Chapter 2 Structure and function of muscarinic receptors

S.-M. Aquilonius and P.-G. Gillberg (Eds.) Progress in Brain Research, Vol. 84 0 1990 Elsevier

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Science Publishers B.V. (Biomedical Division)

CHAFTER 2

Structure and function of muscarinic receptors Michael I. Schimerlik Department of Biochemistry and Biophysics, Oregon State Universiy, Coruallis, OR 97331 -6503, U.S.A.

Introduction Early pharmacological studies by Sir Henry Dale (1914) resulted in the characterization of cholinergic responses as either nicotinic or muscarinic in nature. This was followed by Otto Loewi’s discovery of Vagusstoff (Loewi, 1921), demonstrating that acetylcholine (ACh) was the neurotransmitter which mediated the negative chronotropic and negative inotropic effects of vagal stimulation on the heartbeat. More recently, muscarinic receptors (mAChRs) have been shown to couple to physiological responses in the central and peripheral nervous system, smooth muscle, secretory glands and several clonal cell lines in addition to those in the heart (Schimerlik, 1989). At this time there appear to be at least five distinct subtypes of mAChR. Although this article will attempt to summarize results from several laboratories regarding the structure and function of the five mAChR subtypes, specific examples, particularly concerning physical characterization of the receptor protein, will be taken from work done in the author’s laboratory utilizing the M, subtype from porcine atria. Physiological responses Depending on the particular system under consideration, muscarinic receptors can couple to physiological responses which are either excitatory or inhibitory in nature. In heart preparations, addition of ACh caused a reduction in the level of

adenosine 3’,5’-cyclic monophosphate (CAMP) (Murad et al., 1962). Muscarinic inhibition of adenylyl cyclase was shown to require guanosine triphosphate (GTP) (Jacobs et al., 1979) and is now known to be mediated by one or more of a class of signal-transducing proteins called guanine nucleotide binding proteins (G-proteins) (Gilman, 1987). G-proteins that regulate adenylyl cyclase are heterotrimers consisting of differing G, subunits and similar GB, subunits. The role of the hormone-receptor complex is to promote the release of a tightly bound molecule of guanosine diphosphate (GDP) from the G, subunit of the heterotrimer (Gilman, 1987). The binding of GTP to the vacant guanine nucleotide binding site results in the dissociation of the heterotrimer into G, - GTP plus GBy subunits which then regulate various cellular effector systems or ion channels. Activity is terminated by the hydrolysis of GTP to GDP and phosphate by the GTPase activity of the G, subunit followed by subunit reassociation to give the G-protein - GDP complex. The stimulatory G-protein, Gs, activates adenylyl cyclase, while the inhibitory G-protein, Gi, or the ‘other’ G-protein, Go, inhibits the enzyme. Hormonal inhibition of adenylyl cyclase may occur by the liberation of excess GB, subunits from Gi or Go which promote Gsnreassociation to form the inactive heterotrimer (Katada et al., 1984), by competition of Gi, with Gsn for a binding site on the enzyme or by direct binding of Giayto a site on the enzyme (Katada et al., 1986). Pertussis toxincatalysed ADP-ribosylation of Gi functionally un-

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couples mAChR-mediated inhibition of adenylyl cyclase in the heart (Kurose and Ui, 1983). In 1321 N1 human astrocytes, however, mAChRs attenuate CAMP levels by a different mechanism: activation of a calcium-sensitive phosphodiesterase (Tanner et al., 1986). Since astrocytes contain the M, mAChR subtypes while the M2 subtype is found in heart, it can be seen that different mAChR subtypes may couple to different physiological effector systems (cloning and expression of mAChR genes, below). A second biochemical response initiated by muscarinic agonists is the stimulation of inositol phospholipid (IP) metabolism. The activation of phospholipase C by mAChRs is guanine nucleotide-dependent (Haslam and Davidson, 1984) and is mediated by an as yet unisolatd G-protein(s). That the identity of this G protein may depend on the system under consideration has been demonstrated by differing susceptibilities to uncoupling by treatment with pertussis or cholera toxins. Chick heart (Masters et al., 1985) and 1321 N1 astrocytes (Hepler and Harden, 1986) PI responses were not sensitive to pertussis toxin while the PI response of mast cells (Nakamura and Ui, 1985) was pertussis toxin-sensitive. The PI response of Flow 9000 pituitary tumor cells was sensitive to cholera toxin treatment but not treatment with pertussis toxin (Lo and Hughes, 1987). Phospholipase C acts on phosphatidylinositol 4,5-bisphosphate to give inositol 1,4,5-triphosphate (IP,) and diacylglycerol (DG). IP, can act to release Ca2+ from the endoplasmic reticulum (Streb et al., 1983), while DG activates protein kinase C (Nishizuka, 1988). DG may then be metabolized by the action of diacylglycerol lipase to release arachidonic acid, an eicosanoid precursor. Arachidonate can also arise from phospholipase A, action in a process mediated by a pertussis toxin-sensitive G-protein (Nakashima et al., 1988). Although muscarinic agonists have been shown to cause arachidonate release in cerebellar cortex slices (Reichman et al., 1987), it is not yet known whether this occurred by one or both of the above-mentioned pathways.

