Synoptic receptor function

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70-74 35 Monyer, H. et al. (1992) Science 256, 1217-1221 36 Ikeda, K. et al. (1992) FEBS Lett. 313, 34-38 37 Kein/inen, K. et al. (1990) Science 249,

Receptor receptor

556-560 38 Bettler,B. et al. (1990)Neuron 5, 583-595 39 Egebjerg, J., Bettler, B., HermansBorgmeyer, I. and Heinemann, S. (1991) Nature 351, 745-748 40 Bettler,B. et al. (1992)Neuron 8, 257-265

and drug classification: the need behaviour in membranes

for analysis of

Synoptic receptor function Until the past decade, pharmacologists have been restricted to complex natural systems in their studies of receptors. The heterogeneity of such systems, while being ideal for fine physiological control, is an obstacle to the definitive classification of receptors and drugs. With the advent of molecular biology techniques that allow the selective isolation, expression and reconstitution of receptors, this obstacle is removed and relatively 'pure' populations of receptors can be produced in surrogate systems. This letter discusses the potential importance of post-translational membrane effects and receptor behaviour for receptor classification. The major thesis is that receptors, being proteins predisposed to proteinprotein interactions, may take on various roles when released into membrane environments. There are three ideas that should be considered when evaluating receptor classification data from expression systems: (1) receptors exist in "active' and 'inactive' forms with respect to G protein interaction, and receptors can couple to G proteins in the absence of agonist; (2) receptor promiscuity determines which G proteins they form complexes with; and (3) some antagonists can destabilize ternary complexes and thus have G protein-dependent affinity (negative efficacy). It is well known that agonists promote the formation of ternary complexes with seven transmembrane domain receptors and G proteins. This behaviour is described by the classicaP and allosteric 2 ternary complex models. Studies with %-adrenoceptors 3, o~2-adrenoceptors 4, and ~2-adrenoceptors 2 have shown that

mutation-induced receptors activate G proteins (i.e. the 'active' state of the receptor) and that these have increased affinity for agonists but not antagonists 5. This idea also extends to the formation of spontaneous receptor--G protein complexes in the absence of agonists 6, some of which can be isolated biochemically. For example, immunoprecipitation of Gs produces precipitates of calcitonin gene-related peptide receptor-Gs complexes in rat cerebellum in the absence of agonist 7. There are two important consequences to the formation of such complexes in membranes. The first is that the affinity of agonists and negative antagonists can depend upon the extent of complex formation and, secondly, that the propensity of receptors to exhibit such behaviour depends upon the properties of systems, not receptor type. Thus it can be shown that the extent of spontaneous coupling can depend upon ionic conditions 6 or G protein content 8. These latter conditions may vary between expression systems thus introducing a cellular factor that can affect affinity. Another phenomenon, increasingly evident in receptor function, is the ability of receptors to interact with multiple G proteins. This bebaviour is well documented in reconstituted systems and strongly suggested to occur in natural systems as well (reviewed in Ref. 9). For example, three activated G proteins can be observed upon opioid receptor activation by the agonist DADLE in NG108-15 X neuroblastoma cells ~°. In bovine brain, adenosine A~ receptors have been shown to complex with Gi~, Gi2, and Go (Ref. 11). This makes

