalization and ERK1/2 signalling pathway activation. The Receptor C-Terminus While for most GPCRs the C-terminal tail constitutes the major recognition and anchoring motif for soluble intracellular accessory proteins (more than 50 interacting proteins have been identified so far) [19, 19, 108, 109] for mAChRs only a few partners have been described [110], and thus the main mAChR interacting domain still the 3IL (see above). Interestingly, some of the proteins involved in the post-endocytic sorting of GPCRs have been also shown to interact with the C-terminal tail of mAChRs [111]. For instance, an interaction with N-ethylmaleimide-sensitive factor (NSF), which is responsible for the β2-adrenergic receptor recycling [112], was identified in all members of the mAChR family [111, 112]. In contrast, sorting nexin 1 (SNX1), which was originally demonstrated to be required for the lysosomal sorting of the epidermal growth factor receptor, was suggested only to be involved in the lysosomal sorting of M1 R, M4R and M5R [111]. Finally, the GPCRassociated sorting protein (GASP), which was suggested to be involved in the preferential lysosomal sorting of the δopioid receptor [113], was confirmed to bind to all subtypes of muscarinic receptors [111]. Despite the mAChR C-terminal tail slight role of in the recruitment and anchoring of adaptor and scaffold proteins, it is well established that the C-terminal tail of the M3 R is an essential structural element for signalling to the antiapoptotic pathway [33]. Interestingly, it has been shown that the removal of the distal portion of the C-terminal tail results in a receptor that normally couples to the Gq/11/phospholipase C pathway and the mitogen-activated protein kinase pathway, but unable to couple to the anti-apoptotic pathway [114]. Furthermore, it has been demonstrated that a polybasic region (CDKRKRRKQ) conserved within the Cterminal tail of the Gαq/11-coupled muscarinic receptor subtypes (Table 1) appears to be the structural determinant for the coupling to the anti-apoptotic pathway [33]. Today it is still unclear what protein or proteins are implicated in the connection of the muscarinic receptor with the anti-apoptotic pathway, and also which anti-apoptotic signalling is implicated. THE mAChR RECEPTORSOME Until a few years ago it was thought that the interactions between GPCRs, G proteins and the corresponding effectors will explain the entire GPCR signal transduction cycle. However, we currently know that this simple stoichiometry is a bit more complicated since many other accessory proteins that are able to bind GPCRs also impinge in their functioning [115]. In addition, several reports have shown that a named multiprotein complex not necessarily has an invariable composition as some of its building bricks might be dynamically shared among different complexes. Under these premises, one receptor complex may be the result of not only the physical direct interaction between the receptor and its partner proteins, but also the result of many other mAChR Interacting Proteins non-“direct” associations that integrate the receptor in a network of interconnected pathways, thus determining the final receptor outcome [116]. Interestingly, this is a phenomenon that has currently received several names: interactome, signallosome or receptosome; but that essentially denotes the level of complexity of the receptor activation and signalling and its regulation throughout the formation of multiprotein complexes [117]. Therefore, bounding of each specific receptor interactome (i.e. mAChRsome) might become extremely important since these can be considered the minimal computational unit controlling the receptor functional outcome. A well-documented example of networks and their receptor-specificities is the described for the agonistmediated mAChRs internalization. Thus, agonist challenge produces mAChRs phosphorylation and internalization [118], as happens for most GPCRs. Interestingly, alanine substitution of the putative phosphorylable residues within the third intracellular loop of the human M1R, M2R and M3 R robustly decreases receptor internalization [119, 120]. In addition, three threonine residues within the C-terminus of the human M3 R (T550, T553, and T554) have been shown to play a critical role in the internalization and desensitization processes of the receptor [121]. On the other hand, the role of the various GPCR kinases (GRKs) play in the mAChR internalization has been shown to be extremely dependent of the receptor subtype and of the cell type where the receptor is expressed. For instance, while the M1 R internalization is enhanced by the co-expression of the GRK2K220W mutant in COS-7 cells the M3R internalization is unaltered under the same experimental conditions and in the same cell line [122]. In contrast, expression of the same GRK2 mutant reduced M2R internalization in COS-7 cells [123] but not in BHK-21 and CHO cells [122, 123]. GRK-mediated arrestin binding also initiates receptor internalization/sequestration, which occurs via the association of the receptor-arrestin