- Email: [email protected]
Is the R and R* dichotomy real?
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References 1 Communi, O. and Boeynaems, J. M. (1997) Trends Pharmacol. Sci. 18, 83–86 2 Seifert, R. and Schultz, G. (1989) Trends Pharmacol. Sci. 10, 365–369 3 Connolly, G. P., Harrison, P. J. and Stone, T. W. (1993) Br. J. Pharmacol. 110, 1297–1304 4 Connolly, G. P. and Harrison, P. J. (1995) Br. J. Pharmacol. 116, 2764–2770 5 Rees, D. et al. (1996) Blood 7, 2761–2767 6 Connolly, G. P., Simmonds, H. A. and Duley, J. A. (1996) Trends Pharmacol. Sci. 17, 106 7 Lazarowski, E. R. and Harden, T. K. (1994) J. Biol. Chem. 269, 11830–11836
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T G. P. Connolly Purine and Pyrimidine Research Laboratories, Division of Chemical Pathology, United Medical and Dental Schools, Floor 18, Guy’s Tower, Guy’s Hospital, London Bridge, London, UK SE1 9RT, N. J. Abbott and C. Demaine Physiology Group, Biomedical Sciences Division, King’s College
M
Is the R and R* dichotomy real? Observations of allosteric phenomena on G protein-coupled receptors point to problems in the interpretation of agonist–receptor–G protein interactions. Stanislav Tucˇek In previous issues of TiPS, recent reviews by Kenakin1,2 and Schwartz and Rosenkilde3, in addition to a debate (Refs 4–7), pointed (indirectly) to serious unresolved questions in the presently prevailing interpretations of agonist–receptor–G protein interactions. The extended ternary complex model8,9, the two-state model10 and the cubic ternary model11 all describe the receptor as being either active (R*) or inactive (R). Such distinction of two qualitatively different states (an ‘all-or-none’ distinction in the two-state model) is convenient for modelling the ligand–receptor–G protein interactions, but does it help to understand what really happens? I would like to direct attention to two observations made during studies of the effects of allosteric modulators on muscarinic receptors which appear to justify the search for an alternative, more plastic and more allosteric (!) model of agonist– receptor–G protein interactions.
Observation 1 If the antagonist N-methylscopolamine (NMS) associates with the 414
TiPS – November 1997 (Vol. 18)
London, Strand, London, UK WC2R 2LS, and J. A. Duley, Purine and Pyrimidine Research Laboratories, Division of Chemical Pathology, United Medical and Dental Schools, Floor 18, Guy’s Tower, Guy’s Hospital, London Bridge, London, UK SE1 9RT.
muscarinic M2 receptor, the affinity of the receptor for the allosteric modulator alcuronium becomes severalfold higher, while that for the allosteric modulator gallamine is lowered. If, however, the antagonist quinuclidinyl benzilate (QNB) associates with the M2 receptor, the affinities for both alcuronium and gallamine become diminished12–15. Similar variability can be observed in interactions between other classical antagonists and allosteric modulators15–18, and also between the agonists and allosteric modulators19,20. For example, if acetylcholine binds to the M2 receptor, the affinity of the receptor for the allosteric modulators eburnamonine and brucine is increased, but if oxotremorine-M binds to the same receptor, the receptor’s affinity for eburnamonine is diminished and that for brucine is enhanced. Different affinities for the same allosteric ligand are best explained by different receptor conformations. Apparently, the conformation for the M2 receptor induced by the binding of NMS is different from that induced by the binding of QNB,
and the conformation induced by the binding of acetylcholine is different from that induced by the binding of oxotremorine-M. Similarly, Schimerlik and colleagues21,22 have shown that the affinities of muscarinic receptors for G proteins are different if the receptors are associated with different agonists, confirming that different agonists induce different receptor conformations. An important consequence is that individual transduction pathways may be differentially activated by different agonists, as theoretically deduced by Kenakin2 and Gudermann et al.23, and shown practically by (for example) Spengler et al.24, Gurwitz et al.25, Robb et al.26 and Perez et al.27 A wealth of data thus confirm the multiplicity of receptor conformations, be it in their ‘active’ or ‘inactive’ states.
