Introduction

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Dysregulation of serotonin receptor function may contribute to various peripheral and central nervous system disorders (Glennon and Westkaemper, 1993). The 5-HT_1B/1D receptor subtypes are, among other 5-HT receptors, involved in the regulation of 5-HT release and have gained particular interest because of their potential role in migraine, depression, and diseases involving the basal ganglia (Hoyer et al., 1994). These receptors may serve a presynaptic autoreceptor function inasmuch as their activation acts to inhibit 5-HT release (Middlemiss et al., 1988; Hamblin et al., 1992). They also seem to function as heteroreceptors as indicated by studies of nonserotonergic neurons in which 5-HT inhibits the release of acetylcholine, glutamate, dopamine, norepinephrine, and -aminobutyric acid (Hen, 1992). Similarly, 5-HT_1B/1D receptors have been suggested to inhibit peptide release from trigeminal nerve endings in the dura mater (Buzzi et al., 1991). The precise function of each receptor subtype is still controversial. Available data favor the view that vasoconstriction is mediated primarily by 5-HT_1B receptors, whereas neuroinhibition in the trigeminovascular system involves predominantly the 5-HT_1D receptor subtype (Longmore et al., 1997). It can be put forward that the expression of the 5-HT_1D receptor is less abundant than the 5-HT_1B receptor. Molderings et al. (1996) suggested norepinephrine release to be inhibited by 5-HT_1D receptors located on the noradrenergic axon terminals in human atrial appendages. Regulation of [³H]5-HT release in raphé nuclei of 5-HT_1B receptor gene knockout mice seems to be mediated by a 5-HT_1D-like receptor (Piñeyro et al., 1995). In these mice, 5-HT_1B, but not 5-HT_1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus (Trillat et al., 1997).

The h5-HT_1B and h5-HT_1D receptor subtypes show a relatively low (63%) overall amino acid identity with 77% identity within the TMDs (Weinshank et al., 1992). TMD I is most divergent (59% identity), whereas the six other TMDs share between 71% (TMD V) to 96% (TMD III) amino acid identity. Notwithstanding this low homology, the h5-HT_1D and h5-HT_1B receptor subtypes first were reported to display similar binding profiles (Weinshank et al., 1992). Both receptor subtypes can be pharmacologically differentiated using the 5-HT₂ receptor antagonists ketanserin and ritanserin (Peroutka, 1994; Pauwels et al., 1996). Both compounds show potent binding affinity for and are silent antagonists at cloned 5-HT_1D receptors of human, rat, and guinea pig (Wurch et al., 1997b). Surprisingly, they show micromolar affinity for canine 5-HT_1D receptors (Zgombick et al., 1997). A series of benzanilides, such as GR 125743 (N-[4-methoxy-3-(4-methyl-piperazin-1-yl)phenyl]3-methyl-4-(4-pyridyl)-benzamide) and GR 127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5- methyl-1,2,4-oxadiazol-3-yl)[1,1'-biphenyl]-4-carboxamide), have been reported as examples of mixed 5-HT_1B/1D receptor antagonists (Clitherow et al., 1994). However, they also display agonist properties both in vitro and in vivo (Pauwels, 1997). Recently, some antagonists have been communicated as selective for 5-HT_1B receptors [SB 216641 (N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide) (Price et al., 1997) and SB 224289 (1'-methyl-5-(2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro [furo[2,3f]indole-3,4'-piperidine) (Roberts et al., 1997)] and for 5-HT_1D receptors [BRL-15572 (3-[4-(3-chlorophenyl)piperazin-1-yl]1,1-diphenyl-2-propanol) (Price et al., 1997)]. Hence, several ligands are available that distinguish between 5-HT_1B and 5-HT_1D receptor subtypes.

We are interested in the ligand binding divergence between h5-HT_1D and h5-HT_1B receptors and the molecular correlates that underlie their pharmacological specificity. Therefore, a study was undertaken to identify the domain or domains of 5-HT_1B and 5-HT_1D receptors that determine ligand binding specificity. This was performed using a chimeric receptor approach by combining different parts of h5-HT_1D and h5-HT_1B receptors. The binding profile of the various chimeric receptors was determined on transient expression in Cos-7 cells with two different radioligands (i.e., the agonist [³H]5-CT and the putative antagonist [³H]GR 125743). The current report summarizes our findings on the selective interaction of ketanserin and ritanserin with a 5-HT_1D receptor domain restricted to the second ECL and the fifth TMD.