complex with components of clathrin-coated pits [124, 125]. Interestingly, several groups have confirmed the role of β-arrestins and clathrin-coated vesicles in mAChR internalization. Thus, expression of a dominant-negative β-arrestin-1 mutant, which binds with high affinity to clathrin, but with impaired ability to interact with phosphorylated GPCRs, significantly suppresses internalization of M1 R, M3R, and M4R in HEK293 cells [105, 126]. Similarly, expression of another dominantnegative β-arrestin mutant, the β-arrestinS412D, which binds to phosphorylated GPCRs but not to clathrin, inhibits M1R in HEK293 cells [126]. These results are consistent with the observations that expression of a dominant-negative clathrin mutant strongly inhibits M1R, M3 R, and M4R internalization [105]. Interestingly, in HEK293 cells, M2R internalize in a βarrestin- and clathrin-independent manner [127, 128]. These experiments suggest that phosphorylated M2R do not readily interact with β-arrestin in HEK293 cells. Indeed, Wu et al. proved that a peptide sequence derived from the 3IL of the M2R, which contains the GRK2 phosphorylation sites and a putative β-arrestin binding site, did not bind to β-arrestins derived from a brain cytosolic fraction. In contrast, a similar peptide from the 3IL of the M3R was able to do so [129]. Overall, these observations suggested that other cytosolic Current Drug Targets, 2011, Vol. 12, No. 13 9 adaptor proteins associate with phosphorylated M2R in order to mediate non-clathrin-mediated internalization of the receptor. Interestingly, the β-arrestins-mediated GPCR internalization is not only due to its ability to bind clathrin as it has been shown that the binding of arrestins to clathrin is, by its own, not enough to trigger receptor internalization. In addition, GPCR endocytosis and trafficking are enhanced by the fact that arrestin recruits phosphoinositides, the adapter molecule AP-2, which is another endocytic protein, and intracellular trafficking proteins such as the N-ethylmaleimide-sensitive factor (NSF), the ADP-ribosylation factor ARF6, and its exchange factor ARNO [130]. ARF6 regulates vesicle budding by recruiting vesicle-coat proteins including COP1 coatomers. Therefore, it is feasible that mAChR internalization requires the interaction of the receptors with a specific set of non-canonical internalization related proteins. For instance, it has been shown that the Gβγ subunit is able to bind to a discrete peptide (C289-D329) belonging to the 3IL of the M3 R and while receptor mutants lacking the Gβγ binding domain still functional they are unable to internalize after agonist challenge [29]. A possible explanation for this might lay in the fact that downstream the Gβγ binding motif there are some putative GRK2 phosphorylation sites (332SSS-334 and 349-SASS-352). Thus, it has been proposed that the marked reduction in internalization of the Gβγ subunit binding-defective M3R mutants may be due to impaired phosphorylation of the receptor by GRK2 [131]. Overall, the network of proteins that interact with GPCRs in general and with mAChRs in particular may be extremely important for signalling, trafficking and targeting these receptors to particular subcellular compartments. Thus, different types of connections to different receptor subtypes will allow both positive and negative crosstalk, thus synchronizing and tuning cellular responses in response to a common ligand (i.e. acetylcholine) in a named cellular environment. Therefore, the fact that we have been so far unable to model this complexity may explain why we still are unable to reliably design GPCR-targeted drugs that will be free of side effects. Recently, specific interacting proteins for each mAChR subtype were systematically identified by means of tandemaffinity purification and mass spectrometry. Thus, the so called ILoopTAP approach, which represents a major methodological advance in the identification of GPCRinteracting protein complexes, was used to isolate mAChRs partners. Hence, once the limitations associated with receptor solubilization (i.e. the use of detergents) were overcome then a plethora of proteins were shown to specifically interact with the different mAChR subtypes. Interestingly, many new interactions belonging to diverse signalling pathways were established which together with the reported in the literature revealed a high degree of receptor connectivity. Thus, this allowed for the first time the building of a complex mAChR network of interacting proteins or mAChR interactome (Fig. (4)) [132]. Overall, with the establishment of the mAChR interactome the authors envisaged new signalling roles for mAChR interacting partners that were not previously imagined and also they postulated novel and genuine ways for muscarinic therapeutic intervention [132]. 