Observation 2 Allosteric modulators bind to muscarinic receptors at sites that are different from the orthosteric binding site, and several modulators have been shown to compete for one and the same binding domain14,28,29. Contrary to the original expectation that the allosteric modulators only modify the binding properties of the orthosteric binding sites, we have found that they also affect the interaction of the receptors with the G proteins. On Chinese hamster ovary (CHO) cells expressing individual subtypes of muscarinic
Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0165 – 6147/97/$17.00 PII: S0165-6147(97)01123-1
F receptors, alcuronium, gallamine and strychnine have agonist-like but QNB-resistant effects on the activities of adenylate cyclase and of phosphoinositidase30. In liposomes incorporating purified M2 receptors and G0 proteins, alcuronium either increases or inhibits the activity of G0 proteins (measured by the binding of [35S]GTPγS), depending on the stoichiometric ratio of receptors to G proteins31. Apparently, the muscarinic receptor can be activated not only from the orthosteric binding site, but also from an allosteric site on its extracellular surface. The activation of muscarinic receptors by antibodies directed against them32 is also probably initiated in a domain that is different from the orthosteric binding site. In a recent review, Schwartz and Rosenkilde3 enumerated situations in which receptors for peptides are activated from different sites on their surface. Although they believe that various agonists stabilize one and the same active conformation of the receptor, independently of where they attach to its surface, the assumption of multiple active conformations seems more likely to me in situations where different sites of attachment are used by different ligands, and an argument favouring this view follows in the next paragraph. It is a noteworthy feature of the agonist-like actions of allosteric modulators on muscarinic receptors30 that the activation of the receptors (or, more accurately, receptor-mediated activation of the G proteins) is achieved by agents (alcuronium and gallamine) that are known to diminish the affinity of receptors for acetylcholine19,20,32–34. Consequently, the active conformations of the receptor induced by alcuronium and gallamine are different from the active conformation induced by acetylcholine. While the former are characterized by a high affinity for the G protein and a low affinity for acetylcholine, the latter is characterized by a high affinity for both the G protein and acetylcholine. Interestingly, independent changes in the affinities
for the agonist and for the G protein have recently been induced by site- directed mutations of α1B-adrenoceptors35 and muscarinic receptors36. It should be remembered, however, that mutations (even single-point mutations) create new molecules, with new conformational properties. This is illustrated by recent data on single point mutants of α1B-adrenoceptors that display significantly higher constitutive activities than is the agonist-stimulated activity of their wild-type counterpart37. The question of the multiplicity of receptor conformations and of their dependence on the ligands is not academic and its solution has profound consequences. The terms R and R* imply that there is a principal change of quality between R and R*. The impression one has from observations of allosteric interactions on muscarinic receptors is that the receptors are highly plastic and adopt a large number of conformations depending on the attached ligands (including the G proteins). The richness of conformational states has recently been recognized35. The term R* loses its meaning if there are many R*s, with activities that differ by orders of magnitude. Important questions are obscured by its use. For example, are the active state and conformation of the ‘constitutively active’ molecules of the muscarinic receptor the same as those of the acetylcholine-activated receptor molecules? Or is each receptor molecule endowed with a low degree of activity (i.e. propensity to activate the G protein), so that the inactive state of the receptor can only be induced by its association with an inverse agonist? Are the active state and the conformation of the muscarinic receptor induced by the partial agonist pilocarpine the same as those induced by the full agonist acetycholine? Does the binding of an agonist to the receptor only change the receptor’s affinity for the G protein, or does it also induce an independent change in the rate of the receptor’s catalytic activity, responsible for the GDP–GTP exchange on Gα? It might be easier to consider such questions in terms
