The muscarinic M 5 receptor: a silent or emerging subtype?

The muscarinic acetylcholine receptors belong to the super-family of seven TM domain receptors that interact with G-proteins to initiate intracellular responses. Evidence from molecular cloning indicates that there are separate intronless human genes that encode five muscarinic receptor glycoproteins. Muscarinic receptor sequences have significant homologies with other members of this large super-family and the genes are very similar across mammalian species (Caulfield 1993; Felder 1995). Despite over a decade following their molecular identification, the therapeutic exploitation of this crucial family of receptors remains disappointing. This results from a relative inability to pharmacologically distinguish between the subtypes, markedly hindering their investigation in native mammalian tissues. In particular, this has hampered investigation of the last-identified subtype, the muscarinic M5 receptor. The inability to clearly distinguish it from the M3 receptor has, moreover, led to confusion of its physiological role. Finally, determination of the precise distribution of M5 receptors within tissues is complicated by inadequate selectivity of radioligands as well as the low sensitivity/selectivity of polyclonal antisera in immunocytochemical studies (Caulfield 1993; Reever et al., 1997). Despite these problems, this receptor has recently been assigned an upper case M5 nomenclature (Caulfield & Birdsall, 1998) presumably reflecting recognition of its presence and function in native tissues despite the current incomplete characterization. In this respect the identification of a human A2058 melanoma cell that endogenously expresses the M5 receptor (Kohn et al., 1996) should facilitate its investigation in endogenous tissues, although extensive use of these cells have not been reported to date. Consequently, the majority of the current information on the functional properties and regulation of coupling of this subtype still arises from their expression in model cells following cDNA transfection. The purpose of this short review is to critically evaluate current data on the muscarinic receptor M5 subtype from several standpoints. Hopefully, this critique will stimulate further studies on the M5 receptor that may raise it from a ‘relatively ephemeral’ or ‘fact or fiction’ status, described in recent reviews (Reever et al., 1997; Caulfield & Birdsall 1998). The muscarinic M5 receptor was the last of the muscarinic receptor family to be cloned in the human and is mapped to chromosome 15q26 (Bonner et al., 1988; Liao et al., 1989). The receptor sequence conforms to a predicted seven transmembrane glycoprotein consisting of 531 residues in the human (GeneBank accession number PO8912) and 532 in the mouse (PO8911; 89% homologous to human). Structurally, the M5 receptor is the next largest muscarinic receptor to the M3 subtype with both these subtypes possessing a large third intracellular loop. Differences in this cytoplasmic loop account for the sequence diversity between muscarinic receptor subtypes and also between muscarinic receptors from different species. However, of the five muscarinic receptors, the M5 subtype demonstrates the least homology in this region when comparisons are made between human and rat sequences. Wess and colleagues (Wess et al., 1992; Pittel & Wess, 1994; Wess, 1997) have explored the nature of ligand binding and G-protein coupling by using chimeras of muscarinic M2 and M5 receptors. Most M2/M5 constructs are inactive but the presence of the M2 sequence in TMVII and M5 in TMI agonist activation of G-protein coupling is restored. Pittel & Wess (1994) argued that these data supported the bacteriorhodopsin model in which the seven transmembrane helices are arranged in a ring, such that TMI is adjacent to TMVII. A series of M2/M5 chimeras in which regions of the M5 receptors have been systemically replaced by homologous regions of the M2 receptor, indicated the higher affinity of the antagonist UH-AH 37 for the M2 over the M5 receptor was dependent upon a short stretch of 31 residues in TMVI as well as a short region of the third intracellular loop. This however contrasts to the antagonist AQ-RA 741 which also preferentially binds to the M2 receptor suggesting that different receptor epitopes may be involved in conferring different ligand specificities. In a series of studies, Brann and colleagues also attempted to identify key residues associated with agonist activation of M5 receptors. Initially using random saturation mutagenesis they identified the amino acids 439, A440, A441 towards the C-terminal end of the third intracellular loop of the M5 muscarinic receptor critical for G-protein coupling (Burstein et al., 1995). In a more recent paper, this group (Burstein et al., 1998a) constructed a further series of point mutants at each of