10 Current Drug Targets, 2011, Vol. 12, No. 13 Borroto-Escuela et al. Fig. (4). The M5R interactome. Graphs were automatically generated by organic algorithm using Cytoscape software. Line thickness represents the number of experiments describing a given interaction and illustrates the connection between proteins. Lines are color-coded according to: ILoop-TAP experimental results (red) [132], reported in the literature (black). M5R node is highlighted in red. Each protein is labelled using BIND annotation. Adapted from [132]. Compartimentalization of mAChRs within the Cell Context Much has been written about the influence of receptors on cell function. However, the reverse relationship, namely the influence of the cellular context on receptor functioning has been discussed far less often. Interestingly, the spatial organization of mAChRs complexes into specific membrane microdomains has been shown to play a critical role on receptor-mediated signal transduction [133, 134]. Thus, the cell background imposes phenotypic selectivity, by concentrating components of the cellular signalling cascade within microdomains and, in some cases, determining the spatial and temporal-relationship of interacting proteins with the resulting change in receptor pharmacology properties [135]. The targeting of GPCRs and their downstream signalling machinery into specific membrane lipid compartments, generally called “lipid rafts” (i.e. caveolae), might constitute a widespread pattern for receptor signalling regulation [133, 134]. An interesting property of caveolae is that they selectively accumulate various signal components, including GPCRs, G protein and second messenger-regulated kinases, prompting the assumption that they might regulate signal transduction [136-138]. Interestingly, in cardiac myocytes, as well as in specialized conduction and pacemaker cells, the M2R has been shown to translocate out and into caveolae upon receptor challenge, thus demonstrating that multiprotein complex composition and regulation may be tightly linked to their localization in the lipid microdomain [139, 140]. In addition, in the airway smooth muscle cells, mAChR Interacting Proteins Current Drug Targets, 2011, Vol. 12, No. 13 11 Fig. (5). The influence of cellular context and indirect mechanism of crosstalk on receptor function and regulation. (A) Localization of muscarinic receptor and their downstream signalling partners to specific membrane lipid compartments (lipid raft). In cardiac myocytes and MDCK cells, muscarinic receptors have been shown to translocate out and into caveolae upon agonist stimulation. (B) Transactivation of Receptor tyrosine kinases by muscarinic receptors as a mechanism of crosstalk. (C) Transcriptional regulation of the receptor themselves. For instant stimulation of muscarinic receptor induced both down- and up-regulation of histamine H1 receptor. EGFR: epidermal growth factor receptor; H1R: histamine type 1 receptor; MMP: matrix metalloproteinases; PLC: phospholipase C; ROCK: Rho-associated protein kinase. caveolae helped the mAChR-mediated intracellular Ca2+ mobilization and cell contraction [141], and the loss of caveolin-1 has been associated with disruption of M3R activity in bladder [141]. Also, it has been demonstrated that caveolin-2 has an active role in regulating M1R endocytosis and trafficking in MDCK epithelial cells. Thus, association of the M1 R with caveolin-2 either inhibits receptor endocytosis through the clathrin-mediated pathway or retains the receptor in an intracellular compartment [142]. Interestingly, this intracellular association and the attenuation of receptor trafficking is rescued by the co-expression of caveolin-1 [142]. Finally, one of the most striking examples of the effect of cellular environment on muscarinic receptosome is the one observed in striatal neurons. Thus, the intraneuronal trafficking and the abundance of membrane-bound M4 R have been shown to be, in vivo, under the regulation of the cholinergic environment [143]. Thus, the M4R subcellular compartmentalization and functioning will depend on the phenotype of the cholinoceptive neuron and on its neurochemical environment. In addition, acute and chronic acetylcholinesterase inhibition has been shown to regulate in vivo the M2R and M4 R expression [144]. Thus, treatment with metrifonate, an acetylcholinesterase inhibitor, induced a general decrease of M4 R in the striatum without modifying mRNA levels and with a concomitant increment of mAChRs in the cytoplasm at the sites of synthesis and maturation (i.e., endoplasmic reticulum, nuclear nuclear membrane and Golgi apparatus). Overall, it is suggested that these types of regulations may play key role in the response of target neurons following drug treatment and related to the nature and the duration of treatment. mAChRs CROSSTALK As mentioned above, cellular function and the coordination of cells within tissues is an orchestrated process in which a large array of proteins, ions and metabolites are involved. The extracellular signals are sensed by the cell and processed to intracellular signals that ultimately determine a cellular response to the stimulus perceived. Given the large amount of extracellular molecules and target receptors, the potential for interactions between different networks is significant. However, there is a high