Hydropathicity analysis of 39 G-protein-coupled receptors (GPCR) reveals seven hydrophobic stretches corresponding to membrane spanning alpha-helices. The alignment of the primary sequences shows a high degree of homology in the GPCR transmembrane regions. 3D models of 39 GPCRs were generated using the refined model of bacteriorhodopsin as a template. Five cationic neurotransmitter receptors (serotonergic 5-HT2, dopaminergic D2, muscarinic m2, adrenergic alpha 2 and beta 2 receptors) were taken as prototypes and studied in detail. The 3D models of the cationic neurotransmitter receptors, together with their primary structure comparison, indicate that the agonist binding site is located near the extracellular face of the receptor and involves residues of the membrane-spanning helices 3, 4, 5, 6, and 7. The binding site consists of a negatively-charged Asp located at the middle of transmembrane helix 3 and a hydrophobic pocket containing conserved aromatic residues on helices 4, 5, 6, and 7. To define the precise receptor-ligand interactions, the natural neurotransmitters were docked into the binding sites. Residues responsible for the affinity, selectivity, and eventually stereospecificity of dopamine, adrenaline, noradrenaline, serotonin, and acetylcholine for their receptors were identified. The ligands are involved in electrostatic interactions as well as hydrogen bonds and specific hydrophobic aromatic interactions. All the GPCRs possess invariant hinge residues, which might be responsible for a conformational change during agonist binding and therefore influence dissociation and association of G-proteins to the receptors. The role of hydrophobic interactions and hydrogen bonds in the conformational change of the receptors, modulating the coupling to the G-protein, is discussed with regard to these residues. The models are in agreement with published data obtained from mutagenesis and labeling studies and represent important working hypotheses to direct future mutagenesis studies. They also enable structure-activity relationship studies and more rational drug design. The 3D models of other G-protein-coupled receptors have been generated in a similar way.

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The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[125I]tyrosinamide (abbreviated [125I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [125I]GTI binding sites was largely comparable to that of [125I]iodocyanopindolol ([125I] ICYP) which labels 5-HT1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT1A and beta-adrenoceptor binding sites), although a detailed analysis revealed differences. The pharmacology of the [125I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT1B and 5-HT1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (-)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1- piperazinyl]ethyl]benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [125I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT1B compound CP 93129 and (-)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT1B sites with 100 nM CP 93129, the remaining population of [125I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT1B type: 5-CT > CP 93129 > or = (-)pindolol > sumatriptan > or = PAPP > rauwolscine. The profile of the minor component of [125I]GTI binding is best characterised as that of a 5-HT1D site: 5-CT > PAPP > or = sumatriptan > rauwolscine > (-)pindolol > or = CP 93129. The localisation of the non 5-HT1B [125I]GTI binding sites was characterised by blocking the 5-HT1B receptors with 100 nM CP 93129. Low densities of the 5-HT1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT1B sites were always observed in the same structures.(ABSTRACT TRUNCATED AT 400 WORDS)

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1 Dopamine D 4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-eects. Two such compounds, L-745,870 and U-101958 have been recently introduced. ]-spiperone binding was observed with spiperone (pK i 9.6+0.1, n=3), clozapine (pK i 7.4+0.1, n=4), L-745,870 (pK i 8.5+0.1, n=3) and U-101958 (pK i 8.9+0.1, n=3). By contrast, raclopride was very weak (pK i 55, n=3). 4 Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D 4 cells in a concentration-dependent fashion (E max 71+2% inhibition of forskolin-stimulated levels, pEC 50 8.7+0.1, n=10). This eect was mimicked by the dopamine D 2 -like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).