these residues and characterized their functional phenotypes in order to find structure function relationship for G-protein coupling to the M5 receptor. Their evidence suggests that residue 439 participates in G-protein activation through an ionic mechanism and that A440 fulfils more of a structural role, perhaps forming part of the G-protein coupling pocket. Further, A441 apparently contributes to receptor affinity for G-proteins. Collectively, these data suggest that the third intracellular loop of the M5 receptor forms a G-protein coupling pocket comprised of a positively charged lip and a hydrophobic core. Brann's group (Spalding et al., 1998) also investigated a potential switch between active and inactive conformations of the M5 muscarinic receptor. There is much evidence from several G-protein coupled receptors to suggest that G-protein receptors exhibit constitutive activity (i.e. activation in the absence of agonist) and that agonists stabilize active whereas antagonists stabilise inactive conformations (Kenakin 1996; 1997). In a search for residues that participate in receptor function, several regions of the M5 receptor were randomly mutated and tested for their functional properties. Mutations spanning the face of TMVI were found to induce high levels of constitutive activity of the receptor. The same face of TMVI contained several residues crucial to receptor activation by agonists and one residue was identified as a contact site for both agonists and antagonists. These results suggest that within TMVI of the M5 receptor is a switch that defines the activation state of the receptor and the ligand interactions with TMVI stabilizing the receptor in either active or inactive conformations. In a further study (Burstein et al., 1998b) this group completed a systematic search of the intracellular loops in an attempt to identify further domains that govern G-protein coupling. A feature of the second intracellular loop was an ordered cluster of residues where substitutions also cause constitutive activation of the M5 receptor. A second group of residues in the second intracellular loop have been identified where mutations compromise receptor/G-protein coupling. The residues of each group appear to alternate and are spaced three to four positions apart, perhaps suggesting an α-helical structure where the groups form opposing faces of the helix. The authors suggest that the constitutively activating face normally constrain the receptor in the off state while the other face couples to G-proteins with the receptor being in the on state. It is generally accepted that muscarinic M1, M3 and M5 receptors couple preferentially via the pertussis toxin insensitive Gq/11 protein to phosphoinositide C-β (PLC-β) (Caulfield, 1993). Agonist activation of these subtypes therefore accelerates the rate of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis leading to the formation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) and these products act as second messengers by mobilizing Ca2+ from intracellular stores and activating protein kinase(s) C (PCK) respectively (Berridge, 1997). Bonner et al. (1988) were the first group to observe that recombinant muscarinic M5 receptors expressed in CHO-cells coupled to this signalling pathway. Confirmation of effective coupling of M5 receptors to phosphoinositide hydrolysis-linked signalling has been reported in CHO cells (Jones et al., 1991; Wang & El Fakahany, 1993; Richards & van Giersbergan, 1995; Watson et al., 1999) as well as murine L-cells (Liao et al., 1990), A9L cells (Richards & van Giersbergan, 1995) and insect SF-9 cells (Kukkonen et al., 1996). The assumption underlying these observations i.e. that the M5 receptor activates a phospholipase C-β via Gq/11 proteins, is based upon the fact that responses are insensitive to pertussis toxin (Liao et al., 1990). Direct evidence (as reported for the M1 receptor; Berstein et al., 1992) for this pathway of M5 receptor signalling remains to be established, although efficient coupling of this subtype to Gq/11 in CHO cells using [35S]-GTPγS binding and immunoprecipitation protocols (Smith, Eglen & Nahorski, unpublished, see below) or an antibody capture technique (De Lapp et al., 1999) has recently been observed. In contrast, Gusovosky et al. (1993) reported that M5 receptors expressed in CHO cells stimulated tyrosine phosphorylation of PLC-γ and that tyrosine kinase inhibitors suppressed agonist activation of phosphoinositide hydrolysis. It is unlikely, however, that this response is a subtype-specific effect of M5 receptors, as both M1 and M3 receptor activation of IP3 generation in CHO cells is suppressed by the tyrosine kinase inhibitor, genistein (Umemori et al., 1997). Palmier et al. (1999) have recently reported very similar observations in rat myometrium, recently shown to contract by activation of the M3 receptor (Choppin et al., 1999a). Other reports that the muscarinic M5 receptor shows subtype selective signal transduction have also proven inconclusive. Wang et al. (1993; 1994; 1996) demonstrated that M5 receptors efficiently couple to neuronal nitric oxide synthase (NOS) in CHO cells and Wotta et al. (1998) demonstrated that agonist activation of CHO M5 cells leads to accelerated phosphoinositide hydrolysis and MAP kinase activation. However, there is no evidence that activation of NOS or MAPK is selective for M5 receptors and these responses are almost certainly secondary to changes in either intracellular Ca2+ and/or protein kinase C activation in response to phospholipase C mediated PIP2 hydrolysis. On the other hand, the identification and characterization of an endogenous M5 receptor in the A-2058 human melanoma cell line (Kohn et al., 1996) could provide evidence of unusual transmembrane signalling. Activation of M5 receptors expressed in these cells resulted in no evidence of phospholipase C-β or γ activation but both a robust Ca2+ mobilization from intracellular stores (InsP3 independent?) and the resulting Ca2+ influx was accompanied by marked phospholipase A2 activation and inhibition of forskolin-stimulated cyclic AMP accumulation. Alternatively these data may reflect the very low expression of M5 receptors in A-2058 cells resulting in undetectable phosphoinositide hydrolysis with subsequent amplified Ca2+ mobilization and PLA2 responses. Further studies on this endogenous M5 receptor are clearly However, there is evidence that the M5 receptor from M1 and M3 receptors in its to couple to in order to et al. that expression of M5 and M3 receptors in CHO cells a robust phosphoinositide hydrolysis but M3 receptors were more active at cyclic AMP accumulation. Liao et al. to observe M5 mediated activity in studies have using expression levels in CHO that there are in the coupling of M1 and M3 receptors to and the activation of et al., et al., 1995; & Nahorski, see also et al., 1993; et al., 1994; 1996). these studies it to a In there are both subtype and agonist of activation. These data suggest that the muscarinic receptor subtypes are in their interactions with different G-proteins and that there are on the coupling to different species. different agonists may or receptors to different G-proteins is of much to it suggests that not is the and of an agonist but that the nature of the agonist the and also the of an response by different G-protein mediated signalling There are several of (Kenakin 1996; et al., 1996) but as not data with muscarinic receptors. However, studies on muscarinic receptor subtypes that such as or selective activation of M1 over M3 receptors in cells in which these subtypes are expressed at similar et al., 1996). Richards and van also the relative of agonists at M1, M3 and M5 receptors expressed in CHO was the that while M5 receptors were efficiently coupled to phospholipase C M1 and M3 receptors, agonists and were more at activating M5 receptors. This suggests that this receptor may different G-proteins and/or different phospholipase to stimulate phosphoinositide hydrolysis. in the relative of agonists at M1, M3 and M5 receptors & El 1993; et al., 1991; Richards & Giersbergan, 1995) may not to receptor but also the of phosphoinositide hydrolysis. there is evidence that muscarinic receptor subtypes other seven TM active to selective G-protein coupling in response to different it will be to such in it the potential to receptor active to or an by or conformations of the receptor This may information on the selectivity of M5 receptor signalling and provide to its physiological role. There is evidence that the activation of protein coupled receptors is by receptor from G-proteins and by These appear to of signal transduction such as and of signalling 1998). Muscarinic receptors are no to although of the regulation of coupled subtypes that act via and to the activity of while well that M1 and M3 receptors agonist mediated phosphorylation and that this the of of the relative of the involved coupled receptor kinase in cells remains to be 1997). one study has the of M5 receptors et al. (1998) the of M5 muscarinic receptors expressed in M5 muscarinic receptors were by following with a of was in cells that were also but not by expression of a These data suggest that both and may be involved in the of the M5 subtype and further studies are In to of receptor for G-proteins a further for signal diversity via G-protein coupled receptors 1998). to the evidence that muscarinic receptors may active that selective G-protein coupling in response to different by receptor phosphorylation could provide a further of The of the M5 receptor was first by the use of recombinant cell a series of antagonist to be at a receptor subtype (Bonner et al., 1988; et al., 1991; et al., cloned over and in marked to the other