degree of specificity within the crosstalk events occurring in a named cell, not only about which receptors are involved but also in which cell-type it is occurring. Although receptor crosstalk has been largely demonstrated [145], the significances and outcomes of mAChRs crosstalk begin to be considered in a physiological and pharmacological context. Therefore, mAChR interacting proteins and the concomitant indirect mechanisms of receptor crosstalk confer another level of complexity in the cholinergic signalling transduction process. Thus, these processes can influence the amplitude and duration of the receptormediated cell response as well as the rates of receptor desensitization and trafficking [146, 147]. One of the most described mechanisms of crosstalk involving mAChRs constitutes the transactivation of receptor tyrosine kinases (RTKs) (Fig. (5)) [148, 149]. RTKs are transmembrane proteins with a ligand-binding extracellular domain and a catalytic intracellular kinase domain [150]. These kinds of receptors are emerging targets for cancer 12 Current Drug Targets, 2011, Vol. 12, No. 13 therapy as they play a key role in cellular growth, differentiation, survival and oncogenesis [151]. They are commonly activated by growth factors, which induce dimerization of the receptors, resulting in trans- and auto-phosphorylation events that provide docking sites for the recruitment of the signalling machinery [152]. mAChR-mediated transactivation of both upstream and downstream signalling components of growth factor-induced RTK pathways have been described and related to the growth and proliferationinducing properties of different mAChRs subtypes [153156]. Interestingly, it has been demonstrated that the epidermal growth factor receptor (EGFR) is able to dimerize and become catalytically active by a M1R-mediated pathway which is EGF-independent and that requires protein kinase C [157]. In addition, this M1R-mediated EGFR transactivation is determinant for the M1R modulation of Kv1.2 potassium channel [157]. Overall, these results demonstrate a M1Rmediated ligand-independent mechanism of EGFR transactivation and reveal a novel role for these growth factor receptors in the regulation of ion channels by GPCRs. Similarly, it has been shown that M3R activation is also able to transactivate EGFR in H508 human colon cancer cells [158, 159], a mechanisms that accounts for the cholinergic-mediated proliferation of M3 R expressing colon cancer cells. However, this M3R-mediated EGFR transactivation was PKC-independent but matrix metalloproteinase (MMP7)-dependent [160]. Interestingly, ACh-induced activation of EGFR and downstream ERK signalling also regulated transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplasic cell proliferation (Fig. (5)) [160]. In addition, M1R challenge induced EGFR tyrosine phosphorylation by a M1R-mediated release of heparin-binding EGF-like growth factor (HB-EGF) [161]. In contrast, M2R-mediated EGFR transactivation is independent of matrix metalloproteinases, thus resulting in an incomplete EGFR signalling including ERK and Akt but not PLC-γ1 in the COS-7 cells [155]. Overall, it can be established that Gαq/11 coupled mAChRs (i.e. M1R and M3 R; see Table 1) transactivate the EGFR in a matrix metalloproteinase-dependent manner whereas the Gαi/o coupled mAChRs (i.e. M2 R; see Table 1) transactivate the EGFR in a mode that is independent of matrix metalloproteinase. Muscarinic receptor crosstalk is certainly not limited to interactions with receptor tyrosine kinases. Various other pathways involving, but not limited to, other GPCRs and immune cell receptors can crosstalk and integrate with muscarinic receptor signalling pathways. For instance, another mechanism whereby muscarinic signalling can transactivate other receptors is at the transcriptional regulation level. Thus, increased rates of histamine H1 receptor (H1R) transcription after M3R challenge have been shown in U373 astrocytoma cells (Fig. (5)) [162]. The increase in H1R mRNA levels and protein synthesis after M3R challenge was mimicked by the PKC activating phorbol ester, PMA, suggesting that a PKC-dependent process was involved in the up-regulation of H1 R mRNA and protein. In addition, M3R-mediated up-regulation of H1R was completely inhibited by Ro 31-8220, a PKC inhibitor. Interestingly, the same authors showed that activation of M3 R caused down-regulation of H1R in CHO cells permanently expressing these receptors, a process that was shown to be dependent of M3 R-mediated PKC activation [162]. Overall, Borroto-Escuela et al. it is suggested that M3R activation regulates H1R levels, firstly the M3 R challenge promotes up-regulation of H1 R protein by inducing H1R mRNA synthesis, and secondly the M3R induced H1R down-regulation by accelerating H1 R degradation. Thus, these apparently contradictory results suggest that H1 R expression may depend on the balance between these two processes. Interestingly, other examples of mAChR-mediated transcriptional regulation have been described. For instance, it has been showed that the M3 R