In addition to activating G-protein-dependent second-messenger systems, G-protein subunits may act directly on ion channels. The system most extensively studied in this respect has been the mAChR-activated inward rectifying potassium channel in the heart. Initial results from patch clamping experiments were contradictory, with one group suggesting that the a subunit of Gi activated the inward rectifying potassium channel (Yatani et al., 1987) and a second proposing a role for the P y subunits (Logothetis et al., 1987). As of this time, the controversy is not yet completely resolved; however, the a subunit is active at lower concentrations (Codina et al., 1987) than By, antibodies against Gi, block channel activation in insideout patches (Yatani et al., 1988a), and recombinant Gi, subunits open the channel (Yatani et al., 1988b). These results strongly suggest that the Gi, subunit can directly interact with and activate the inward rectifying potassium channel in atria. More recently, it has been proposed that the f l y subunit may activate the inward rectifying potassium channel in heart by activation of a phospholipase A,. This results in increased levels of an arachidonic acid metabolite(s) which may act as a second messenger (Kim et al., 1989; Kurachi et al., 1989) to open the channel. Ligand binding properties of muscarink receptors Interactions of ligands with mAChRs have been characterized utilizing radiolabeled antagonists such as L-quinuclidinyl benzilate (Yamamura and Synder, 1974) or labeled agonists such as cismethyldioxolane (Galper et al., 1987). Antagonist binding appears to be consistent with a two-step kinetic mechanism (Jarv et al., 1979; Schimerlik and Searles, 1980), while agonists and GTP appeared to add in a random manner to form the agonist . mAChR . G protein GTP complex (Galper et al., 1987). Agonist binding properties have been interpreted in terms of a ternary complex model (Ehlert, 1985) where the free mAChR binds agonist with low affinity while the mAChR G-protein complex has high affinity for agonists.

-

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Guanine nucleotide binding to the G-protein results in uncoupling of the mAChR from the Gprotein with a return to the low-affinity agonkt state. The interconversion of the mAChR from low- to high-affinity form for agonists has not been observed (Schreiber et al., 1985; Galper et al., 1987), suggesting that the mAChR and G-protein are either pre-coupled in the membrane or that the interaction between them was too fast to be observed. Muscarinic receptor subtypes were first identified by the binding properties of the antagonist pirenzepine (Hammer et al., 1980), which permitted classification into high-affinity (M,) and low-affinity (M,) subtypes. The M, subtype was further subclassified into cardiac and glandular mAChRs based on the binding of AF-DX116 (Hammer et al., 1986), which binds more tightly to the cardiac subtype, and p-F-HHSiD (Lambrecht et al., 1988), which has a higher affinity for the smooth muscle and glandular M, mAChRs. Studies with cell lines expressing the five recombinant subtypes have shown, however, that it is not possible to differentiate between all five subtypes based on ligand binding properties alone (Peralta et al., 1987b; Buckley et al., 1989).

Purification and characterization of muscarinic receptors Muscarinic receptors have been purified from porcine atria (Peterson et al., 1984), porcine cerebellum (Haga and Haga, 1985), rat forebrain (Berrie et al., 1985) and chick heart (Kwatra and Hosey, 1986). The procedures were somewhat different for each group; however, all laboratories utilized affinity chromatography on 3-(2’-aminobenzhydryloxy) tropane agarose, a method invented by Haga and Haga (1983). The results of hydrodynamic and compositional analysis of the purified porcine atrial mAChR are summarized in Table I. In either Triton X-405 or dodecyl pmaltoside the protein appeared roughly spherical with a frictional ratio of 1.2 and bound large quantities of detergent (1-2.5 g of detergent per

TABLE I Properties of purified porcine atrial muscarinic receptor Parameter

Stokes radius (A) Frictional ratio Bound detergent (g/g) Molecular mass (daltons) Carbohydrate by weight (W) Molecular weight of polypeptide chain

Dodecyl

Triton x-405

/3-D-

43 1.21 1.01 68 OOO

53 1.22 2.54 72 OOO

maltoside

26% 55 OOO

Data are summarized from Peterson et al. (1986, 1988).

gram of protein), confirming the hydrophobic nature of the molecule. The molecular mass of protein plus carbohydrate was about 70 kDa and compositional analysis showed that the molecule was about 26% carbohydrate by weight, resulting in an estimation of the molecular mass of the protein portion of the molecule to be about 55 kDa. The mAChR is an acidic molecule with a PI of about 4.6 (Repke and Matthes, 1980).