41 Werner, P., Voigt, M., Keinfinen, K., Wisden, W. and Seeburg, P. H. (1991) Nature 351, 742-744 42 Herb, A. et al. (1992) Neuron 8, 775-785 CNQX: 6-cyano-7-nitro-,~,:inoxaline-2,3dione ligand binding in expression systems hazardous with ligands that predispose receptor-G protein complexes, since both the quantity and type of G protein present can affect affinity. The effects of receptor-G protein ratios was recently demonstrated in transfected Chinese hamster ovary cells for the oc2-adrenoceptor 12. With increasing levels of receptor from 0.3-10pmolmg -1 protein, this receptor concomitantly activated a cholera toxin- and pertussis toxinsensitive G protein to activate and inhibit adenylyl cyclase. Also, surprising results can occur if receptors are brought into systems containing G proteins they normally do not encounter. For example, the mouse 5-hydroxytryptamine 5-HT2c receptor is known to normally couple to phospholipase C but when transfected into Syrian hamster tumour cells, an inhibition of cAMP was observed 13. Similarly Duzic and Lanier 14 have shown that expression of o¢2-adrenoceptors in NIH 3T3 and DDT1 MF-2 cells produces adrenaline-mediated inhibition of cAMP, while transfection of the same receptor in PC12 cells produces adrenalineinduced elevation of cAMP. Finally, the obvious complications with agonists generally steer classification-minded pharmacologists towards using antagonists. However, the simplistic criterion of lack of observed response to a ligand may not be sufficient to ensure that these ligands bind only to receptors and do not affect subsequent interaction of the receptor with G proteins. Cellular amplification systems control observed response and drugs can demonstrate activities from full agonism to full antagonism in a range of tissues by activating the same receptor 15-17. These effects can b e striking in receptor expression systems, especially if receptors are over-expressed. For example, when high densities of 5-HTIDa and 5HTIDI~ receptors were transfected into cells, antagonists such as yohimbine and dihydroergotamine behaved as agonists 18"~9. Therefore, the absence of measurable

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response may be a very inaccurate criterion upon which to classify an antagonist. A more sinister p h e n o m e n o n may be operative within the class of known 'antagonists' for ligands that destabilize the formation of ternary complexes (negative antagonists) 6"2°,2I. The practical consequence of these data is that, in systems where significant receptor-G protein coupling occurs, the affinity of negative antagonists is affected by the same factors as those that affect the affinity of agonists. At present, with no knowledge of possible negative efficacy, all antagonists are considered equal in correlations of antagonist affinity in natural and expression systems. If some of these ligands are indeed negative antagonists, then they may tend to obscure otherwise good correlations. This raises the question of whether pharmacologists should reclassify the system or the ligand. These ideas suggest that the post-translational pharmacological study of expression systems should be a necessary part of the receptor classification process and that data in expression systems need not necessarily trump data from natural ones. Rather, a 'synoptic' view (i.e. affording a general view of a whole) of receptor function considering the m e m b r a n e milieu of receptor and coupling proteins might be more appropriate. In view of the complex interconnections between receptors and effector systems it should not be expected that the physiological situation will readily be modelled in an expression system and in fact, with receptor over-expression, artifactual systems m a y result 22,23. The expression of a receptor in a surrogate system could be likened to putting Hamlet on an empty stage and expecting the grandeur of Shakespeare to emerge. In some cases it may, while in others the Prince may take on a different role. The recognition of these different roles m a y be a critical factor in receptor classification processes. TERRY KENAKIN

Department of Cellular Biochemistry, Giaxo Research Institute, 5 Moore Drive, Research Triangle Park, NC 27709, USA.

References 1 De Lean, A., Stadel, J. M. and Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 7108-7117 2 Samama, P., Cotecchia, S., Costa, T. and

Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625-4636 3 Kjelsberg, M. A., Cottechia, S., Ostrowski, J., Caron, M. G. and Lefkowitz, R. J. (1992) J. Biol. Chem. 267, 1430-1433 4 Ren, Q., Kurose, H., Lefkowitz, R. J. and Cotecchia, S. (1993) J. Biol. Ctzem. 268, 16483-16487 5 Lefkowitz, R. J., Cotecchia, S., Samama, P. and Costa, T. (1993) Trends Pharmacol. Sci. 14, 303-307 6 Costa, T. and Herz, A. (1989) Proc. Natl Acad. Sci. USA 86, 7321-7325 7 Chatterjee, T. P., Moy, J. A., Lee, J. J. H-C. ahd Fisher, R. (1993) Mol. Pharmacol. 43, 167-175 8 Senogles, S. E., Spiegel, A. M., Pardrell, E., lyengar, R. and Caron, M. (1990) J. Biol. Chem. 265, 4507-4514 9 Kenakin, T. P. (1990) Drugs 40, 666-687 10 Roerig, S. C., Loh, H. H. and Law, P. Y. (1992) Mol. Pharm,col. 41, 822831 11 Munshi, R., Pang, I-H., Sternweis, P. C. and Linden, J. (1991) J. Biol. Chem. 266, 22285-22289 12 Eason, M. C., Kurose, H., Holt, B. D., Raymond, J. R. and Liggett, S. B. (1992) ] Biol. Chem. 267, 15795-15801

