The molecular basis of muscarinic receptor diversity

The molecularbasis of muscarMc receptordiversify Tom

I. B o n n e r

TomL Bonneris at The cloning of cDNAs and genes for five different the Laboratoryof Cell muscarinic acetylcholine receptors provides a new basis Biology, National for characterizing muscarinic receptorfunction. Studies Institute of Mental of the cloned receptors when introduced into cells not Health, Bethesda,MD expressing endogenous receptors have allowed the initial 20892, USA

were named ml and m2; these were cloned as cDNAs from porcine brain and heart using the classic approach of purifying enough receptor to obtain a partial amino acid sequence, synthesizing a corresponding oligonucleotide probe, and using it to screen a identification of two classes of functional response. The cDNA library by DNA hybridization 2-4. cDNAs for the ml, m3 and m5 receptors belong to a class charac- ml, m3 and m4 receptors were isolated from a rat terized by agonist-induced stimulation of phosphatidyl- cerebral cortex library by hybridization to an oligoinositol metabolism and are structurally more related to nucleotide probe derived from the porcine ml cDNA each other than they are to the m2 and m4 receptors, sequence under conditions that allowed the detection which belong to a class associated with agonist-induced of similar but not identical DNA sequences 5. When inhibition of adenylate cyclase. While functional the rat ml, porcine m2 and rat m3 genes were cloned differences within these classes may yet be found, it and sequenced 4'5, it became apparent that all three appears likely that much of the difference between genes have an exon which contains all of the coding functionally similar receptors will befound to lie in their sequence and 3' untranslated sequence of the cDNAs, regulation. as well as a small amount, 19-69 bases, of 5' untranslated sequence. In all three cases it is clear It has long been known that there are two that there must be additional exons containing more 5' pharmacologically distinguishable types of acetylcho- untranslated sequence. The m4 and m5 genes as well line receptor, the nicotinic receptors which are now as several, but not all, genes for structurally similar known to form an ion channel and the muscarinic receptors have also proved to lack introns in their receptors which act through GTP-binding proteins (G coding sequences. If there are no introns in the coding proteins) to activate a variety of second messenger sequence, one does not need a cDNA to define fully systems ~. In recent years, a number of drugs have the structure of the receptor protein or conveniently been identified that can discriminate between muscar- to insert the coding sequence into an expression inic receptor types, most notably pirenzepine which vector. Furthermore, unlike cDNA cloning, one does has formed the basis for the classification of the not need to know where the gene is expressed to receptors as M~ or M2. Mx receptors have high clone it from a genomic library. Using this approach affinity for pirenzepine, are found in brain, and are with probes derived from the cDNAs, the rat gene for generally associated with metabolism of phosphatidyl- a frith receptor (m5), all five human genes, and the inositol (PI), whereas M2 receptors have low affinity porcine m3 gene have been cloned5-7'9'1°. A rat m2 for pirenzepine and are associated with inhibition of cDNA has also been cloned n. The identity of each of the five receptors as adenylate cyclase in heart. Although these pharmacologically defined receptor types have different distri- muscarinic receptors has been established by butions and functional responses, it was unclear expression of the clones (see Table I). For the ml whether they represented one or more proteins until receptor this was first done by injecting RNA the cloning of cDNAs and genes encoding muscarinic transcribed from the cDNA into Xenopus oocytes and receptors established that there are at least five observing an electrophysiological response to genes each of which encode distinct receptors z-7. acetylcholine 12. For the other receptors, mammalian Through the characterization of these cloned recep- cells that do not express endogenous muscarinic tors, it has become apparent that most of the tissues, receptors were transfected with mammalian expresexcept perhaps heart, used for pharmacological sion vectors containing the cloned cDNAs or genes characterization of muscarinic receptors contain more and then tested for binding of muscarinic ligands 4-7. than one receptor type. Fortunately, a more rational Both expression systems have since been used to pharmacological classification based on the properties characterize further the properties of the receptors. of the individual proteins is now possible. The In particular, it is clear that the ml and m2 receptors, nomenclature used here 5 is based on a simple respectively, have the high and low affinities for numbering of the proteins in chronological order of pirenzepine associated with the M1 and M2 pharmacodiscovery, using lower case m's to denote the logical classification 5-7'1°'12. However, the m3, m4 proteins, to reduce the confusion with the pharmaco- and m5 receptors have intermediate or high (for m4 in logical types denoted with capital M's. However, some assay systems) affinities and may have been some authors 7'8 have reversed the numbering of the considered to be either M1 or M2 by different authors. third and fourth receptors and have used capital M's to label the clones. Since the first two receptors to be The s t r u c t u r e of the receptors As is the case with all other G protein-coupled sequenced correspond most closely to the M~ and M2 pharmacological types, there is some similarity receptors that have been sequenced, the amino acid between the numbering of the receptor proteins and sequences (Fig. 1) derived from the various clones the previously defined pharmacological subtypes. contain seven hydrophobic segments of 20-30 amino The first two muscarinic receptors to be cloned acids which are assumed to be transmembrane