challenge induced a robust matrix metalloproteinase gene expression in human colon cancer cells [160] and in a human T-cell line (i.e. Jurkat cells) the activation of mAChRs induced interleukin-2 production and transcription factor AP-1 expression [163]. Overall, several modes of mAChRs crosstalk has been already described in different experimental models. THE UGLY FACE OF mAChRs We discussed earlier the importance of accessory protein in receptor specificities and signal efficiency/efficacy, and therefore we concluded that the molecular understanding of the mechanism underlying receptor-protein interaction is likely to provide previously unrealized opportunities to achieve greater muscarinic pharmacological intervention. On the other hand, mAChRs interacting proteins not always are related to normal -non-pathological- conditions of cholinergic physiology. Thus, there are pathological conditions associated to mAChR interacting proteins. Accordingly, evidence collected over the last decade provided enough proofs for the existence of circulating antibodies in different autoimmune diseases. Interestingly, these autoantibodies bind to mAChRs and transform the target cells in pathologically active cells. For instance, one of these examples is the Chagas disease which is caused by a parasite widely distributed in Latin America, the Trypanosoma cruzi [164]. Chagasic cardiopathy or digestive damage is the most frequent complication of chronic T. cruzi infection. In addition, it was reported that more than 30% of T. cruziinfected patients develop the disease, leading to heart failure and sudden-death [165]. Interestingly, the existence of circulating autoantibodies against β-adrenergic receptor and M2R has been reported in Chagas disease and it has been postulated that these antibodies may play a key role in the pathogenesis of the dilated cardiomyopathy associated to the disease [166]. Hence, chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (2EL) of the cardiac M2R which is involved in the binding of various allosteric agents. Interestingly, the M2R autoantibody binding triggers an agonist-like response that alters the physiological behaviour of the target organs, thus leading to tissue damage. Hence, the binding of the partial-agonist autoantibody on the myocardial M2 Rs produce a decrease in the intracellular cAMP, an increase of cGMP, and an impairment of L-type Ca2+ currents. Interestingly, all these effects are blocked by preincubating the CChP-IgGs with a peptide corresponding to the M2 R 2EL. Finally, the chronic autoantibody M2 R allosteric modulation can lead to a progressive blockade of the receptor that results in a sympathetic and parasympathetic denervation [167]. mAChR Interacting Proteins Sjögren syndrome (SS), the second most common autoimmune rheumatic disease, refers to keratoconjunctivitis sicca and xerostomia resulting from immune lymphocytes that infiltrate the lacrimal and salivary glands [168]. Interestingly, by means of several experimental approaches (i.e. SDS-PAGE, immunoblotting, radioligand binding assay and ELISA) autoantibodies against M3R have been found in the sera of SS patients [169]. Increasing experimental evidences support the idea that the immunoglobulin G (IgG) of SS patients affects M3R function. Thus, the infusion of SS patient IgGs into to non-obese diabetic IgM null mice resulted in the loss of secretory function. In addition, antibodies stimulating M3R, M4 R and M1R increased the COX-2 mRNA without affecting COX-1 mRNA expression and also increased the prostaglandin E2 (PGE 2) production [170], thus resulting in the acute inflammation and cognitive dysfunction characteristic of SS patients [171]. Interestingly, Kovacs et al. mapped a discrete region within the M3 R 2EL as a potential target of the SS patient IgGs. Indeed, the molecular interaction between this acid peptide with autoantibodies from SS patients was shown by means of plasmon surface resonance-based optical biosensor and microspectrofluorimetry [172]. Finally, incubation of the SS patient sera with a peptide containing the amino acid sequence of the C-terminus of the 2EL precluded the autoantibody activity [173, 174]. A further example is the case of the existence of circulating autoantibodies against M1R in schizophrenic patients. These autoantibodies are able to interact with and activate the M1R expressed in the cerebral frontal cortex. Interestingly, by using a synthetic peptide in dot blot and ELISA experiments it has been possible to demonstrate that the autoantibodies reacted against the second extracellular loop of the human cerebral M1R. In addition, the corresponding affinity-purified anti-peptide antibody displayed an agonistic-like activity associated to specific receptor activation, thus increasing cyclic GMP production and inositol phosphate accumulation. Also, these autoantibodies promoted both M1R and neuronal nitric oxide synthase gene expression. Similarly, circulating autoantibodies against neonatal heart M1Rs in the sera of children correlated well with congenital heart block [175]. Finally, these