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Agonist activation of muscarinic acetylcholine receptors
Cellular Signalling Vol. 5, No. 6, pp. 687-694, 1993. Printed in Great Britain.
0898-6568/93 $6.00 + 0.00 © 1993 Pergamon Prcu Ltd
MINI REVIEW AGONIST
ACTIVATION
OF MUSCARINIC RECEPTORS
ACETYLCHOLINE
EDWARD C. HULME,* CAROL A. M. CURTIS, KARL~ M. PAGE and PHILIP G. JONES Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K. (Received 18 July 1993; and accepted 2 August 1993) Key words: Muscarinic, acetylcholine, GTP, G-protein, mutagenesis.
INTRODUCTION
STRUCTURAL MODELS OF THE mAChRs
Tim muscarinic acetylcholine receptors (mAChRs) are important members of the supeffamily of G-protein-coupled receptors (GPCRs). Five distinct mammalian mAChR genes have been identified. Broadly, these are divided into two categories: (i) the ml, m3 and m5 mAChRs, which activate G-proteins of the Gq/G 11 class stimulating phosphoinositide (PI) breakdown and Ca 2+ mobilization; (ii) the m2 and m4 mAChRs which preferentially couple to pcrtussis toxin-sensitive G-proteins of the Gi and Go class, so mediating a variety of processes, including inhibition of adenylyl cyclase, activation of inward-rectifier potassium currents, and inhibition of voltage-sensitive Ca 2+ channels. For recent reviews of mAChR physiology, pharmacology and structure, see Refs 1-4. The purpose of this review is to outline how recent mutagenesis and protein chemical studies have advanced our perceptions of the way in which agonists bind to, and activate, the mAChRs, in the context of recent progress in our understanding of the structure and mechanism of GPCRs.
The protein fold of the mAChRs is a 7-transmembrane helical bundle The idea that the protein fold of the GPCRs is an antiparallel 7-transmembrane (TM) helical bundle topologically similar to that found in bacteriorhodopsin with an extracellular N-terminus, and an intracellular C-terminus, is well established [5] (Fig. l a). Crude models of the TM structures of these receptors have relied on the analysis of sequence variation to position non-conserved residues on the lipid facing surfaces of the putative helices [3]. Fourier transform analysis has contributed an extra cycle of refinement [6, 7], confirming the probable a-helical nature of the hydrophobic transmembrane sequences, and allowing optimal orientation vectors to be computed. Detailed 3dimensional models have been built, based on the proposed analogy with the TM structure of bacteriorhodopsin [8-10]. The first low-resolution projection structure of a GPCR, bovine rhodopsin, has been a milestone [11]. The TM region is indeed a bundle of 7 a-helices, but their distribution and tilt differs from that in bacteriorhodopsin. A revised generic model of the TM structure of the GPCRs has been composed, taking into
* Author to whom correspondenceshould be addressed. 687
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E.C.HULME
et al.
(a) Extracellular 1
2
3
4
5
6
7
Intracellular
(b)
T A14
T 1 7 1 ~ ' ~ ¼ L.
v,~-'-"~
LllP
FIG. 1. (a) The transmembrane sequences of the ml mAChR plotted as helical nets, in antiparallel orientation, as in a 7-transmembrane helical bundle. Amino acids (numbered with respect to the N-terminus of each helix) which may contribute to the acetylcholine binding site are shaded. They are (i) Asp6 (TM3) and Tyr7 (TM7), which are characteristic of cationic amine receptors, (ii) Ala7 and Alal0 (TM5) which are homologous to the Ser residues thought to "gate the catechol OH groups in the fl-adrenergic receptor binding site and (iii) a series of conserved amino acids with potentially H-bonding side-chains mostly specific to the mAChR sequences. Of these residues, all but TrP2(TM3), Ala7 and Alal0 (TM5) and Ash 16 (TM6) have been studied by site-directed mutagenesis [see (b)]. (b) An oriented helical wheel model of the transmembrane region of the ml mAChR,
Agonist activationof muscarinicacetylcholinereceptors account the rhodopsin projection map [12]. Its most obvious feature is an almost hexagonal packing of TM helices 2,4,5,6,7 around TM3. TM3 is predicted to be the structural core of the molecule. TM1 is more exposed. Figure lb shows a similarly adapted model of the TM region of the rat ml mAChR. The bundle is proposed to be very tightly packed at the inner cytoplasmic surface, where G-proteins bind, but to open out somewhat to create a ligand binding pocket within the outer leaflet of the phospholipid bilayer. A tight structure incorporating the membrane-proximal portions of the intracellular loops would provide a plausible basis for the conformational constraint which has to be broken, utilizing the binding energy of an agonist, to expose the epitopes needed for G-protein recognition and activation.
A disulphide bond stabilizes the protein fold A vital element of the structure of virtually all GPCRs, the mAChRs included [13], is a disulphide bond which links the outer end of TM3 to the second outside (o2) loop between TM4 and 5. Comparing the mAChRs with the other cationic amine receptors, it is notable that a nearly invariant number of amino acids provides the link between the C-terminal end of this bond and specific polar residues in TM5 which are thought to contribute to the ligand binding pocket. The disulphide-bonded cysteines cannot be deleted without preventing the folding and expression of the mAChRs [14]
689
and rhodopsin [15]. Models based on the rhodopsin projection map rationalize the role of the disulphide link, suggesting it to be critical for packing TM helices 4 and 5 against TM3.