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© 1989, ElsevierSciencePublishersLtd, (UK) 0166 2236/89/$02.00 -

TINS, Vol. 12, No. 4, 1989

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(R) 2.3.5. .6. 11 12. Identical sequences for Pm2 (Ref. 4), Hml (Ref. 10), Hm2-4 (Ref. 7) have been reported. Another Hml sequence 7 has/v1173 rather than V173. A second Rm3 cDNA 8 encodes R516 rather than C516 and A'1556rather than 7"556. The transmembrane domains I-VII and the inner, i.e. cytoplasmic (il-i4) and outer, i.e. extracellular, domains (ol-o4) assumed by analogy to bacteriorhodopsin are indicated above the sequences. Stars above the sequence indicate amino acids that are invariant for all the receptors. Symbols below the sequence indicate conservative (o) or non-conservative (x) substitutions for at least one receptor subtype when compared across species. (B) A schematic diagram of the structure of the muscarinic receptors with regions labelled as above. Portions shown in black are well conserved among the different subtypes while the diagonally shaded region following transmembrane region V is well conserved among ml, m3 and m5 or between m2 and m4 but not among all five subtypes. TINS, Vol. 12, No. 4, 1989

149

TABLE I. Characteristics of cloned muscarinic receptors

conditions that allow the detection of genes that differ in their DNA ml m2 m3 m4 m5 sequence by roughly 25-30% indiCalculated mol. 51 387 51 681 66085 53 014 60120 cate that there are nine to ten wt of human detectable restriction fragments in receptoP rat and five to six in human S. Pirenzepine High Low Intermediate Intermediate/ Intermediate Although there is no reason to affinity high expect only a single fragment per gene, hybridization of similar blots Stimulates No effect b Stimulates No effect b Stimulates PI response using the homologous portions of cAMP response Stimulates Inhibits Stimulates Inhibits Stimulates the five cloned receptors as probes Arachidonic Stimulates No effect Stimulates No effect Not tested and hybridization conditions allowacid ing the detection of only nearly response perfectly matched genes indicates that each of the cloned receptors Ca2+Opens No effect Opens No effect Not tested accounts for only one of the redependent striction fragments 5'6. Thus there K+ channel in A9 and is at least one related human gene NG-108 cells and as many as five related rat genes yet to be characterized. M-current in Inhibits No effect Inhibits No effect Not tested Whether they represent functional NG-108 genes for muscarinic receptors or Ca2+None Stimulates None Stimulates Not tested other receptors or even non-funcindependent tional genes remains to be seen. currents in Even five muscarinic receptor Xenopus genes are substantially more than oocytes was expected from the existing mRNA Brain, glands Heart, smooth Brain, glands, Brain, Unknown pharmacology. Why are there so distribution muscle, smooth NG-108 many? One possibility is that the brain muscle, cells different l:eceptors could be 132N1 specialized for different functions; cells another is that the existence of aDue to glycosylation, sizes are expected to be substantially larger. different genes for functionally bWeak stimulation at high receptor levels or high agonist doses. equivalent receptors allows domains. The receptor subtypes are well conserved greater regulatory flexibility. The regulatory (89-98% amino acid sequence identity) in mammalian differences could be based on transcription from species with most of the substitutions occurring in the different promoters leading to tissue or developamino terminal (ol) or large cytoplasmic region (i3) mental specificity of the receptors or could be based connecting transmembrane regions V and VI. The on differences in structure which serve as the different receptor subtypes are also quite similar to substrate for post-translational mechanisms such as each other in having 160 invariant residues. However, phosphorylation. The evidence to date indicates that there is a complete lack of sequence similarity in the i3 both reasons apply to muscarinic receptors. region except for the first and last 15-20 amino acids. Excluding the ol and i3 regions, the ml, m3 and m5 Distribution of the receptors Northern blot analysis of pig and rat tissues 1'2'7'16 receptors are very similar to each other but less similar to the m2 and m4 receptors which are closely and in situ hybridization to rat brain 5 ' 17 ' 18 indicate that related to each other. In particular this pattern there is tissue specificity in the distribution of extends to the first 20 amino acids of the i3 region. receptor mRNAs. RNA for m2 is abundant in heart Since this region has been implicated in coupling to and present, but in low abundance, in restricted second messengers through deletions of the [32- regions of brain, whereas ml, m3 and m4 are adrenergic receptor 13, it is not surprising that second abundant and broadly distributed in brain but absent messenger preferences of the receptors fall into the from heart. Although the distributions of ml, m3 and same pattern as discussed below. Mutational studies m4 in brain are largely overlapping, there are of the [32-receptor also suggest that the second, third differences in their relative abundances in hippoand seventh transmembrane regions determine the campus, striatum and dentate gyrus, mRNAs for ml ligand-binding properties of the receptor 13-15. and m3 have been observed in exocrine glands (lacrimal, parotid and submandibular) while m2 and m3 mRNAs are found in smooth muscle tissues (small and How many receptors are there? Although the only definitive answer will be provided large intestines, trachea and urinary bladder). Comby the cloning and characterization of additional parable abundance of m5 RNA in brain and heart has genes, clues can be obtained by examining Southern not been observed and peripheral tissues have not blots of genomic DNA. In principle the most reliable been extensively examined 6. The m5 receptor thus information would be provided by using a probe has an as yet unknown distribution but is a candidate containing as much as possible of the sequence that is for either a primarily peripheral distribution or a conserved between different muscarinic receptors developmental specificity. However, there are as yet and as little as possible of the sequence that is not no data that directly address developmental regulation conserved. Data using such a probe and hybridization of expression of the individual receptors. 150

