146s Biochemical SocietyTransactions ( 1992) 20 Structural analysis of tho q d o p u i n o rocaptor. U R S U U M. D'SOUZA

and PHILIP G. STRANGE

Biological Laboratory, The University, Canterbury, Kent CT2 7NJ, U.K. The D, dopamine receptor binds to dopamine an important neurotransmitter in the brain and periphery, and it is also a key site of action of antiparkinsonian and antischizophrenic drugs. This receptor belongs t o the family of receptors coupled t o GTP binding proteins (G proteins) and has seven putative transmembrane alpha helices. Recently two forms of the D, dopamine receptor (D-, and D-) were identified by gene cloning and are produced by alternative splicing [I]. The two subtypes differ by a 29 amino acid insert in the third intracellular loop. Amino acid residues that are important for binding ligands t o the D, dopamine receptor can be identified from site directed mutagenesis studies and, from the effects of pH changes and chemical modification. The information will be useful f o r the design of more selective drugs that bind t o the receptor. It has been previously shown [ 2 J that a shift in pH from 7.4 t o 6.0 affects the binding of antagonists such as the substituted benzamide drugs t o a greater extent than the classical antagonists such as spiperone in binding to bovine caudate nucleus membranes, a rich source of D, dopamine receptors. Recently, data have been published on the pH dependency of ( - ) sulpiride binding t o D, dopamine receptors in bovine brain 131, where it was shown that the binding of this substituted benzamide drug involved an ionising group of the receptor whose pK, was 7.3. Similar studies were carried out here with a range of substituted benzamide drugs D0710, clebopride, raclopride and YM 09151-2, and also with the classical antagonists of the D, dopamine receptor, spiperone and haloperidol. This was performed t o compare the binding of the classical antagonists with the substituted benzamide drugs which appear t o behave differently. The pH dependency of [%I] spiperone binding was determined by performing saturation experiments at different pH values (8.0 t o 5.5). This involved incubating approximately 1OOpg of bovine caudate nucleus membranes with a range of [%IJspiperone concentrations and determining the amount of ligand bound at each concentration. Incubation was achieved at 25OC for 45 minutes before the membranes were harvested by filtration using GFfB filters. Non specific binding was defined by the addition of 3pM (+) butaclamol and mianserin was also added t o block serotonin 5HT, receptors. The pH dependency of the substituted benzamides and haloperidol was determined by performing Competition assays with ['HJspiperone at the different pH values. In this case, the membranes were incubated with a range of drug concentrations and a single concentration o f ['HJspiperona (ligand). As the concentration of drug increases, the amount of ligand bound decreases and the Ki (inhibition constant) value is obtained from The pK, of the ionising the competition curve [2-3 J groups is determined by usingthe following equation: Ki,ob, = Ki ( 1 + [H+J/K, ) [21 The results indicate that the substituted benzamide drugs D0710, clebopride, raclopride and YM 09151-2 bind t o bovine caudate nucleus membranes, where the interaction involves an ionising group whose pK, is 7.02, 6.98, 6.74 and 6.88 respectively The binding of the classical antagonists spiperone and haloperidol t o the same membranes involved an ionising group whose pK. is 6.17 and 6.33 respectively In summary, the binding of substituted benzamide drugs to the D, dopamine receptor involves an ionising group whose pK, is approximately 7.0, whereas the binding of the classical antagonists, spiperone and haloperidol t o t h e receptor involves an ionising group whose pK, is about 6.0. From site directed mutagenesis studies of the padrenergic [ 4 ] and ml muscarinic acetylcholine receptors 151, conserved aspartic acid residues have been shown to be involved in ligand binding. In particular an aspartic acid residue in the third putative transmembrane region has been found t o be important for ligand binding. This would correspond

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t o aspartic acid 114 (asp 114) of the D, dopamine receptor, and may be a candidate for interacting electrostatically with the classical antagonists, and may correspond to the ionising group of pK, about 6.0. The nature of the group of pK, of about 7.0 that seems to affect the binding of the substituted benzamide drugs is unclear. However, mutation of aspartic acid 80 (asp 80) in the second transmembrane region of the D, dopamine receptor decreases the regulation of the affinity of the D, receptors for substituted benzamide antagonists by sodium and pH 161. This suggests that aspartic acid 80 may correspond t o the ionising group. We acknowledge financial support from the Medical Research Council. 1. Giros, B., Sokoloff, P., Martres, M . , Riou, J., Emorine, L.J. & Schwartz, J. (1989) Nature. 341. 923-926 2. Williamson, R.A. & Strange, P.G. (1990) J.Neurochem. 55, 1357-1365 3. Presland, J.P. & Strange, P.G. (1991) Biochem. Pharmacol. 41, R9-Rl2 4. Strader, C.D., Sigal, I.S. & Dixon, R.A.F. (1989) FASEB J. 3, 1825-1832 5. Fraser. C.M., Wang, C., Robinson, D.A., Gocayne, J.D. & Venter, J.C. (1989) M o l . Pharmacol. 36, 840-847 6. Neve, K.A., Cox, B.A., Henningsen, R.A., Spanoyannis, A. h Neve, R.L. (1991) M o l . Pharmacol. 39, 733-739

Structural analysis of the D2 dopamine receptor.

146s Biochemical SocietyTransactions ( 1992) 20 Structural analysis of tho q d o p u i n o rocaptor. U R S U U M. D'SOUZA and PHILIP G. STRANGE Biol...
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