Transmembrane helices 1-5 and 6-7 may fold as independent domains When TM 1-5 and TM6-7 of the m3 and m2 mAChRs were co-expressed as separate polypeptides, receptor activity was reconstituted in vivo, suggesting that these two parts of the molecule, which are separated by the very long third intracellular (i3) loop, fold as independent N-terminal and C-terminal domains, which possess a specific affinity for one another [16]. A novel suggestion, based on the recovery of a low level of activity following the co-expression of otherwise inactive m3(1-5)/~t2(6-7) and ~t2(l-5)/m3(6-7) chimaeras, is that domains from different receptor molecules may be able to associate, leading to the formation of active dimers or higher oligomers [17], although proteolytic nicking, followed by association of the cognate domains, appears not to have been ruled out as an explanation for the observations. At present, it is not clear how these findings impinge upon the proposal, based on deletion mutagenesis studies of rhodopsin [18], that the extracellular loops of the GPCRs fold to form a globular domain which is essential for positioning and packing the TM helices. However, they suggest the possibility of a more dynamic struc-
viewed from the extracellular side. The apparent helical pitch angles, and orientation vectors (arrows) were calculated by Fourier transform power spectral analysis of sequence variation, as described in Ref. 7. Fully conserved residues are printed in bold. The helices are arranged to match the apparent distribution of the TM helices in the projection structure of rhodopsin, as described by Baldwin [12], with the non-conserved faces adjacent to the phospholipid bilayer. The location of the disulphide bond linking Cys98 two residues from the top of TM3 to Cys178 in the second extracellular loop is shown diagrammatically. The effects of mutations (Asp-Asn/Giu; Thr-Ala; Tyr-Phe; Trp-Phe; Pro-Ala; Cys-Ser) on the binding properties of the mAChRs are indicated as follows: light/dark shaded residues--agonist (ACh or carbachol) affinity reduced by more than 5fold; *antagonist affinity reduced by more than 5-fold. Note that Trp9 and Prol8 (TM4), and Prol4 (TM5) are additional to the set of residues shown in (a). Mutation of Thrl I (TM6) and Thrl I (TM7) was without major effects on binding. Trp2 (TM3), Ala7 and Alal0 (TM5) and Asnl6 (TM6) have not yet been studied by mutagenesis. Mutation of Asp6 (TM3), Thr6 (TM5) and Cys6 (TM7) (dark shading) strongly reduced the maximum PI response to carbachoi [39, 49-51].
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ture than that implied by the Baldwin model [121.
Modelling allows the rational prediction of binding-site residues Residues thought to be important in ligand binding occur on the inner surfaces of the TM helices, one to three helical turns from their predicted extracellular termini, at a depth similar to that of the protonated Schift's base of the retinal chromophore in rhodopsin. Of primary importance is an Asp in TM3 [D6(TM3)] which is found in all GPCRs whose ligands are cationic amines. Ala7 and Alal0 in TM5 are homologous to two serine residues in the fl-adrenergic receptors (flARs) which are thought to H-bond to the catechol OH groups of fl-agonists [19]. These alanines may participate in binding the ACh methyl group. Conserved H-bond donors/acceptors, several of them unique to the mAChRs, also populate this level in the TM structure. Prominent amongst these are Thr3/Thr6 in TM5, Tyrl5/Asnl6 in TM6, and Tyr3/Tyr7 in TM7 (Fig. lb). As discussed below, several of these amino acids may be important in the ligation of the ACh ester group. Indeed, it seems extremely likely that some of these polar side-chains may have a dual role, participating in a network of intramolecular H-bonds which constrains the ground-state structure, until rearranged by the interpolation of the agonist to create the conformationally rearranged activated state. Site-directed mutagenesis has allowed a start to be made in evaluating a number of these possibilities.
TERNARY COMPLEX MODELS CAN BE USED TO DISSECT THE EFFECTS OF MUTATIONS ON AGONIST BINDING AND G-PROTEIN RECRUITMENT In analysing the effects of mutations, it is important to extract the maximum amount of
mechanistically relevant information. Some years ago [20, 21], we showed that antagonists (which bind without activation) yield simple binding curves. In contrast, agonists, which cause conformational induction leading to G-protein recruitment, give complex, multicomponent binding curves. In these, the lowaffinity component represents binding to monomeric receptors, and the high-affinity component(s) arise mainly or totally from the formation of agonist-receptor-G-protein (ARG) ternary complexes [22-26]. Ternary complex formation is promoted by millimolar concentrations of magnesium ions [27], whose action is essential to link agonist binding to GDP dissociation [28, 29]. GTP binding appears to be succeeded by a receptor-catalysed ~activation step, in which a conformational change in the GTP-liganded G-protein is a necessary preliminary to the separation of ~GTP from the ~7 subunit [30, 31]. The break-up of the A R G - G T P quaternary complex causes the reversion of tlie receptor to its low-affinity AR form. The ternary complex model [32-34] is therefore the minimal scheme suitable for the analysis of the equilibrium binding properties of GPCRs in the absence of guanine nucleotides. The analysis of binding data using this model effectively provides three independent parameters: (i) the affinity constant K for agonist binding to free receptor to yield the AR binary complex; (ii) the apparent affinity constant governing formation of the ARG ternary complex--in fact, not a true bimolecular association constant, but a composite constant K*.KG, consisting of the product of the affinity, KG, of the G-protein for the free receptor and the affinity, K*, of the agonist for the receptorG-protein complex and (iii) an estimate of the apparent ratio of total G-protein to total receptor in the system, Gt:Rt. The ratio K*KG/K estimates the affinity of the G-protein for the AR binary complex and is a measure of the efficacy of the agonist in inducing formation of the catalytically active ARG ternary complex. The ternary complex model can readily be extended to allow for the presence of
Agonist activationof muscarinicacetylcholinereceptors multiple G-proteins. Recently, it has been expanded to take account of the complex interplay of cooperativity between agonist, receptor, G-protein ct and fly subunits, and guanine nucleotides, and now describes the characteristic phenomenology in a satisfying fashion, at least at the qualitative level [35]. It has also been extended explicitly to incorporate the agonist-induced conformational change in the receptor [36], so clarifying the relations which exist between agonist affinity and efficacy. The virtue of the ternary complex model of agonist-receptor-G-protein interaction is that it enables the effects of mutations on formation of the binary agonist-receptor complex to be distinguished from their effects on stabilization of the ternary complex. Measurements of functional activity, for instance, stimulation of the GTPase activity of the G-protein in membranes, or of second messenger production in whole cells, then allow the catalytic activities of ternary complexes formed by different mutant receptors to be compared.