TINS, VoL 12, No. 4, i989

Functional responses

The individual expression of different cloned receptors in the same host cell has allowed the responses of the receptors to be examined in a controlled environment in which it is clear that differences in responses are due to the receptors. The first report of differential responses of the cloned receptors compared the electrophysiological responses of Xenopus oocytes injected with RNA transcribed from the cloned porcine ml and m2 cDNAs z2. Acetylcholine-stimulated ml receptors exhibited a Ca2+-dependent chloride conductance, whereas m2 receptors gave a much weaker response primarily via Ca2+-independent Na + and K + conductances. Recently it has been shown that similarly expressed pig m3 receptors give the same response as ml receptors, and rat m4 receptors give the same response as m2 receptors z9. In both A9 cells 2°'21 (mouse fibroblasts which do not express endogenous muscarinic receptors) and NG-108 cells 22 (neuroblastoma-glioma hybrid cells which express m4 receptors7'22), the ml and m3 receptors activate a Ca2+-dependent K + channel, whereas the m2 and m4 receptors do not. The ml, m3 and m5 receptors transfected into mammalian cells stimulate PI metabolism and cAMP accumulation while m2 and m4 receptors inhibit cAMP6'22-24. Stimulation of cAMP is not a response commonly associated with muscarinic receptors but has been observed in association with PI metabolism in SK-N-SH neuroblastoma cells25. The stimulation of cAMP in these cells is pertussis toxin-insensitive as is the PI stimulation by ml and m3 receptors in NG-108 cells 22. The mechanism for cAMP stimulation has not been determined but may be a secondary response to PI stimulation. The ml and m3 receptors also stimulate arachidonic acid release while m2 and m4 receptors do not 26. Since the PI response is inhibited by phorbol esters whereas the arachidonic acid response is stimulated by them, it appears that arachidonic acid release is not secondary to PI stimulation and that the two responses must be mediated by different phospholipases. In general the responses define two mutually exclusive classes. This agrees with studies of cell lines such as NG-108 and 132N1 (which contain m4 and m3 mRNA, respectively TM)in which muscarinic stimulation is coupled exclusively to either stimulation of PI or inhibition of cAMP27. However, there are indications that the m2 and m4 receptors can couple weakly to PI stimulation at least at high receptor levels per cell and at high doses of agonist 24. Whether this lack of exclusivity of second messenger systems reflects an unusual stoichiometry in these cells or reflects a normal mode of coupling in cells with endogenous receptors is unclear. A recent report also suggests that exogenous ml receptors couple more efficiently to inhibition of adenylate cyclase than to PI turnover in RAT-1 cells28, but this is difficult to evaluate without more data. Most of the mammalian cells used for the characterization of the receptors were chosen because they lack endogenous muscarinic receptors so that muscarinic responses can be clearly ascribed to the exogenous cloned receptors. However, such cells cannot be expected to display all of the potential responses of muscarinic receptors in their normal TINS, VoL 12, No. 4, 1989