	Experimental Procedures

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Construction of chimeric 5-HT_1D/1B receptors and point-mutated 5-HT_1D receptors. Chimeric 5-HT_1D/1B receptors and point-mutated 5-HT_1D receptors were constructed by a modified PCR-based overlap extension technique. It allows the construction of chimeric receptors without generating a frame-shift or insertion or deletion of amino acids. Briefly, each chimeric receptor is realized by a three-step PCR-based method that allows the fusion of two or three PCR fragments corresponding to the respective segments that represent the chimeric receptor. The first PCR step corresponds to the amplification of the different fragments of the chimeric receptor, which will be fused together in a second PCR step. A third PCR step amplifies the obtained fusion product. The corresponding PCR-primers were designed according to the reported h5-HT_1B and h5-HT_1D receptor gene sequences (Weinshank et al., 1992) such that they possess a 5' extension that is complementary to the adjacent PCR fragment that has to be fused. These primer sequences are listed in Table 1. For each series of primers, the first PCR mixture (50 µl each) contained 10 ng of purified full-length receptor gene fragment, 25 µM concentration of each dNTP, 400 nM concentration of each primer, and 1.25 units of Taq DNA polymerase in PCR buffer (50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris·HCl, pH 8.3). The PCR program consisted of 10 repetitive cycles with a strand separation step at 94° for 30 sec, an annealing step at 58° for 1, min and an elongation step for 1 min at 72°. The PCR products were separated by 2% agarose gel electrophoresis and purified using a Geneclean II kit. For the second PCR step, an equimolecular amount of each fragment (~10 nmol each) was mixed with 50 µM concentration of each dNTP and 2.5 units of Taq DNA polymerase in the absence of additional primers (each DNA strand serves as a primer) in PCR buffer. The PCR program consisted of 10 repetitive cycles with a denaturation step at 94° for 30 sec, an annealing step at 50° for 25 min (to improve base-pairing between the complementary ends of each fragment that must be fused), and an elongation step for 5 min at 72°. The fusion product was subsequently amplified: 0.5 or 5 µl of the PCR reaction was mixed with 25 µM concentration of each dNTP, 400 nM concentration of primers located at the start and stop codons of the h5-HT_1B or h5-HT_1D receptor gene sequences (Weinshank et al., 1992), and 1.25 units of Taq DNA polymerase in PCR buffer. The PCR program was identical to that of the first PCR reaction, except that the elongation step was prolonged to 1.5 min at 72°. The PCR products were separated and purified as described above before ligation into 50 ng of a pCR3.1 expression vector. Sequencing was performed manually on denatured double-stranded plasmid DNA using a Sequenase quick-denature sequencing kit.

Chimeric receptor nomenclature. Extramembrane loops and TMDs were determined according to the hydrophobicity plots of the h5-HT_1D and h5-HT_1B receptors (Weinshank et al., 1992). Constructs were defined by two letters: the first letter (D for h5-HT_1D or B for h5-HT_1B receptor) corresponds to the receptor that represents the majority of the chimeric receptor, and the second letter denotes the exchanged TMD of the second receptor indicated by a roman number, the exchanged ECL indicated by an arabic number, or both.

Expression of receptor genes. Ten micrograms of plasmid was used to transfect 5 × 10⁶ Cos-7 cells by electroporation using a BioRad (Hercules, CA) gene pulser apparatus at 250 mV and 250 µF. Cells were grown for 48 hr in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum. Each transfection experiment with chimeric receptor plasmids was performed in parallel with WT h5-HT_1D and h5-HT_1B receptor plasmids.