MUTAGENESIS STUDIES OF mAChRs
The conserved aspartic acid residue in TM3 of the mAChRs is vital for ternary complex formation and functional activation, but is less important for ligand docking The conserved Asp in TM3 (Asp6, TM3) seems certain to be involved in ligand binding. We have shown that the carboxylate side-chain is labelled by the aziridinium headgroup of the alkylating antagonist analogue [3H]PrBCM [13], and by the corresponding acetylcholine derivative, [aH]acetylcholine mustard [37, 38]. In the ml mAChR, the Aspl05Asn mutation, which deletes the charge while partially preserving the polarity and H-bonding potential of the carboxylate side-chain, greatly reduces the receptor's affinity for the antagonist [3H]quinuclidinyl benzilate, and virtually abolishes the PI response to carbachol [39]. These findings, in conjunction with muta-
691
genesis studies on the catecholamine receptors [40, 41], have encouraged the understandable assumption that the role of the Asp residue in mAChRs and related receptors is simply to dock the cationic headgroup of agonists and antagonists in a more or less equivalent fashion. However, our investigations suggest its function to be more subtle. We have studied the properties of the ml and m2 mAChRs with Asp6 (TM3) mutated to Glu, in an attempt to disrupt the headgroup interaction while maintaining the negative charge in the TM domain. This gives a surprisingly modest (3-fold) decrease in the binding of the potent antagonist [3H]N-methylscopolamine [42] by comparison with the 10,000-fold reduction in antagonist affinity imposed by the homologous mutation in the flAR [43]. Because the antagonist affinity remains high, we have been able to carry out detailed binding studies on the Glu mutants. These have been complemented by functional studies. The Asp6-Glu (TM3) mutation exerts a large effect on acetylcholine binding, reducing the binary affinity constant K by 20-fold (ml) to 70fold (m2) and the composite constant K*.KG governing ternary complex formation by 40fold (ml) to 250-fold (m2). Strong destabilization of the ternary complex has been seen for all potent agonists studied, but, unexpectedly, binary complex formation by several pharmacologically potent, high-efficacy agonists (e.g. oxotremorine-M) was discovered to be relatively unperturbed [42]. In the case of ACh at the ml mAChR, the affinity of the G-protein for the agonist-receptor complex is reduced by a factor of two, but residual ternary complex formation is still adequate to support a good phosphoinositide response; in contrast, the phosphoinositide response to agonists such as oxoM, which lose the ability to stabilize the ARG complex at the expense of the AR complex, is almost obliterated by the mutation (P.G. Jones, unpublished data). In general, analysis of the binding data using ternary complex models has proved reasonably predictive of the functional responses, for both the wild-type and Glul05 ml mAChRs.
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These results show that the interactions made
by Asp6 (TM3) are more important for ternary complex formation, and receptor activation, than for binary complex formation, and binding. Pharmacophoric models of the activated state of the mAChR binding site assume a separation of about 3 A between the onium headgroup and the carboxylate oxyanion [44]. Such a bond length would be consistent with the disruptive effect of the Asp6-Glu mutation on formation of the ternary complex. In contrast, the headgroups of many agonists appear to make a relatively loose association with Asp6 (TM3) in the binary complex, consistent with a greater ionic bond length in ligand docking. The structure-activity relations suggest that the docking site may be predominantly apolar, and that desolvation of the ligand headgroup may contribute strongly to the binding energy realised in the initial interaction. In this context, it is interesting that neutron scattering studies suggest the hydration shell of the tetramethylammonium cation to be that of an apolar molecule, rather than a small ion [45]. Bonding between the onium group and the n-electrons of aromatic residues might contribute to such a desolvation-driven binding process [8, 9, 46].The agonist docking site probably represents a primary binding site for muscarinic antagonists. Our results accord with a classical pharmacological investigation by Burgen [47], from which he concluded that the headgroup of the antagonist benzilylcholine interacts less strongly with the anionic subsite in smooth muscle mAChRs than does the headgroup of acetylcholine. ACh itself is a member of a small group of closely related agonists whose headgroups appear to make a tight interaction with Asp6 (TM3) in the binary complex, analogous to that formed in the activated state. This may entail some rearrangement of the transmembrane structure of the receptor, even in the binary complex, creating a conformer primed for G-protein recognition. Such a form would find an analogy in the long-lived metarhodopsin II intermediate which is detectable spectroscopically in the rhodopsin photocycle [48].