autoantibodies by reacting with the second extracellular loop of the human M1R decreased contractility, activated nitric oxide synthase activity, and increased production of cyclic GMP [176]. mAChRs INTERACTING PROTEINS AS A DRUG TARGETS Approximately 40-50% of currently marketed drugs target GPCRs [177], a fact that underlines the role of GPCR signalling in treating disease. However, there are many cases in which the current drugs targeted to GPCRs are inadequate. A good example is targeting of M3 R. Indeed, M3R selective antagonists are widely used in the clinical management of overactive bladder (OAB) [6, 178]. As the M3 R are involved in the control of a large array of physiological processes (i.e. salivation, gastrointestinal motility, papillary constriction, etc.) the receptor blockade in OAB therapy can lead to significant side effects [179]. Thus, at least 50% of patients Current Drug Targets, 2011, Vol. 12, No. 13 13 taking oxybutynin experience severe dry mouth, which is the most common cause to suspend medication (25% of oxybutynin treated patients) [180]. Overall, the significant overlap in the functional muscarinic receptor expression profiles of the detrusor smooth muscle and the salivary glands has opened the search for pharmacological agents with selectivity to inhibit detrusor smooth muscle versus salivary secretion. Another example can be found in the mAChR-based therapy of psychosis and cognitive disorders. Interestingly, both acetylcholine esterase inhibitors and selective mAChR agonists improved neuropsychiatric symptoms and cognitive function in Alzheimer’s disease patients [181, 182]. However, the lack of selective drugs for the different mAChR subtypes has prevented an unambiguous determination of the role of each receptor subtype in human behaviour [183]. Thus, mAChRs are important candidates for developing allosteric modulators to fill an unmet clinical need [184-186]. The knowledge of the mAChR interacting proteins effects and the cellular and phenotypic context met by endogenously expressed receptors have led to some fairly dramatic changes in the way GPCRs in general and mAChR in particular are viewed and studied. As well as posing a great challenge, these new aspects of muscarinic biology are also of prompt interest because there is enormous potential for drug target and discovery. Thus, to take advantage of the knowledge gained these new mAChRs features should be taken into consideration when developing strategies to screen for new muscarinic-based compounds. Indeed, the above mentioned multiprotein signal complexes are dynamic structures which specifically assemble and disassemble from mAChRs, thus providing a potential exciting rationale for allowing receptor display tissue-dependent therapeutic intervention. This intervention can be performed by developing new allosteric modulators -i.e. small organic molecules- or altered/blocking peptides ligand that mimic binding sites and thus compete with the original receptor-protein interaction. Certainly, the development of peptide mimetic intracellular ligands (pepducins) is evolving into an exciting research area within the GPCR field [187]. The ability to form homo- and heterodimers involving diverse GPCR subtypes for the same or different transmitters has potentially far reaching implications on drug discovery [35, 36]. Thus, of all the novel aspects surrounding the mAChR biology the receptor dimerization/oligomerization is probably the one that will most likely have high impact in muscarinic drug discovery on the near future. Consequently, once a new mAChR oligomer is described the known muscarinic pharmacology should be revisited, firstly to catalogue that particular receptorial entity and secondly to shed light on those unexplained muscarinic effects of the classical pharmacology. For instance, experimental evidence suggested that two allosteric agents and one orthosteric ligand might be able to bind to the M2R simultaneously [188, 189], thus it has been postulated that the existence of a receptor homodimer is likely the structural support that account for this complex pharmacological behaviour [43, 189]. On the other hand, the development of allosteric ligands acting on mAChRs interacting proteins might be of special interest for these mAChRs in which the design of selective orthosteric agonists or antagonists has been elusive. 