environments. Unlike the A9 cells which lack voltagedependent channels, NG-108 cells contain an Mcurrent, a voltage-dependent K + current, which is known to be inhibited by stimulation of an endogenous muscarinic receptor 29. This M-current is also inhibited by stimulation of exogenous ml and m3 receptors. This response is consistent with the expectation that M-current inhibition is mediated by a pertussis toxin-insensitive G protein which activates PI metabolism29. However, the interpretation of Mcurrent inhibition by the endogenous receptor in NG108 cells is unclear. Is it mediated by the m4 receptor which is encoded by a reasonably abundant mRNA? If so, then the m4 receptor has a similar response to that of the ml and m3 receptors, although perhaps requiring a greater dose of agonist since Fukuda et al. observed no significant M-current inhibition in control NG-108 cells22. Or does it reflect the presence of another muscarinic receptor whose mRNA has not been detected, an inevitable question with cells containing endogenous receptors? To characterize fully the possible responses of the different receptor subtypes it will be necessary to examine the responses of the cloned receptors in a variety of cells both containing and lacking endogenous receptors. It is clearly possible that further investigation will reveal heterogeneity within or clear overlap between what now appear to be two homogenous classes of distinct functional responses.

Acknowledgements I thank my collaborators, especiallyNoel Buckle),and Mark &ann, for numerous discussionswhich have helpedto form my viewson the muscarinicreceptors.

Selected references 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Nathanson, N. M. (1987) Annu. Rev. Neurosci. 10, 195-236 Kubo, T. etal. (1986) Nature 323,411-416 Kubo, T. et al. (1987) FEB5 Lett. 209, 367-372 Peralta, E. G. et al. (1987) Science 236, 600-605 Bonner, T. I., Buckley, N. J., Young, A. C. and Brann, M. R. (1987) Science 237, 527-532 (1556 and 1628) Bonner, T. I., Young, A. C., Brann, M. R. and Buckley, N. J. (1988) Neuron 1,403-410 Peralta, E. G. etal. (1987) EMBOJ. 6, 3923-3929 Braun, T. et aL (1987) Biochem. Biophys. Res. Commun. 149, 125-132 Allard, W. J., Sigal, I. S. and Dixon, R. A. F. (1987) Nucleic Acids Res. 15, 10604 Akiba, I. etal. (1988) FEBS Lett. 235, 257-261 Gocayne, J. et al. (1987) Proc. Natl Acad. 5ci. USA 84, 8296-8300 Fukuda, K. etal. (1987) Nature 327, 623-625 Strader, C. D. etal. (1987)J. BioL Chem. 262, 16439-16443 Strader, C. D. etal. (1988)J. BioL Chem. 263, 10267-10271 Kobilka, B. K. etal. (1988)Science 240, 1310-1316 Maeda, A., Kubo, T., Mishina, M. and Numa, S. (1988) FEB5 Lett. 239, 339-342 Brann, M. R., Buckley, N. J, and Bonner, T. I. (1988) FEB5 Lett. 230, 90-94 Buckley, N. J., Bonner, T. I. and Brann, M. R. (1988) J. Neurosci. 8, 4646-4652 Bujo, H. et al. (1988) FEBSLett. 240, 95-100 Jones, S. V. P. et al. (1988) Proc. Natl Acad. 5ci. USA 85, 4056-4060 Jones, S. V. P. etal. (1988) MoL PharmacoL 34, 421-426 Fukuda, K. etal. (1988) Nature 335, 355-358 Peralta, E. G. etal. (1987) Science 238, 672-675 Peralta, E. G. etal. (1988) Nature 334, 434-437 Baumgold, J. and Fishman, P. H. (1988) Biochem. Biophys. Res. Commun. 154, 1137-1143 Conklin, B. R. et al. (1988) Proc. Natl Acad. 5ci. USA 85, 8698-8702 Harden, T. K. et al. (1986) Trends PharmacoL 5ci. 7 (Suppl.), 14-18 Stein, R., Pinkas-Kramarski, R. and Sokolovsky, M. (1988) EMBO J. 7, 3031-3035 Brown, D. (1988) Trends Neurosci. 11,294-299 151