Several polar residues in the T M region of the mAChRs may contribute to binding the sidechain of A Ch The concept of a partial separation between agonist docking and activation sites has more general implications for attempts to use mutagenesis to identify residues which contribute to the ACh binding site. Systematic studies have been done on Tyr, Thr, Trp and Pro residues in the m3 and of Cys in the ml mAChRs [49-51]. A typical outcome has been an overall reduction in ACh or carbachol potency, both in binding, and in stimulating a PI response, of 5-60-fold, often without any major change in antagonist affinity (Fig. lb). This underlines the idea that agonist and antagonist binding exploit distinct as well as common epitopes, a perception reinforced by the discovery of a mutation [Serl7-Thr (TM2; m3 mAChR)] [49]; Fig. lb) which reduced antagonist affinity without affecting agonist binding. Two of the mutations, namely Thr6-Ala (TM5; m3 mAChR) and Cys6-Ser (TM7; ml mAChR), strongly reduce the maximum PI response evoked by carbachol as well as inhibiting agonist binding. These amino acids, unique to the mAChRs, are predicted to be on the inner surfaces of the TM helices in the ligand-binding plane (Fig. 1), indeed the Cys is predicted to be a near neighbour of Asp6 (TM3). These amino acids are outstanding candidates for direct involvement in ACh activation of the mAChRs. It needs to be emphasized that site-directed mutagenesis affects the overall energetics of agonist binding and receptor activation. Changes may be mediated by alteration of either the bond energy between receptor and ligand, or the free energy of the conformational change, or both [52]. Mutations may also have a global effect on the protein fold. This is probably the explanation for the dramatic reduction (greater than 200-fold) in both agonist and antagonist affinity imposed by the mutation Prol8-Ala (TM4) [50]. It is actually very difficult to design mutagenesis experiments to support the hypothesis that a given side-chain directly contacts the
Agonist activationof muscarinicaeetylcholinereceptors ligand. A possible strategy is to try to match the effects of mutations in the receptor with specific deletions of functional moieties from the ligand, an approach successfully applied to the flAR [19]. In the case of the mAChRs, similar tactics have been attempted by the replacement of the ester oxygen, and carbonyl group of ACh by methylene groups or sulphur [50], and the use of the resulting analogues to probe point mutants of the Thr and Tyr residues. However, the study failed to identify selective interactions of these amino acid side-chains with the ester function of ACh. Using the same compounds to probe the Glu mutants, we have found a contributory factor to be lowered polarity of the analogues relative to the parent molecule. This leads to an enhancement of their affinity for the docking site at the expense of the primed/ activation site, obscuring the effects of the mutations on the binding of the analogues. In general, however, it seems that both the Asp6Glu (TM3) and the Thr6Ala (TM5) mutations produce similarly disabled activation sites, without affecting ligand docking. In contrast, the properties of the Tyr7Phe (TM7) mutation [49] suggest that it may selectively affect the initial docking interaction. CONCLUSION An emerging theme is that antagonists and agonists exploit significantly different sets of epitopes in binding to the GPCRs [40, 53]. In the mAChRs, the archetypal ionic interaction between the agonist headgroup and the binding-site carboxylate, pinpointed by afffinitylabelling, seems to be more important in stabilizing the agonist-promoted activated state than the ground state of the receptor. Mutagenesis studies have highlighted several polar amino acid side-chains which may be candidates for ligating the ester group of ACh, but definitive results have not been obtained. It seems likely that a combination of scanning mutagenesis with chemical modification, as pioneered for the transmembrane channel of the nicotinic receptor [54] will be needed to identify specific bonds. In this way, we should eventually be
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able to deduce which parts of the TM structure of the receptor undergo relative movement when the agonist binds, and the activated state is induced, and whether the activation process involves the liberation of the G-protein binding site from a tight structure designed to occlude it, or its creation by the agonist-mediated coordination of disparate epitopes in an otherwise relatively dynamic molecule. Acknowledgements---We are grateful to the Medical Research Council (U.K.) for the provision of a Research Studentship (to K.M.P.) and a Training Fellowship (to P.G.J.).