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of allosterically induced multiple receptor conformations and multiple degrees of activity, without the assumption of an R versus R* dichotomy; a model avoiding such dichotomy has already been applied21. References 1 Kenakin, T. (1995) Trends Pharmacol. Sci. 16, 188–192 2 Kenakin, T. (1995) Trends Pharmacol. Sci. 16, 232–238 3 Schwartz, T. and Rosenkilde, M. M. (1996) Trends Pharmacol. Sci. 17, 213–216 4 Kenakin, T. (1995) Trends Pharmacol. Sci. 16, 256–258 5 Kenakin, T. (1996) Trends Pharmacol. Sci. 17, 190–191 6 Gardner, B. (1995) Trends Pharmacol. Sci. 16, 259–260 7 Bruns, R. F. (1996) Trends Pharmacol. Sci. 17, 189 8 Samama, P., Cotecchia, S., Costa, T. and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625–4636 9 Scheer, A. and Cotecchia, S. (1997) J. Rec. Sign. Transd. Res. 17, 57–73 10 Leff, P. (1995) Trends Pharmacol. Sci. 16, 89–97 11 Weiss, P., Morgan, P. H., Lutz, M. W. and Kenakin, T. P. (1996) J. Theor. Biol. 178, 151–167 12 Tucˇek, S. et al. (1990) Mol. Pharmacol. 38, 674–680 13 Birdsall, N. J. M., Cohen, F., Lazareno, S. and Matsui, H. (1995) Biochem. Soc. Trans. 23, 108–111 14 Prosˇka, J. and Tucˇek, S. (1995) Mol. Pharmacol. 48, 696–702 15 Tucˇek, S. and Prosˇka, J. (1995) Trends Pharmacol. Sci. 16, 205–212 16 Tucˇek, S., Prosˇ ka, J., Hejnová, L. and El-Fakahany, E. E. (1995) Life Sci. 56, 1009 17 Lazareno, S. and Birdsall, N. J. M. (1995) Mol. Pharmacol. 48, 362–378 18 Prosˇka, J. and Tucˇek, S. (1996) Eur. J. Pharmacol. 305, 201–205 19 Jakubík, J., Bacˇáková, L., El-Fakahany, E. E. and Tucˇek, S. (1997) Physiol. Res. 46, 21P 20 Jakubík, J., Bacˇáková, L., El-Fakahany, E. E. and Tucˇek, S. (1997) Mol. Pharmacol. 52, 172–179 21 Vogel, W. K., Mosser, V. A., Bulseco, D. A. and Schimerlik, M. I. (1995) J. Biol. Chem. 270, 15485–15493 22 Bulseco, D. A. and Schimerlik, M. I. (1996) Mol. Pharmacol. 49, 132–141 23 Gudermann, T., Kalkbrenner, F. and Schultz, G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 429–459 24 Spengler, D. et al. (1993) Nature 365, 170–175 25 Gurwitz, D. et al. (1994) Eur. J. Pharmacol. 267, 21–31 26 Robb, S. et al. (1994) EMBO J. 13, 1325–1330 27 Perez, D. M. (1996) Mol. Pharmacol. 49, 112–122 28 Ellis, J. and Seidenberg, M. (1992) Mol. Pharmacol. 42, 638–641 29 Waelbroeck, M. (1994) Mol. Pharmacol. 46, 685–692 30 Jakubík, J., Bacˇáková, L., Lisá, V., El-Fakahany, E. E. and Tucˇek, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8705–8709
TiPS – November 1997 (Vol. 18)
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31 Jakubík, J., Haga, T. and Tucˇek, S. (1997) Life Sci. 60, 1170 32 Fu, M. L-X., Schulze, W., Wallukat, G., Hjalmarson, A. and Hoebeke, J. (1995) J. Mol. Cell. Cardiol. 27, 427–436 33 Clark, A. L. and Mitchelson, F. (1976) Br. J. Pharmacol. 58, 323–331
34 Gragey, A. and Ellis, J. (1996) Biochem. Pharmacol. 52, 1767–1775 35 Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. and Cotecchia, S. (1996) EMBO J. 15, 3566–3578 36 Liu, J., Thin, N., Conklin, B. R. and Wess, J. (1996) J. Biol. Chem. 271, 6172–6178
Agonist-specific receptor conformations Ligands create bias in already complex systems Terry Kenakin I agree entirely with Dr Tucˇek with respect to the idea that the pharmacological world of agonism cannot be accommodated by a single receptor active state. The concept that receptor proteins traverse an energy landscape to adopt a number of conformations and that different agonists select different spectra of conformations is one possibility1,2. There is now evidence to show that some