14 Current Drug Targets, 2011, Vol. 12, No. 13 In the chronic chagasic cardiopathy the positive allosteric cooperativity between the endogenous ligand carbachol and autoantibodies acting at the cardiac M2R has been suggested to account for the major symptoms reported for this illness [190]. Thus, the pharmacological intervention in all chronic Chagasic patients with sinus node dysfunction should in theory target (i.e. block) this allosteric interaction [190]. Therefore, a pharmacological intervention addressed to deregulate this allosteric interaction might be therapeutically effective as a co-adjuvant treatment of chagasic disease. Indeed, in line with this hypothesis, Matsui et al. showed that the treatment with AF-DX 116, a specific M2R allosteric antagonist, had a protective effect on experimental dilated cardiomyopathy induced in rabbits immunized with an M2 R 2EL peptide [191]. Similarly, in the generation of Sjögren's syndrome (SS), CD4 positive αβ T cells play a key role [192]. Thus, some studies have provided evidence about the role of the T cell receptor (TCR) Vβ and Vα genes on these T cells, and the sequence analysis of the CDR3 region suggested the presence of some conserved amino acid motifs, thus supporting the notion that infiltrating T cells recognise relatively few epitopes on autoantigens [193]. Accordingly, candidate autoantigens recognized by T cells that infiltrate the labial salivary glands of SS patients have been analyzed [194, 195]. As mentioned earlier, Gordon et al showed that autoantibodies against the 2EL of M3 R occurred in SS and were associated with the sicca (dryness) symptoms [196, 197]. In addition, based in the knowledge that the mechanism by which a peptide is recognised by a TCR is flexible, thus if the amino acid residue of the peptide ligand for TCR is substituted by a different amino acid can still binding to the major histocompatibility complex molecule (altered peptide ligand), some authors proposed that such an altered peptide ligand could regulate the activation of T cells [198]. Accordingly, several studies have provided evidence that VPPGECFKQFLSEPT (M3 R 223I→K) and VPPGECFIAFLSEPT (M3R 224Q→A) are candidate altered peptide ligands of the 2EL of M3R [199]. And those finding may provide the basis of a potentially useful antigen specific treatment for SS using altered peptide ligands of autoantigens recognised by autoreactive T cells. Another example of potential therapeutic intervention based on mAChR interacting proteins is at the level of the G protein activation. Thus, previous results using a synthetic peptide containing 16 amino acid residues matching the junction between the C-terminus of the 3IL and the sixth transmembrane helix (TM-VI) of the human M3R (Fig. (3)) showed that at micromolar concentrations was able to activate Gαq but not Gαi2 [200]. Interestingly, the Gq protein is an heterotrimeric guanine nucleotide-binding protein that plays important roles in cell functioning (i.e. regulation of calcium mobilization and cell proliferation) [201] and is considered a promising drug target for the treatment of cardiac hypertrophy [202]. Overall, this peptide is the first small compound that selectively activates Gαq but not Gαi2 and would be useful for explaining the role of Gq protein in a given signalling pathway, particularly when employed in combination with YM-254 890 compound, a Gαq-selective blocker [203]. Finally, as described previously, mAChR could transactivate growth factor receptor and modulate their function. Tyrosine kinases receptor activators have not met with Borroto-Escuela et al. clinical success owing to their poor pharmacokinetic behaviour, whereas small-mimetic molecules have shown promise for future clinical treatment of neurodegenerative diseases [204, 205]. Thus, given the difficulty of designing direct TKR agonists a coherent alternative approach consists in designing allosteric modulators that can either act directly on the TKR or indirectly through another receptor (i.e. GPCR). Interestingly, experimental results revealed that two staurosporine-like compounds, K-252a and K-252b, which are allosteric modulators of mAChRs [206], were found, depending on the concentration used, either to inhibit or to potentiate the action of the TKR [204]. Overall, the indirect pharmacological intervention with small molecules could open new avenues for manipulating TKRs. CONCLUSIONS The classical view of mAChRs as simple heterotrimeric G protein activators is now being replaced by a new view that poses these receptors as complex modulators of several cellular signalling pathways, a process that is mastered by a complex network of protein-protein interactions or interactome. However, and although this mechanistic new concept, there are still many remaining and open questions about the mAChR signalling. For instance, the physiological relevance of many mAChR-binding partners identified so far for which no clear function in the downstream muscarinic signalling have yet been proved. On the other hand, new evidences suggested that the signalling efficiency/specificity for mAChRs is determined by a microdomain that allows colocalization or even direct interaction of the receptors and their effectors. Overall, a great number of proteins have been identified to interact with mAChRs, including GPCRs, kinases, scaffolding proteins (i.e. arrestin), oncogenic proteins, ion channels, regulatory G-protein signalling and small G protein. However, more work is needed to clearly ascertain the functional meaning of these interactions and how these are integrated in a spatio-temporal framework giving up both the heterotrimeric G protein-dependent and G protein-independent muscarinic signalling pathway. 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