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37. Spalding T. A., Birdsall N. J. M., Corrie J. E. T., Curtis C. A. M. and Hulme E. C. (1993) Life Sci. 52, 561. 38. Spalding T. A. 0992) Ph.D. Thesis, Council for National Academic Awards, U.K. 39. Fraser C. M., Wang C.-D., Robinson D. A., Gocayne J. D. and Venter J. C. (1989) Molec. Pharmac. 36, 840-847. 40. Strader C. D., Sigal I. S. and Dixon R. A. F. (1989) FASEB J. 3, 1825-1832. 41. Savarese T. M. and Fraser C. M. (1992) Biochem. J. 283, 1-19. 42. Page K. M., Curtis C. A. M. and Hulme E. C. (1993) Life Sci. 52, 560. 43. Strader C. D., Candelore M. R., Hill W. S. and Dixon R. A. F. (1989) J. biol. Chem. 264, 16,470-16,477. 44. Schulman J. M., Sabio M. L. and Disch R. L. (1983) J. Med. Chem. 26, 817-823. 45. Turner J., Soper A. K. and Finney J. L. (1990) Molec. Physics 70, 679-700. 46. Dougherty D. A. and Stauffer D. A. (1990) Science 250, 1558-1560. 47. Burgen A. S. V. (1965) Br. J. Pharmac. 25, 4-17. 48. Hofmann K. P. (1986) Photobiochem. Photobiophys. 13, 309-327. 49. Wess J., Gdula D. and Brann M. R. (1991) EMBO J. 10, 3729-3734. 50. Wess J., Maggio R., Palmer J. R. and Vogel Z. (1992) J. biol. Chem. 267, 19313-19319. 51. Savarese T. M., Wang C.-D. and Fraser C. M. (1992) J. biol Chem. 267, 11439-11448. 52. Shortle D. (1992) Quart. Rev. Biophys. 253, 205-250. 53. Ho B. Y., Karschin A., Branchek T., Davidson N. and Lester H. A. (1992) FEBS Lett. 312, 259-262. 54. Akabas M. H., Stauffer D. A., Xu M. and Karlin A. (1992) Science 258, 307-310.
0898-6568/93 $6.00 + 0.00 © 1993 Pergamon Prcu Ltd
MINI REVIEW AGONIST
ACTIVATION
OF MUSCARINIC RECEPTORS
ACETYLCHOLINE
EDWARD C. HULME,* CAROL A. M. CURTIS, KARL~ M. PAGE and PHILIP G. JONES Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K. (Received 18 July 1993; and accepted 2 August 1993) Key words: Muscarinic, acetylcholine, GTP, G-protein, mutagenesis.
INTRODUCTION
STRUCTURAL MODELS OF THE mAChRs
Tim muscarinic acetylcholine receptors (mAChRs) are important members of the supeffamily of G-protein-coupled receptors (GPCRs). Five distinct mammalian mAChR genes have been identified. Broadly, these are divided into two categories: (i) the ml, m3 and m5 mAChRs, which activate G-proteins of the Gq/G 11 class stimulating phosphoinositide (PI) breakdown and Ca 2+ mobilization; (ii) the m2 and m4 mAChRs which preferentially couple to pcrtussis toxin-sensitive G-proteins of the Gi and Go class, so mediating a variety of processes, including inhibition of adenylyl cyclase, activation of inward-rectifier potassium currents, and inhibition of voltage-sensitive Ca 2+ channels. For recent reviews of mAChR physiology, pharmacology and structure, see Refs 1-4. The purpose of this review is to outline how recent mutagenesis and protein chemical studies have advanced our perceptions of the way in which agonists bind to, and activate, the mAChRs, in the context of recent progress in our understanding of the structure and mechanism of GPCRs.
The protein fold of the mAChRs is a 7-transmembrane helical bundle The idea that the protein fold of the GPCRs is an antiparallel 7-transmembrane (TM) helical bundle topologically similar to that found in bacteriorhodopsin with an extracellular N-terminus, and an intracellular C-terminus, is well established [5] (Fig. l a). Crude models of the TM structures of these receptors have relied on the analysis of sequence variation to position non-conserved residues on the lipid facing surfaces of the putative helices [3]. Fourier transform analysis has contributed an extra cycle of refinement [6, 7], confirming the probable a-helical nature of the hydrophobic transmembrane sequences, and allowing optimal orientation vectors to be computed. Detailed 3dimensional models have been built, based on the proposed analogy with the TM structure of bacteriorhodopsin [8-10]. The first low-resolution projection structure of a GPCR, bovine rhodopsin, has been a milestone [11]. The TM region is indeed a bundle of 7 a-helices, but their distribution and tilt differs from that in bacteriorhodopsin. A revised generic model of the TM structure of the GPCRs has been composed, taking into
* Author to whom correspondenceshould be addressed. 687
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et al.
(a) Extracellular 1
2
3
4
5
6
7
Intracellular
(b)
T A14
T 1 7 1 ~ ' ~ ¼ L.
v,~-'-"~
LllP
FIG. 1. (a) The transmembrane sequences of the ml mAChR plotted as helical nets, in antiparallel orientation, as in a 7-transmembrane helical bundle. Amino acids (numbered with respect to the N-terminus of each helix) which may contribute to the acetylcholine binding site are shaded. They are (i) Asp6 (TM3) and Tyr7 (TM7), which are characteristic of cationic amine receptors, (ii) Ala7 and Alal0 (TM5) which are homologous to the Ser residues thought to "gate the catechol OH groups in the fl-adrenergic receptor binding site and (iii) a series of conserved amino acids with potentially H-bonding side-chains mostly specific to the mAChR sequences. Of these residues, all but TrP2(TM3), Ala7 and Alal0 (TM5) and Ash 16 (TM6) have been studied by site-directed mutagenesis [see (b)]. (b) An oriented helical wheel model of the transmembrane region of the ml mAChR,
Agonist activationof muscarinicacetylcholinereceptors account the rhodopsin projection map [12]. Its most obvious feature is an almost hexagonal packing of TM helices 2,4,5,6,7 around TM3. TM3 is predicted to be the structural core of the molecule. TM1 is more exposed. Figure lb shows a similarly adapted model of the TM region of the rat ml mAChR. The bundle is proposed to be very tightly packed at the inner cytoplasmic surface, where G-proteins bind, but to open out somewhat to create a ligand binding pocket within the outer leaflet of the phospholipid bilayer. A tight structure incorporating the membrane-proximal portions of the intracellular loops would provide a plausible basis for the conformational constraint which has to be broken, utilizing the binding energy of an agonist, to expose the epitopes needed for G-protein recognition and activation.