no agonist
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50 II III
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Frequency
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agonist A
II III
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I
agonist B
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0 Conformations Fig. 1. A frequency distribution of a range of receptor conformations. Three shaded bars represent three ‘active’ receptor states (I, II, III) that can activate G proteins. In the absence of ligands, the relative abundance of these states is low. Agonist A shifts the frequency distributions of all the conformations and enriches states II and III, while agonist B produces another distribution enriching state I.
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agonists ‘traffic’ stimulus to different G proteins; this may be the next frontier for agonist selectivity in therapeutics. There is a theoretical rationale3 linked to experimental findings4 to indicate that different setpoints of receptor systems can unveil agonist-selective receptoractive states. There are also more direct data to show that different agonists produce different receptoractive states5. Finally, there are indications that ligands may induce conformations that do not reveal their presence by a physiological response, but in other ways. For example, the CCK receptor antagonist D-Tyr-Gly[(Nle28,31,DTrp30)cholecystokinin-26–32] phenylethyl ester, does not produce an acute physiological response (positive or negative), but nevertheless does accelerate CCK receptor internalization. This effect is consistent with the production of a conformation by ligand binding that directs the receptor into the clathrin-dependent endocytotic pathway6. So where does this leave us modellers who try to reduce the world to minimal numbers of variables? While the cubic ternary complex model7,8 is a statistically complete version of the extended ternary complex model, it already is heuristic and essentially not useful for fitting purposes. Although the computer program can accommodate up to three different G proteins (see Fig. 5 of Ref. 7) and therefore can handle three different activation reactivities,
37 Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. and Cotecchia, S. (1997) Proc. Natl. Acad Sci. U. S. A. 94, 808–813
Stanislav Tucˇek Institute of Physiology, Academy of Sciences,14220 Prague, Czechia.
this leads to systems where it is difficult to visualize what is happening in the Euclidean space. More importantly, such models result in a plethora of unverifiable constants. Any number of receptor states (see Ref. 9) can be built into the ternary complex model and this leads to a bewildering array of possible interconversions. However, such models quickly lose their ability to be effectively predictive of pharmacological receptor behaviour. In the end, if a model is to be useful and predictive, Occam’s razor must be applied and when the model fails to describe the experiment, then grudgingly, new pieces must be added. However, for agonist-selective states, it is difficult to see how conventional models will be useful because agonist-specific phenomena will cease to be consistent with unique sets of parameter values. Perhaps a better interpretation of the energy landscape spectrum for receptors can be obtained from Onaran and Costa’s ‘unitary probabilistic view of allosteric transition’10. Here, a spectrum of conformations is predicted and agonists, by selective affinity for the different conformations, redistribute these conformations. From this standpoint, the histograms describing stimulus trafficking2 might be better visualized as shown in Fig. 1. As agonists interact with the system, different receptor-active states are enriched and this leads to different signalling to G proteins. Thus, models rooted in statistics of probability and not conventional equilibrium kinetics may hold the key to moving this branch of receptor pharmacology forward. I view such a prospect as exciting and look forward to applications of new mathematical techniques to older pharmacological problems.
Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0165 – 6147/97/$17.00 PII: S0165-6147(97)01127-9
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References 1 Communi, O. and Boeynaems, J. M. (1997) Trends Pharmacol. Sci. 18, 83–86 2 Seifert, R. and Schultz, G. (1989) Trends Pharmacol. Sci. 10, 365–369 3 Connolly, G. P., Harrison, P. J. and Stone, T. W. (1993) Br. J. Pharmacol. 110, 1297–1304 4 Connolly, G. P. and Harrison, P. J. (1995) Br. J. Pharmacol. 116, 2764–2770 5 Rees, D. et al. (1996) Blood 7, 2761–2767 6 Connolly, G. P., Simmonds, H. A. and Duley, J. A. (1996) Trends Pharmacol. Sci. 17, 106 7 Lazarowski, E. R. and Harden, T. K. (1994) J. Biol. Chem. 269, 11830–11836
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T G. P. Connolly Purine and Pyrimidine Research Laboratories, Division of Chemical Pathology, United Medical and Dental Schools, Floor 18, Guy’s Tower, Guy’s Hospital, London Bridge, London, UK SE1 9RT, N. J. Abbott and C. Demaine Physiology Group, Biomedical Sciences Division, King’s College
M
Is the R and R* dichotomy real? Observations of allosteric phenomena on G protein-coupled receptors point to problems in the interpretation of agonist–receptor–G protein interactions. Stanislav Tucˇek In previous issues of TiPS, recent reviews by Kenakin1,2 and Schwartz and Rosenkilde3, in addition to a debate (Refs 4–7), pointed (indirectly) to serious unresolved questions in the presently prevailing interpretations of agonist–receptor–G protein interactions. The extended ternary complex model8,9, the two-state model10 and the cubic ternary model11 all describe the receptor as being either active (R*) or inactive (R). Such distinction of two qualitatively different states (an ‘all-or-none’ distinction in the two-state model) is convenient for modelling the ligand–receptor–G protein interactions, but does it help to understand what really happens? I would like to direct attention to two observations made during studies of the effects of allosteric modulators on muscarinic receptors which appear to justify the search for an alternative, more plastic and more allosteric (!) model of agonist– receptor–G protein interactions.
Observation 1 If the antagonist N-methylscopolamine (NMS) associates with the 414
TiPS – November 1997 (Vol. 18)
London, Strand, London, UK WC2R 2LS, and J. A. Duley, Purine and Pyrimidine Research Laboratories, Division of Chemical Pathology, United Medical and Dental Schools, Floor 18, Guy’s Tower, Guy’s Hospital, London Bridge, London, UK SE1 9RT.
muscarinic M2 receptor, the affinity of the receptor for the allosteric modulator alcuronium becomes severalfold higher, while that for the allosteric modulator gallamine is lowered. If, however, the antagonist quinuclidinyl benzilate (QNB) associates with the M2 receptor, the affinities for both alcuronium and gallamine become diminished12–15. Similar variability can be observed in interactions between other classical antagonists and allosteric modulators15–18, and also between the agonists and allosteric modulators19,20. For example, if acetylcholine binds to the M2 receptor, the affinity of the receptor for the allosteric modulators eburnamonine and brucine is increased, but if oxotremorine-M binds to the same receptor, the receptor’s affinity for eburnamonine is diminished and that for brucine is enhanced. Different affinities for the same allosteric ligand are best explained by different receptor conformations. Apparently, the conformation for the M2 receptor induced by the binding of NMS is different from that induced by the binding of QNB,
and the conformation induced by the binding of acetylcholine is different from that induced by the binding of oxotremorine-M. Similarly, Schimerlik and colleagues21,22 have shown that the affinities of muscarinic receptors for G proteins are different if the receptors are associated with different agonists, confirming that different agonists induce different receptor conformations. An important consequence is that individual transduction pathways may be differentially activated by different agonists, as theoretically deduced by Kenakin2 and Gudermann et al.23, and shown practically by (for example) Spengler et al.24, Gurwitz et al.25, Robb et al.26 and Perez et al.27 A wealth of data thus confirm the multiplicity of receptor conformations, be it in their ‘active’ or ‘inactive’ states.