four muscarinic receptor subtypes, there remains a of functional studies a or response mediated by M5 receptor activation. with other potential endogenously expressed M5 receptors be In of an endogenous of the muscarinic M5 receptor, one to recombinant to the affinity data from group et al., et al., reports on the receptor affinity the M5 receptor a low affinity for AQ-RA 741 and an affinity for and no has been reported that a high affinity for the receptor. It also be from antagonists such as or exhibit a low affinity at the receptor, such as and of the in and on their affinity at this receptor and their selectivity also Caulfield & Birdsall, 1998). In several the of antagonist at the M5 subtype that at the muscarinic M3 receptor a by several in the et al., et al., 1991; et al., several AQ-RA and are for the M3 over the M5 receptor et al., et al., 1991; & et al., Watson et al., group has also shown that and similar selectivity (Choppin et al., of these in a endogenous M3 receptor is in the the in M3 and M5 binding it be to of the of such selective tissues to M3 receptors, and to M5 receptors. in this respect is the a that to both M3 and M5 receptors et al., 1997). to the cloning of the muscarinic receptor of this were to the affinity of at the M3 receptor et al., & the majority of studies of antagonists at the M5 receptor have binding to affinity functional studies have been reported in which the antagonist affinity has been & Brann reported an using activation of the and with antagonist affinity from binding studies at the M5 receptor. Watson et al. (1999) have reported an antagonist affinity using agonist mediated phosphoinositide hydrolysis in CHO M5 by these also with in binding studies that the binding are In et al. a constitutively active recombinant M5 receptor in which is antagonists as agonists and responses of agonists of low were This could also in the of antagonists for the receptor. Finally, recently et al. (1999) [35S]-GTPγS to an antibody capture to agonist at M5 receptors in CHO at M5 receptors by this technique were not although it clearly has the potential for use in this of However, the absence of a robust functional for the M5 receptor in an endogenous remains a in the when the potential selectivity of muscarinic such agonists to have been identified to date. use of several with be to pharmacologically the nature of the receptor a these the identification of the M5 receptor when expressed in an endogenous when with the M3 receptor remains A of the is the precise nature of the muscarinic receptors of et al. 1992) reported an antagonist at receptors of this with M1, M3 and receptor with for and was the of M5 receptors in the subsequent in has not these use of an extensive series of antagonists suggests that the is similar to that of the M3 receptor of (Choppin et al., 1998). the from other species remains of in of a functional of the M5 receptor. & demonstrated that a of was selective for muscarinic receptors of over suggesting either in M3 receptors or that of this were mediated by more one receptor, with the from group (Choppin et al., these in the antagonist affinity at muscarinic receptors of is with activation of muscarinic M3 receptors, of M5 receptors. This is with the antagonists AQ-RA 741 and (Choppin et al., in which the by these clearly from that at M3 receptors in observations have been made in human (Choppin et al., The fact however, that in the absence of for the M5 receptor, of its in endogenous tissues is by resulting in The distribution of muscarinic receptor subtypes in mammalian tissues have been investigated using both and These studies, although have and in Caulfield 1993). with sequences to with part of the muscarinic receptor either in from tissues or with in have resulted in a potential of on the of muscarinic receptor subtypes in mammalian tissues. However, it be that such studies may a of receptor when the site of of is from the site of expression of the receptor although the use of receptor and it is still to on receptor The and of the is not and there is a for further with different subtype certainly at higher and with information their these with the M5 receptor, et al. found no receptor immunoprecipitation with an M5 receptor antibody the despite the presence of M5 receptor in and However, et al. (1993) has found low levels of M5 receptor in and studies reported muscarinic receptors in the is apparently of the M5 through more recent using et al., M5 and M3 expressed in et al. have the and binding of several muscarinic antagonists to by the distribution of muscarinic M5 receptors in rat and tissues, this technique Reever et al. demonstrated a the M3 receptor, although this was not in In were in the of the and the was also in the with in the This is with the in although the identification of was not from the immunoprecipitation The group (Reever et al., 1997) that