A disulphide bond stabilizes the protein fold A vital element of the structure of virtually all GPCRs, the mAChRs included [13], is a disulphide bond which links the outer end of TM3 to the second outside (o2) loop between TM4 and 5. Comparing the mAChRs with the other cationic amine receptors, it is notable that a nearly invariant number of amino acids provides the link between the C-terminal end of this bond and specific polar residues in TM5 which are thought to contribute to the ligand binding pocket. The disulphide-bonded cysteines cannot be deleted without preventing the folding and expression of the mAChRs [14]
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and rhodopsin [15]. Models based on the rhodopsin projection map rationalize the role of the disulphide link, suggesting it to be critical for packing TM helices 4 and 5 against TM3.
Transmembrane helices 1-5 and 6-7 may fold as independent domains When TM 1-5 and TM6-7 of the m3 and m2 mAChRs were co-expressed as separate polypeptides, receptor activity was reconstituted in vivo, suggesting that these two parts of the molecule, which are separated by the very long third intracellular (i3) loop, fold as independent N-terminal and C-terminal domains, which possess a specific affinity for one another [16]. A novel suggestion, based on the recovery of a low level of activity following the co-expression of otherwise inactive m3(1-5)/~t2(6-7) and ~t2(l-5)/m3(6-7) chimaeras, is that domains from different receptor molecules may be able to associate, leading to the formation of active dimers or higher oligomers [17], although proteolytic nicking, followed by association of the cognate domains, appears not to have been ruled out as an explanation for the observations. At present, it is not clear how these findings impinge upon the proposal, based on deletion mutagenesis studies of rhodopsin [18], that the extracellular loops of the GPCRs fold to form a globular domain which is essential for positioning and packing the TM helices. However, they suggest the possibility of a more dynamic struc-
viewed from the extracellular side. The apparent helical pitch angles, and orientation vectors (arrows) were calculated by Fourier transform power spectral analysis of sequence variation, as described in Ref. 7. Fully conserved residues are printed in bold. The helices are arranged to match the apparent distribution of the TM helices in the projection structure of rhodopsin, as described by Baldwin [12], with the non-conserved faces adjacent to the phospholipid bilayer. The location of the disulphide bond linking Cys98 two residues from the top of TM3 to Cys178 in the second extracellular loop is shown diagrammatically. The effects of mutations (Asp-Asn/Giu; Thr-Ala; Tyr-Phe; Trp-Phe; Pro-Ala; Cys-Ser) on the binding properties of the mAChRs are indicated as follows: light/dark shaded residues--agonist (ACh or carbachol) affinity reduced by more than 5fold; *antagonist affinity reduced by more than 5-fold. Note that Trp9 and Prol8 (TM4), and Prol4 (TM5) are additional to the set of residues shown in (a). Mutation of Thrl I (TM6) and Thrl I (TM7) was without major effects on binding. Trp2 (TM3), Ala7 and Alal0 (TM5) and Asnl6 (TM6) have not yet been studied by mutagenesis. Mutation of Asp6 (TM3), Thr6 (TM5) and Cys6 (TM7) (dark shading) strongly reduced the maximum PI response to carbachoi [39, 49-51].
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ture than that implied by the Baldwin model [121.
Modelling allows the rational prediction of binding-site residues Residues thought to be important in ligand binding occur on the inner surfaces of the TM helices, one to three helical turns from their predicted extracellular termini, at a depth similar to that of the protonated Schift's base of the retinal chromophore in rhodopsin. Of primary importance is an Asp in TM3 [D6(TM3)] which is found in all GPCRs whose ligands are cationic amines. Ala7 and Alal0 in TM5 are homologous to two serine residues in the fl-adrenergic receptors (flARs) which are thought to H-bond to the catechol OH groups of fl-agonists [19]. These alanines may participate in binding the ACh methyl group. Conserved H-bond donors/acceptors, several of them unique to the mAChRs, also populate this level in the TM structure. Prominent amongst these are Thr3/Thr6 in TM5, Tyrl5/Asnl6 in TM6, and Tyr3/Tyr7 in TM7 (Fig. lb). As discussed below, several of these amino acids may be important in the ligation of the ACh ester group. Indeed, it seems extremely likely that some of these polar side-chains may have a dual role, participating in a network of intramolecular H-bonds which constrains the ground-state structure, until rearranged by the interpolation of the agonist to create the conformationally rearranged activated state. Site-directed mutagenesis has allowed a start to be made in evaluating a number of these possibilities.