Observation 2 Allosteric modulators bind to muscarinic receptors at sites that are different from the orthosteric binding site, and several modulators have been shown to compete for one and the same binding domain14,28,29. Contrary to the original expectation that the allosteric modulators only modify the binding properties of the orthosteric binding sites, we have found that they also affect the interaction of the receptors with the G proteins. On Chinese hamster ovary (CHO) cells expressing individual subtypes of muscarinic
Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0165 – 6147/97/$17.00 PII: S0165-6147(97)01123-1
F receptors, alcuronium, gallamine and strychnine have agonist-like but QNB-resistant effects on the activities of adenylate cyclase and of phosphoinositidase30. In liposomes incorporating purified M2 receptors and G0 proteins, alcuronium either increases or inhibits the activity of G0 proteins (measured by the binding of [35S]GTPγS), depending on the stoichiometric ratio of receptors to G proteins31. Apparently, the muscarinic receptor can be activated not only from the orthosteric binding site, but also from an allosteric site on its extracellular surface. The activation of muscarinic receptors by antibodies directed against them32 is also probably initiated in a domain that is different from the orthosteric binding site. In a recent review, Schwartz and Rosenkilde3 enumerated situations in which receptors for peptides are activated from different sites on their surface. Although they believe that various agonists stabilize one and the same active conformation of the receptor, independently of where they attach to its surface, the assumption of multiple active conformations seems more likely to me in situations where different sites of attachment are used by different ligands, and an argument favouring this view follows in the next paragraph. It is a noteworthy feature of the agonist-like actions of allosteric modulators on muscarinic receptors30 that the activation of the receptors (or, more accurately, receptor-mediated activation of the G proteins) is achieved by agents (alcuronium and gallamine) that are known to diminish the affinity of receptors for acetylcholine19,20,32–34. Consequently, the active conformations of the receptor induced by alcuronium and gallamine are different from the active conformation induced by acetylcholine. While the former are characterized by a high affinity for the G protein and a low affinity for acetylcholine, the latter is characterized by a high affinity for both the G protein and acetylcholine. Interestingly, independent changes in the affinities
for the agonist and for the G protein have recently been induced by site- directed mutations of α1B-adrenoceptors35 and muscarinic receptors36. It should be remembered, however, that mutations (even single-point mutations) create new molecules, with new conformational properties. This is illustrated by recent data on single point mutants of α1B-adrenoceptors that display significantly higher constitutive activities than is the agonist-stimulated activity of their wild-type counterpart37. The question of the multiplicity of receptor conformations and of their dependence on the ligands is not academic and its solution has profound consequences. The terms R and R* imply that there is a principal change of quality between R and R*. The impression one has from observations of allosteric interactions on muscarinic receptors is that the receptors are highly plastic and adopt a large number of conformations depending on the attached ligands (including the G proteins). The richness of conformational states has recently been recognized35. The term R* loses its meaning if there are many R*s, with activities that differ by orders of magnitude. Important questions are obscured by its use. For example, are the active state and conformation of the ‘constitutively active’ molecules of the muscarinic receptor the same as those of the acetylcholine-activated receptor molecules? Or is each receptor molecule endowed with a low degree of activity (i.e. propensity to activate the G protein), so that the inactive state of the receptor can only be induced by its association with an inverse agonist? Are the active state and the conformation of the muscarinic receptor induced by the partial agonist pilocarpine the same as those induced by the full agonist acetycholine? Does the binding of an agonist to the receptor only change the receptor’s affinity for the G protein, or does it also induce an independent change in the rate of the receptor’s catalytic activity, responsible for the GDP–GTP exchange on Gα? It might be easier to consider such questions in terms
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R
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of allosterically induced multiple receptor conformations and multiple degrees of activity, without the assumption of an R versus R* dichotomy; a model avoiding such dichotomy has already been applied21. References 1 Kenakin, T. (1995) Trends Pharmacol. Sci. 16, 188–192 2 Kenakin, T. (1995) Trends Pharmacol. Sci. 16, 232–238 3 Schwartz, T. and Rosenkilde, M. M. (1996) Trends Pharmacol. Sci. 17, 213–216 4 Kenakin, T. (1995) Trends Pharmacol. Sci. 16, 256–258 5 Kenakin, T. (1996) Trends Pharmacol. Sci. 17, 190–191 6 Gardner, B. (1995) Trends Pharmacol. Sci. 16, 259–260 7 Bruns, R. F. (1996) Trends Pharmacol. Sci. 17, 189 8 Samama, P., Cotecchia, S., Costa, T. and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625–4636 9 Scheer, A. and Cotecchia, S. (1997) J. Rec. Sign. Transd. Res. 17, 57–73 10 Leff, P. (1995) Trends Pharmacol. Sci. 16, 89–97 11 Weiss, P., Morgan, P. H., Lutz, M. W. and Kenakin, T. P. (1996) J. Theor. Biol. 178, 151–167 12 Tucˇek, S. et al. (1990) Mol. Pharmacol. 38, 674–680 13 Birdsall, N. J. M., Cohen, F., Lazareno, S. and Matsui, H. (1995) Biochem. Soc. Trans. 23, 108–111 14 Prosˇka, J. and Tucˇek, S. (1995) Mol. Pharmacol. 48, 696–702 15 Tucˇek, S. and Prosˇka, J. (1995) Trends Pharmacol. Sci. 16, 205–212 16 Tucˇek, S., Prosˇ ka, J., Hejnová, L. and El-Fakahany, E. E. (1995) Life Sci. 56, 1009 17 Lazareno, S. and Birdsall, N. J. M. (1995) Mol. Pharmacol. 48, 362–378 18 Prosˇka, J. and Tucˇek, S. (1996) Eur. J. Pharmacol. 305, 201–205 19 Jakubík, J., Bacˇáková, L., El-Fakahany, E. E. and Tucˇek, S. (1997) Physiol. Res. 46, 21P 20 Jakubík, J., Bacˇáková, L., El-Fakahany, E. E. and Tucˇek, S. (1997) Mol. Pharmacol. 52, 172–179 21 Vogel, W. K., Mosser, V. A., Bulseco, D. A. and Schimerlik, M. I. (1995) J. Biol. Chem. 270, 15485–15493 22 Bulseco, D. A. and Schimerlik, M. I. (1996) Mol. Pharmacol. 49, 132–141 23 Gudermann, T., Kalkbrenner, F. and Schultz, G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 429–459 24 Spengler, D. et al. (1993) Nature 365, 170–175 25 Gurwitz, D. et al. (1994) Eur. J. Pharmacol. 267, 21–31 26 Robb, S. et al. (1994) EMBO J. 13, 1325–1330 27 Perez, D. M. (1996) Mol. Pharmacol. 49, 112–122 28 Ellis, J. and Seidenberg, M. (1992) Mol. Pharmacol. 42, 638–641 29 Waelbroeck, M. (1994) Mol. Pharmacol. 46, 685–692 30 Jakubík, J., Bacˇáková, L., Lisá, V., El-Fakahany, E. E. and Tucˇek, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8705–8709
TiPS – November 1997 (Vol. 18)
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31 Jakubík, J., Haga, T. and Tucˇek, S. (1997) Life Sci. 60, 1170 32 Fu, M. L-X., Schulze, W., Wallukat, G., Hjalmarson, A. and Hoebeke, J. (1995) J. Mol. Cell. Cardiol. 27, 427–436 33 Clark, A. L. and Mitchelson, F. (1976) Br. J. Pharmacol. 58, 323–331
34 Gragey, A. and Ellis, J. (1996) Biochem. Pharmacol. 52, 1767–1775 35 Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. and Cotecchia, S. (1996) EMBO J. 15, 3566–3578 36 Liu, J., Thin, N., Conklin, B. R. and Wess, J. (1996) J. Biol. Chem. 271, 6172–6178
Agonist-specific receptor conformations Ligands create bias in already complex systems Terry Kenakin I agree entirely with Dr Tucˇek with respect to the idea that the pharmacological world of agonism cannot be accommodated by a single receptor active state. The concept that receptor proteins traverse an energy landscape to adopt a number of conformations and that different agonists select different spectra of conformations is one possibility1,2. There is now evidence to show that some