the receptor via a on In contrast, studies have indicated that a of M5 receptors were associated with these and the majority of these were expressed on on et al., Muscarinic M5 receptors are also expressed in murine from an cell line et al., from which it has been that the expression could be This with the neuronal of the receptor and its in as There is data the expression and function of the M5 receptor in tissues. et al. by the presence of muscarinic M5 receptor in rat and although the functional of these data is cells M3 receptors through which nitric In of data no evidence to the presence of a M5 receptor & 1990). Consequently, the of the M5 receptor in the remains to be It has been for that the of the expresses M3 receptors, through which it is the Eglen et al., for binding studies in human cells also suggests the presence of M3 receptors et al., 1993). immunoprecipitation data from this group et al., 1997) a M3 but also indicates expression of the M5 receptor in human of the muscarinic M3 receptor is in or & 1998). This of et al. is of it suggests that selective antagonists could have a for an over current & 1998). However, the study of et al. has not been and it is such receptors are et al. (1999) in of human cells to muscarinic agonists M3 receptor data were reported for the expression of the M5 receptor et al. have shown by binding M5 receptors are in rat with an by Watson & (1994) demonstrated that the from rat of M1 and M3 receptors. However, functional studies of have been in or have the of muscarinic antagonists. Consequently, of the muscarinic has not been over group et al., 1998) has reported affinity data for several antagonists in a of rat cells These data were by using and subsequent to the nature of the subtype the in the of the as a for muscarinic receptor The results a series of antagonist to in CHO M3 or M5 It is these data from activation of both subtypes, in which case it is that both be or the the of a muscarinic receptor. is from the studies the function of the M5 receptor is Muscarinic M5 receptors are in the and of rat suggesting that they may have a in the of (Reever et al., 1997). In of cells from muscarinic receptor activation stimulated This to be via a receptor coupled to inositol via a pertussis toxin insensitive extensive was these are with activation of an M5 receptor et al., 1993). a functional of a receptor in the be in of changes in expression or has been in this the M5 receptor, although one et al., 1995) has changes in the subtype The subtype M5 expression was in to the marked in M2 and of The of the M5 receptor has been reported by et al. in which it was found that the expression levels of the receptor in rat were low of the receptor at It is that M5 receptors are expressed in cell of which could model M5 receptors are expressed in cells & 1995). with γ both the expression of M5 in cells from cells et al., 1996). muscarinic receptor in these it is the M5 receptor the extensive is to the nature of the muscarinic receptor subtype the The of selective M5 receptor antagonists has led to the of to the function of the endogenous M5 receptor. et al. (1999) have reported data a mouse with a in the M5 The of the that the of by was and no other This with the presence of the M5 receptor in rat et al., 1997) and the antagonist by et al. an antagonist in the of is selective for the muscarinic M3 over the M5 receptor et al., may to have on when to antagonists such as while on the which activity is by the muscarinic M3 receptor; et al., 1997). However, extensive and with shows no et al., 1995). A similar may be for in for & 1999) in of its selectivity for the M3 over the M5 receptor. In this however, in at least at low is & although this has been et al., 1997). the muscarinic M5 receptors identification of with affinity for this receptor, but with high M3 receptor a to for in which to be but function A similar be made that the M5 receptor a in the of et al., 1997; 1997). In this case muscarinic selective antagonists may a for at at which inhibition of activity data to this is that also an M3 over M5 selective not in at that et al., 1993). The muscarinic M5 receptor remains the least of the five muscarinic receptors, it is over a decade the identification of the receptor of the receptor in recombinant the use of the receptor as model to study agonist of a that may provide the for its in it is that the receptor, to its has a to in In the the identification of its expression in and suggests a role, but the data is and extensive the distribution of the receptor in tissues associated with of has for it is clearly to the of the receptor in both and The of antisera and use of has this Most a in this is an absence of an antagonist for the subtype, in the of the receptor. the M5 receptor will from the critically on the of its The authors to of and for in the of this review and to and for of