13 Lucaites, V. L., Nelson, D. L., Yu, L. and Baez, M. (1992) J. Cell Biochem. Suppl. 0 (16 Part E), 228 14 Duzic, E. and Lanier, S. M. (1992)J. Biol. Chem. 267, 24045-24052 15 Kenakin, T. P. (1985)J. Cardiovasc. Pharmacol. 7, 208-210 16 Kenakin, T. P. (1987) Trends Pharmacol. Sci. 8, 423--426 17 Hoyer, D. and Boddecke, H. W. G. M. (1993) Trends Pharmacol. Sci. 14, 270-275 18 Adham, N., Ellerbrock, B., Hartig, P., Weinshank, R. and Branchek, T. (1993) Mol. Pharmacol. 43, 427-433 19 Schechter, L. E., Adham, N., Weinshank, R., Hartig, P. and Branchek, T. (1992) in 2nd International Symposium on Serotonin, Houston, September 1992, P29

20 Costa, T., Ogino, Y., Munson, P. J., Onaran, H. O. and Rodbard, D. (1992) Mol. Pharmacol. 41, 549-560 21 Schutz, W. and Freissmuth, M. (1992) Trends Pharmacol. Sci. 13, 376-380 22 Taylor, C. W. (1990) Biochem. J. 272,1-13 23 Milligan, G. (1993) Trends Phmmacol. Sci. 14, 239-244 DADLE: [DAla2,DLeuS]enkephalin

Relevance of airway hyperreactivity and leukocyte phosphodiesterase abnormahties to n o v e l a n t i - a s t h m a

drugs

New aproaches to asthma therapy In asthma, drug therapy is empirical, and expert opinion 1 favours the early use of inhaled glucocorticosteroids supplemented by ~2-adrenoceptor agonists ~0 control symptoms. Despite a large and sustained growth in the use of anti-asthma drugs, both the incidence and severity of the disease a p p e a r to be increasing 2. Clearly, a more fundamental therapeutic strategy is required, and the article by Stewart et al. 3 is to be welcomed since it may p r o m p t p h a r m a cologists to adopt a n e w approach to the discovery of anti-asthma drugs. However, two omissions deserve comment. First, the authors have pres u m e d that hyperresponsivity of asthmatic airways is nonselective. Their presumption has probably been influenced by the finding of an exceedingly close correlation between sensitivity to inhaled histamine and to inhaled methacholine 4. However, concordance of responses was not found with a wider range of spasmogens 5,6 and, since procedures that exacerbate airway hyperreactivity in asthma (e.g. exposure to allergens or allergic mediators) can produce hyperreactivity, isoreactivity and

hyporeactivity according to the test s p a s m o g e n used 7, it cannot be p r e s u m e d that nonselective hyperreactivity to a range of s p a s m o g e n s characterizes asthma. Until this issue has b e e n further clarified by appropriate experimental study of asthmatics, unifying hypotheses will r e m a i n premature. Secondly, in considering defects that might contribute to, or underlie, lung inflammation in asthma, no mention is m a d e of the aberrant profile of p h o s p h o d i e s terase enzymes in the leukocytes of allergic and asthmatic subjects s. This p h e n o m e n o n justifies the evaluation of selective inhibitors of p h o s p h o d i e s t e r a s e isoenzymes 7 as a m e a n s of resolving w h a t m a y be a potential f u n d a m e n t a l molecular defect. A n u m b e r of pharmaceutical companies have a d o p t e d this approach as a basis for selecting novel a n t i - a s t h m a drugs. It is therefore p r e m a t u r e to conclude that other approaches (e.g. inhibition of selective tyrosine kinases) will necessarily be advantageous. J. MORLEY

Mumed Ltd, Chesham House, I50 Regent Street, London, UK WC1 5FA.