Cloning and expression of muscarinic receptor genes At this time, the amino acid sequences of five distinct mAChR subtypes have been deduced from clones isolated from human, rat and porcine cDNA and genomic libraries (Kubo et al., 1986a,b; Peralta et al., 1987a,b; Bonner et al., 1987, 1988). Southern analysis of rat and human genomic DNA has suggested that there may be up to four additional muscarinic subtypes in the rat and one additional human subtype (Bonner et al., 1987). The expression of mAChR subtypes appears to be relatively tissue and/or cell line specific (Bonner et al., 1987; Peralta et al., 1987b). Hydropathy analysis of the amino acid sequence data from the five mAChR subtypes, shown in Fig. 1 for the porcine atrial mAChR, suggested a common structural motif in which the proteins have seven transmembrane regions. By

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I

Amino acid no

Fig. 1. Hydropathy analysis of porcine M, muscarinic acetylcholine receptor. Relative hydropathy of the amino acid sequence was analysed by the method of Kyte and Doolittle (1982) using a nineteen amino acid window. The bars indicate the putative transmembrane sequenm and are numbered one to seven from the amino to the carboxyl terminus of the molecule.

analogy with rhodopsin (Ovchinnikov, 1982), the amino terminus is located outside the cell and the carboxyl terminus intracellularly. Fig. 2 shows the deduced amino acid sequence and proposed topology of the porcine atrial mAChR. Potential sites of N-linked glycosylation, given by the sequence N-X-S or T, are located at the amino terminal and potential phosphorylation sites can be found in the large intracellular loop between transmembrane regions five and six. Cytosolic loops one-two and three-four and the seven transmembrane regions have a high degree of amino acid identity amongst the mAChR subtypes; however, there is little sequence identity in the cytosolic loop connecting transmembrane regions five and six. Therefore this region may play a unique role in coupling mAChR subtypes to specific effector systems and/or in the regulation of the various subtypes. All subtypes contain three aspartic acid

E x t r a c e l l u l a r space

Cytoplasm

Fig. 2. Amino acid sequence and topography of the porcine M, muscarinic receptor. Amino acids are identified using standard oneletter abbreviations.

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residues located just outside or within transmembrane regions two and three (aspartates 69,97 and 103 for the porcine mAChR, Fig. 2). Peptide mapping studies after affinity alkylation with propylbenzilylcholine mustard have led Curtis et al. (1989) to propose that either or both of the two aspartates at the top of transmembrane helix two (aspartate 97 and 103 for the porcine mAChR) may act as the negative counter ion for the positively charged amino group of muscarinic ligands. Expression of the genes coding for the five mAChR subtypes has permitted analysis of the ligand binding properties for the proteins as well as a determination of their specificity for effector systems. Expression of the M, and M, subtypes in Xenopus oocytes resulted in the expected ligand specificity for pirenzepine (M,, high affinity; M,, low affinity) and both mAChR subtypes coupled to a calcium-dependent increase in chloride conductance. In addition, the M, subtype activated a second channel in which current was carried by sodium and potassium ions (Kubo et al., 1986a; Fukuda et al., 1987). Expression of the M, subtype in Chinese hamster ovary cells (Ashkenazi et al., 1987) showed that the protein could couple to both inhibition of adenylyl cyclase and stimulation of IP metabolism. The M, mAChR was more tightly coupled to inhibition of adenylyl cyclase and the responses appeared to be mediated by different G proteins. Analysis of MI-M, mAChRs expressed in embryonic kidney cells (Peralta et al., 1988), NG108-15 cells (Fukuda et al., 1988) and A9 L cells (Conklin et al., 1988) showed that the M, and M, subtypes coupled

