L E T T E R S Schwartz and Rosenkilde reply Dogmas versus data in 7TM 'locks'for 'keys' It is certainly important to empha- size that the binding sites for the small monoamine 'keys' are far from well established in 7rM- (seven transmemblane domain) receptors. Besides the truly conserved Asp- III:08 in TM-III, which, at least in the [3-adrenoceptor, has been addressed by complementary chemi- cal modifications performed in the ligand and the receptor1, the picture of the rest of the binding site(s) is mainly based on interpretations of much weaker data. As previously discussed2 and as clearly underlined by Philip Strange, the data concerning the presumed role of hydrogen-bond dot,ors/ acceptors in TM-V varies signifi- cantly among monoamine receptors and even among closely related sub- types of adrenoceptors. Importantly, substitut',on of the various serines in TM-V frequently has an almost neg- ligible effect on agonist affinity as determined by competition binding assays, e.g. for adrenaline m the C~A- adrenoceptor3. In some cases, even the effect on signal transduction is limited, whereas in other cases more dramatic effects are observed in these assays4. Interestingly, when Asn-VI:17, the presumed crucial hydrogen-bond interaction point for acetylcholine in TM-VI of the mus- carinic receptor after many years of molecular modelling5.6 was finally addressed by Ala-substitution, only limited effects (two- to threefold) were observed also at this positionL In other words, if it was not already 'known' to be part of the binding site for acetylcholine it would most certainly have been undetected in mutational mapping studies. When data from a multitude of such stud- ies are analysed, a slightly different interpretation of what is significant and what is not can lead to a very different picture, where the mono- amine agonist is modelled to bind more vertically in a pocket between TM-ll, TM-III and TM-VII rather than horizontally between TM-III, TM-V and TM-VI (Ref. 8). This alter- native binding mode is, in fact, simi- lar to that suggested for agonists at adenosine receptors9. Thus, the binding sites for small monoamine ligands are not as well characterized and as simple as indi- cated in the diagram in our artide. It was our intention there, merely to use the presumed interaction points of the B-adrenoceptor to illustrate the location of a binding site for a small agonist in the deep crevice of the main ligand-binding pocket - ha contrast to the presumea binding site for a peptide such as substance P and the binding site for activating antisera developed against synthetic loop peptides 1° which are located more towards the exterior regions of the receptor. However, it is true that a diagram can be misleading. Much of the information used to characterize the 'locks' in 7rM receptors has come from interpre- tations of kinetic data obtained with chemically modified ligands or receptors. However, actual ligand- binding sites have been character- ized by X-ray crystallography in globular enzymes n. The existence of multiple binding sites and surpris- ingly small, structurally different binding modes for similar ligands having similar affinity have been observed in the relatively rigid active site of a globular enzyme12. Similar phenomena are very likely to occur in 7TM receptors possibly to c greater extent due to the (pre- sumably) more flexible binding sites located in the dynamic interface between the TMs and between the connecting loops. Nevertheless, where the ligand is a very small and well defined metal-ion and the binding site can be systematically built up and moved from one 7TM receptor to another, it is more likely that the predicted binding site may, in b ct, correspond to the actual one12,t3, In order to obtain a greater under- standing of 'locks' for 'keys' in 7TM receptors, it will be necessary to ap- ply other biochemical and especially biophysical techniques such as affinity cross-linking14, fluorescence energy transferis, and site-directed spin labelling~ in combination with mutational mapping. Finally, it will be important to try to perform truly convincing complementary modifi- cations of receptors and ligands by combining molecular biology with medicinal chemistry more closelyL T. W. Schwartz and M. M. Rosenkilde Laboratory for Molecular Pharmacology, Rigshospitalet6321, Blegdamsvej 9. DK-2100 Copenhagen,Denmark. References I Strader C. D. et al. (1991) [. Biol. Chem. 260, 5-8 2 Schwartz, T. W. (1994) Cnrr. Opin. Biotedmol. 5, 434-444 3 Hwa, I. and Perez, D. M. (1996~ J. Biol. Chem. 271, 6322-6327 4 Strange, P. G. (1996J Trends Pharnu~coL 5ci. 17, 238-244 5 Trumpp-Kalmeyer, S., Hoflack, J., Bruinvels, A. and Hibert, M. F. (1992) ]. Med. Gwm. 35, 3448-3462 6 Nordvall, G. and Hacksell, U. (t993) J. Med. Chem. 36, 967-976 7 Blurnl, K., MuLschler, E. and Wess, J. (1994) ]. Biol. Chem. 269,18870-18876 8 Hutchins, C. (1994) Endocrine ]. 2, 7-23 9 lizerman, A. P., van der Wenden, E. M., van Galen, P. J. and Jaconson, K. A. (1994) Eor. J. Pharmacol. 268, 95-104 10 Schwartz, T. W. and Rosenkilde, M. M. (1996) Trends Pharntacol. Sci. 17, 213-216 11 Mattos, C. et al. (1994) Nat. Stract. Biol. 1, 55-58 12 EI]ing, C. E., Nielsen, S. M. and Schwartz, T. W. (1995) Nature 374, 74-77 13 Thirstrup, K., Elling, C. E., Hjorth, S. A. and Schwartz, T. W. (1996) ]. Biol. Chem. 271, 7875-7878 14 Boyd, N. et al. (1996) Proc. Natl. Acad. Sci. U. 5. A. 93, 433-437 15 Turcatti, G. et al. (1990) J. Biol. Cheat. 271, 19991-19998 16 Hubbell,W. L. and Alterbach, C. (19941 Cart. Opin. 8tract. Biol. 4, 566-573 Students Subscribe to TiPS at a 50% discount PI! S0165-6147(96}20039-2 TiPS - October 1996 (Voi. 17) 3 4 7