no agonist
100
50 II III
I
Frequency
0 100
agonist A
II III
50
0 100
I
agonist B
50
0 Conformations Fig. 1. A frequency distribution of a range of receptor conformations. Three shaded bars represent three ‘active’ receptor states (I, II, III) that can activate G proteins. In the absence of ligands, the relative abundance of these states is low. Agonist A shifts the frequency distributions of all the conformations and enriches states II and III, while agonist B produces another distribution enriching state I.
416
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agonists ‘traffic’ stimulus to different G proteins; this may be the next frontier for agonist selectivity in therapeutics. There is a theoretical rationale3 linked to experimental findings4 to indicate that different setpoints of receptor systems can unveil agonist-selective receptoractive states. There are also more direct data to show that different agonists produce different receptoractive states5. Finally, there are indications that ligands may induce conformations that do not reveal their presence by a physiological response, but in other ways. For example, the CCK receptor antagonist D-Tyr-Gly[(Nle28,31,DTrp30)cholecystokinin-26–32] phenylethyl ester, does not produce an acute physiological response (positive or negative), but nevertheless does accelerate CCK receptor internalization. This effect is consistent with the production of a conformation by ligand binding that directs the receptor into the clathrin-dependent endocytotic pathway6. So where does this leave us modellers who try to reduce the world to minimal numbers of variables? While the cubic ternary complex model7,8 is a statistically complete version of the extended ternary complex model, it already is heuristic and essentially not useful for fitting purposes. Although the computer program can accommodate up to three different G proteins (see Fig. 5 of Ref. 7) and therefore can handle three different activation reactivities,
37 Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. and Cotecchia, S. (1997) Proc. Natl. Acad Sci. U. S. A. 94, 808–813
Stanislav Tucˇek Institute of Physiology, Academy of Sciences,14220 Prague, Czechia.
this leads to systems where it is difficult to visualize what is happening in the Euclidean space. More importantly, such models result in a plethora of unverifiable constants. Any number of receptor states (see Ref. 9) can be built into the ternary complex model and this leads to a bewildering array of possible interconversions. However, such models quickly lose their ability to be effectively predictive of pharmacological receptor behaviour. In the end, if a model is to be useful and predictive, Occam’s razor must be applied and when the model fails to describe the experiment, then grudgingly, new pieces must be added. However, for agonist-selective states, it is difficult to see how conventional models will be useful because agonist-specific phenomena will cease to be consistent with unique sets of parameter values. Perhaps a better interpretation of the energy landscape spectrum for receptors can be obtained from Onaran and Costa’s ‘unitary probabilistic view of allosteric transition’10. Here, a spectrum of conformations is predicted and agonists, by selective affinity for the different conformations, redistribute these conformations. From this standpoint, the histograms describing stimulus trafficking2 might be better visualized as shown in Fig. 1. As agonists interact with the system, different receptor-active states are enriched and this leads to different signalling to G proteins. Thus, models rooted in statistics of probability and not conventional equilibrium kinetics may hold the key to moving this branch of receptor pharmacology forward. I view such a prospect as exciting and look forward to applications of new mathematical techniques to older pharmacological problems.
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