preferentially to the stimulation of IP metabolism, while M, and M, were selective for inhibition of adenylyl cyclase. The M, subtype, expressed in Chinese hamster ovary cells, coupled preferentially to the stimulation of IP metabolism (Bonner et d.,1988). The properties of the various subtypes are summarized in Table 11. Reconstitution of muscarinic receptors with Gproteins Reconstitution of purified porcine brain mAChRs (Haga et al., 1985) or bovine brain mAChRs resolved from G-proteins by ion-exchange chromatography (Florio and Sternweis, 1985) with Gi and Go resulted in preparations that showed agonist-stimulated GTPase activity and high-affinity guanine nucleotide-sensitive muscarinic agonist binding, respectively. Purified brain mAChRs reconstituted effectively with Gi and Go (Haga et al., 1986) as well as G, (Haga et al., 1989), a G protein most likely corresponding to Gi2.Studies with resolved subunits of Go (Florio and Sternweis, 1989) showed that both G protein subunits were required for agonist-stimulated guanine nucleotide exchange. Agonists increased the association rate constant for GTP but decreased the rate of GDP association with Go, suggesting a differential effect on the two guanine nucleotides. Reconstitution of purified porcine atrial mAChRs with purified atrial Gi also demonstrated guanine nucleotide-sensitive high-affinity agonist binding (Tota et al., 1987). The data from

TABLE I1

Properties of muscarinic receptor subtypes Subtype

A.A. number

Molecular weight

Physiological response

MI M2 M3 M4 Ms

460 466 479 590 532

51 387 51 681 53 014 66085 60120

Stimulation of PI metabolism Inhibition of adenylyl cyclase Inhibition of adenylyl cyclase Stimulation of PI metabolism Stimulation of PI metabolism

Data are for the human subtypes.

~

16 TABLE 111 Kinetic and thermodynamic analysis of the properties of reconstituted porcine atrial G, and porcine atrial muscarinic receptors ParameterAigand

w-')

k,,, KE"F" (nM) K,GTP(nM) K2Pp (min-') KEDP (nM) KgTP(nM) KgTPrS(pM)

Carbachol 2.2 33 31 4.5

500 10 332

L-Hyoscyamine 0.2 10 0.1 10

10 454

Ratio 11 3.1 45 50

1 0.7

a series of kinetic and thermodynamic measurements on this reconstituted system are summarized in Table 111. In the presence of the antagonist L-hyoscyamine, the dissociation of GDP appeared to be the sole rate-limiting step in the steady-state mechanism for GTP hydrolysis (k,,, = K2pp); however in the presence of the agonist carbachol, k,,, was increased by about 11-fold, while K$pp was increased 45-fold. These results suggest that GDP release is no longer solely rate-limiting in the presence of carbachol. The IC,, for GTP in inducing the conversion of highaffinity mAChR for carbachol to low affinity (~2:) was approximately equal to the K , for GTP (31 nM) for the agonist-stimulated GTPase activity. Finally, the affinity of GDP for Gi in the presence of the mAChR . carbachol complex was decreased by about 50-fold, consistant with the 45-fold increase in the rate of GDP dissociation; however, the affinity of GTP and GTP,S was unaffected by muscarinic ligands. These results suggest that the agonist . mAChR complex selectively weakens the binding of GDP to G I mainly by increasing the dissociation rate constant for that ligand. This would then promote a feed-forward mechanism whereby bound GDP is exchanged for GTP. Additional studies of the reconstituted system showed that the mAChR could be phosphorylated by CAMP-dependent protein kinase A (Rosenbaum et al., 1987). The phosphorylation stoichiometry was altered by muscarinic ligands only when the agonist-occupied mAChR was

phosphorylated in the presence of GI. These results suggested that the mAChR receptor has a unique conformation in the presence of both an agonist and G, such that the extent of phosphorylation by CAMP-dependent protein kinase is increased. The reconstituted system has also been used to define the mechanism of action of partial versus full muscarinic agonists. The GTPase activity of GI was measured at varying levels of mAChR in the presence of saturating concentrations of the full agonist carbachol or the partial agonist pilocarpine. Analysis of the data showed that at saturating receptor concentrations, both ligands gave about the same value for the turnover number of the GTPase activity of G, (5.0 min-' for carbachol, 6.4 min-' for pilocarpine); however, the receptor-agonist complex bound about 4-fold more weakly to GI in the presence of the partial agonist (Kd = 3.1 nM) than in the presence of the full agonist ( Kd = 0.8 nM). These results indicated that the difference between full and partial agonists in this system was due to weaker binding of the agonist . mAChR to GI, resulting in a lower steady-state level of activated G-protein. Conclusions Over the past decade, our knowledge of the structure and mechanism of action of mAChRs has increased enormously. The detailed mechanism of receptor interactions with G-proteins has yet to be elucidated, as has the manner in which the various G-proteins interact with effector systems in the cell. These goals are complicated by the multiplicity of receptor subtypes, G-proteins and effector systems with which they interact and the apparent tissue specificity exhibited by the various components of the muscarinic signaling system.

Acknowledgements The author would like to acknowledge the expert typing skills of Barbara Hanson and the support of his research by USPHS grants HL23632 and ES00210.

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