TERNARY COMPLEX MODELS CAN BE USED TO DISSECT THE EFFECTS OF MUTATIONS ON AGONIST BINDING AND G-PROTEIN RECRUITMENT In analysing the effects of mutations, it is important to extract the maximum amount of
mechanistically relevant information. Some years ago [20, 21], we showed that antagonists (which bind without activation) yield simple binding curves. In contrast, agonists, which cause conformational induction leading to G-protein recruitment, give complex, multicomponent binding curves. In these, the lowaffinity component represents binding to monomeric receptors, and the high-affinity component(s) arise mainly or totally from the formation of agonist-receptor-G-protein (ARG) ternary complexes [22-26]. Ternary complex formation is promoted by millimolar concentrations of magnesium ions [27], whose action is essential to link agonist binding to GDP dissociation [28, 29]. GTP binding appears to be succeeded by a receptor-catalysed ~activation step, in which a conformational change in the GTP-liganded G-protein is a necessary preliminary to the separation of ~GTP from the ~7 subunit [30, 31]. The break-up of the A R G - G T P quaternary complex causes the reversion of tlie receptor to its low-affinity AR form. The ternary complex model [32-34] is therefore the minimal scheme suitable for the analysis of the equilibrium binding properties of GPCRs in the absence of guanine nucleotides. The analysis of binding data using this model effectively provides three independent parameters: (i) the affinity constant K for agonist binding to free receptor to yield the AR binary complex; (ii) the apparent affinity constant governing formation of the ARG ternary complex--in fact, not a true bimolecular association constant, but a composite constant K*.KG, consisting of the product of the affinity, KG, of the G-protein for the free receptor and the affinity, K*, of the agonist for the receptorG-protein complex and (iii) an estimate of the apparent ratio of total G-protein to total receptor in the system, Gt:Rt. The ratio K*KG/K estimates the affinity of the G-protein for the AR binary complex and is a measure of the efficacy of the agonist in inducing formation of the catalytically active ARG ternary complex. The ternary complex model can readily be extended to allow for the presence of
Agonist activationof muscarinicacetylcholinereceptors multiple G-proteins. Recently, it has been expanded to take account of the complex interplay of cooperativity between agonist, receptor, G-protein ct and fly subunits, and guanine nucleotides, and now describes the characteristic phenomenology in a satisfying fashion, at least at the qualitative level [35]. It has also been extended explicitly to incorporate the agonist-induced conformational change in the receptor [36], so clarifying the relations which exist between agonist affinity and efficacy. The virtue of the ternary complex model of agonist-receptor-G-protein interaction is that it enables the effects of mutations on formation of the binary agonist-receptor complex to be distinguished from their effects on stabilization of the ternary complex. Measurements of functional activity, for instance, stimulation of the GTPase activity of the G-protein in membranes, or of second messenger production in whole cells, then allow the catalytic activities of ternary complexes formed by different mutant receptors to be compared.
MUTAGENESIS STUDIES OF mAChRs
The conserved aspartic acid residue in TM3 of the mAChRs is vital for ternary complex formation and functional activation, but is less important for ligand docking The conserved Asp in TM3 (Asp6, TM3) seems certain to be involved in ligand binding. We have shown that the carboxylate side-chain is labelled by the aziridinium headgroup of the alkylating antagonist analogue [3H]PrBCM [13], and by the corresponding acetylcholine derivative, [aH]acetylcholine mustard [37, 38]. In the ml mAChR, the Aspl05Asn mutation, which deletes the charge while partially preserving the polarity and H-bonding potential of the carboxylate side-chain, greatly reduces the receptor's affinity for the antagonist [3H]quinuclidinyl benzilate, and virtually abolishes the PI response to carbachol [39]. These findings, in conjunction with muta-
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genesis studies on the catecholamine receptors [40, 41], have encouraged the understandable assumption that the role of the Asp residue in mAChRs and related receptors is simply to dock the cationic headgroup of agonists and antagonists in a more or less equivalent fashion. However, our investigations suggest its function to be more subtle. We have studied the properties of the ml and m2 mAChRs with Asp6 (TM3) mutated to Glu, in an attempt to disrupt the headgroup interaction while maintaining the negative charge in the TM domain. This gives a surprisingly modest (3-fold) decrease in the binding of the potent antagonist [3H]N-methylscopolamine [42] by comparison with the 10,000-fold reduction in antagonist affinity imposed by the homologous mutation in the flAR [43]. Because the antagonist affinity remains high, we have been able to carry out detailed binding studies on the Glu mutants. These have been complemented by functional studies. The Asp6-Glu (TM3) mutation exerts a large effect on acetylcholine binding, reducing the binary affinity constant K by 20-fold (ml) to 70fold (m2) and the composite constant K*.KG governing ternary complex formation by 40fold (ml) to 250-fold (m2). Strong destabilization of the ternary complex has been seen for all potent agonists studied, but, unexpectedly, binary complex formation by several pharmacologically potent, high-efficacy agonists (e.g. oxotremorine-M) was discovered to be relatively unperturbed [42]. In the case of ACh at the ml mAChR, the affinity of the G-protein for the agonist-receptor complex is reduced by a factor of two, but residual ternary complex formation is still adequate to support a good phosphoinositide response; in contrast, the phosphoinositide response to agonists such as oxoM, which lose the ability to stabilize the ARG complex at the expense of the AR complex, is almost obliterated by the mutation (P.G. Jones, unpublished data). In general, analysis of the binding data using ternary complex models has proved reasonably predictive of the functional responses, for both the wild-type and Glul05 ml mAChRs.
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These results show that the interactions made
by Asp6 (TM3) are more important for ternary complex formation, and receptor activation, than for binary complex formation, and binding. Pharmacophoric models of the activated state of the mAChR binding site assume a separation of about 3 A between the onium headgroup and the carboxylate oxyanion [44]. Such a bond length would be consistent with the disruptive effect of the Asp6-Glu mutation on formation of the ternary complex. In contrast, the headgroups of many agonists appear to make a relatively loose association with Asp6 (TM3) in the binary complex, consistent with a greater ionic bond length in ligand docking. The structure-activity relations suggest that the docking site may be predominantly apolar, and that desolvation of the ligand headgroup may contribute strongly to the binding energy realised in the initial interaction. In this context, it is interesting that neutron scattering studies suggest the hydration shell of the tetramethylammonium cation to be that of an apolar molecule, rather than a small ion [45]. Bonding between the onium group and the n-electrons of aromatic residues might contribute to such a desolvation-driven binding process [8, 9, 46].The agonist docking site probably represents a primary binding site for muscarinic antagonists. Our results accord with a classical pharmacological investigation by Burgen [47], from which he concluded that the headgroup of the antagonist benzilylcholine interacts less strongly with the anionic subsite in smooth muscle mAChRs than does the headgroup of acetylcholine. ACh itself is a member of a small group of closely related agonists whose headgroups appear to make a tight interaction with Asp6 (TM3) in the binary complex, analogous to that formed in the activated state. This may entail some rearrangement of the transmembrane structure of the receptor, even in the binary complex, creating a conformer primed for G-protein recognition. Such a form would find an analogy in the long-lived metarhodopsin II intermediate which is detectable spectroscopically in the rhodopsin photocycle [48].
Several polar residues in the T M region of the mAChRs may contribute to binding the sidechain of A Ch The concept of a partial separation between agonist docking and activation sites has more general implications for attempts to use mutagenesis to identify residues which contribute to the ACh binding site. Systematic studies have been done on Tyr, Thr, Trp and Pro residues in the m3 and of Cys in the ml mAChRs [49-51]. A typical outcome has been an overall reduction in ACh or carbachol potency, both in binding, and in stimulating a PI response, of 5-60-fold, often without any major change in antagonist affinity (Fig. lb). This underlines the idea that agonist and antagonist binding exploit distinct as well as common epitopes, a perception reinforced by the discovery of a mutation [Serl7-Thr (TM2; m3 mAChR)] [49]; Fig. lb) which reduced antagonist affinity without affecting agonist binding. Two of the mutations, namely Thr6-Ala (TM5; m3 mAChR) and Cys6-Ser (TM7; ml mAChR), strongly reduce the maximum PI response evoked by carbachol as well as inhibiting agonist binding. These amino acids, unique to the mAChRs, are predicted to be on the inner surfaces of the TM helices in the ligand-binding plane (Fig. 1), indeed the Cys is predicted to be a near neighbour of Asp6 (TM3). These amino acids are outstanding candidates for direct involvement in ACh activation of the mAChRs. It needs to be emphasized that site-directed mutagenesis affects the overall energetics of agonist binding and receptor activation. Changes may be mediated by alteration of either the bond energy between receptor and ligand, or the free energy of the conformational change, or both [52]. Mutations may also have a global effect on the protein fold. This is probably the explanation for the dramatic reduction (greater than 200-fold) in both agonist and antagonist affinity imposed by the mutation Prol8-Ala (TM4) [50]. It is actually very difficult to design mutagenesis experiments to support the hypothesis that a given side-chain directly contacts the
Agonist activationof muscarinicaeetylcholinereceptors ligand. A possible strategy is to try to match the effects of mutations in the receptor with specific deletions of functional moieties from the ligand, an approach successfully applied to the flAR [19]. In the case of the mAChRs, similar tactics have been attempted by the replacement of the ester oxygen, and carbonyl group of ACh by methylene groups or sulphur [50], and the use of the resulting analogues to probe point mutants of the Thr and Tyr residues. However, the study failed to identify selective interactions of these amino acid side-chains with the ester function of ACh. Using the same compounds to probe the Glu mutants, we have found a contributory factor to be lowered polarity of the analogues relative to the parent molecule. This leads to an enhancement of their affinity for the docking site at the expense of the primed/ activation site, obscuring the effects of the mutations on the binding of the analogues. In general, however, it seems that both the Asp6Glu (TM3) and the Thr6Ala (TM5) mutations produce similarly disabled activation sites, without affecting ligand docking. In contrast, the properties of the Tyr7Phe (TM7) mutation [49] suggest that it may selectively affect the initial docking interaction. CONCLUSION An emerging theme is that antagonists and agonists exploit significantly different sets of epitopes in binding to the GPCRs [40, 53]. In the mAChRs, the archetypal ionic interaction between the agonist headgroup and the binding-site carboxylate, pinpointed by afffinitylabelling, seems to be more important in stabilizing the agonist-promoted activated state than the ground state of the receptor. Mutagenesis studies have highlighted several polar amino acid side-chains which may be candidates for ligating the ester group of ACh, but definitive results have not been obtained. It seems likely that a combination of scanning mutagenesis with chemical modification, as pioneered for the transmembrane channel of the nicotinic receptor [54] will be needed to identify specific bonds. In this way, we should eventually be
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able to deduce which parts of the TM structure of the receptor undergo relative movement when the agonist binds, and the activated state is induced, and whether the activation process involves the liberation of the G-protein binding site from a tight structure designed to occlude it, or its creation by the agonist-mediated coordination of disparate epitopes in an otherwise relatively dynamic molecule. Acknowledgements---We are grateful to the Medical Research Council (U.K.) for the provision of a Research Studentship (to K.M.P.) and a Training Fellowship (to P.G.J.).
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