Radioligand binding experiments to membrane preparations. Membrane preparations of Cos-7 cells were prepared in 50 mM Tris·HCl, pH 7.7, containing 4 mM CaCl₂, 10 µM pargyline, and 0.1% ascorbic acid as described previously (Pauwels et al., 1996). Binding assays were performed with either 3.0 nM [³H]5-CT or 1.0 nM [³H]GR 125743. Incubation mixtures consisted of 0.4 ml of cell membranes, 0.05 ml of radioligand, and 0.05 ml of compound for inhibition or 10 µM 5-HT to determine nonspecific binding. The reactions were stopped after a 30-min incubation at 25° by adding 3.0 ml of ice-cold 50 mM Tris·HCl, pH 7.7, and rapid filtration over Whatman GF/B glass-fiber filters with a Brandel (Montreal, Quebec, Canada) harvester, washed, and counted as described previously (Pauwels et al., 1996). In the case of [³H]GR 125743, 50 mM Tris·HCl, pH 7.7, was used, and filtration was performed over 0.2% polyethyleneimine-treated Whatman (Clifton, NJ) GF/B glass-fiber filters. Data were analyzed graphically with inhibition curves, and IC₅₀ values (concentration of the compounds producing 50% inhibition of specific binding) were derived. K_i values were calculated according to the equation K_i = IC₅₀/(1 + C/K_D), where C is concentration and K_D is the equilibrium dissociation constant of the radioligand. For ligand saturation binding curves, [³H]5-CT and [³H]GR 125743 were used at concentrations of 0.07-20 nM and 0.15-10 nM, respectively. The curves were analyzed by the nonlinear least-squares curve-fitting program Ligand (Biosoft, Cambridge, UK). Control binding experiments were run with nontransfected cells and did not display specific [³H]5-CT or [³H]GR 125743 binding.

Statistical analysis. Statistical analysis was performed on the K_i values of the chimeric 5-HT_1D/1B receptors versus the K_i values of their respective WT receptors using a Student's t test.

Materials. Oligonucleotides were synthesized on an Applied Biosystems DNA/RNA synthesizer (Foster City, USA). Cos-7 cells were from American Type Culture Collection (Rockville, MD). The pCR3.1 vector was from InVitrogen (San Diego, CA). The Geneclean II kit was purchased from Bio 101 (Vista, CA). Taq DNA polymerase and dNTP were from Gibco-Life Technologies (Paisley, UK). The Sequenase quick denature sequencing kit was from Amersham (Les Ulis, France). The protein assay kit was from BioRad Laboratories. [³H]5-CT (61.3 Ci/mmol) and [³H]GR 125743 (69 Ci/mmol) were obtained from New England Nuclear (Les Ulis, France) and Amersham (Les Ulis, France), respectively. 5-HT and ketanserin were from Sigma Chemical (St. Louis, MO). Ritanserin was obtained from RBI (Natick, MA). L 694247 (2-[5-[3-(4-methylsulfonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine) was purchased from Tocris Cookson (Bristol, UK). Zolmitriptan ([4(S)-[3- [2-(dimethylamino)ethyl]-1H-indol-5-yl-methyl]oxazolidin-2-one]), sumatriptan, GR 127935, and SB224289 were synthesized at the Center de Recherche Pierre Fabre. Stock solutions (1 mM) of compounds were prepared in water, ethanol, or dimethylsulfoxide.

	Results

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Construction and expression of chimeric 5-HT_1D/1B receptors compared with WT h5-HT_1B and h5-HT_1D receptors in Cos-7 cells. In a first approach, different parts of the WT h5-HT_1D and WT h5-HT_1B receptor were combined and evaluated for expression of [³H]5-CT and [³H]GR 125743 binding compared with their parental receptors on transient expression in Cos-7 cells. Fig. 1 shows a schematic representation of eight chimeric 5-HT_1D/1B receptors being investigated. The PCR primers designed at the junction of selected TMD sequences (TMDs I, IV, and V) are indicated for each chimeric receptor in Table 1. Sequencing of each chimeric receptor demonstrated full identity with the predicted chimera sequence. Each of these chimeric receptors displayed specific binding for [³H]5-CT and [³H]GR 125743 as observed with the WT h5-HT_1B and h5-HT_1D receptors. With the exception of some variation in the K_D values for [³H]5-CT of the chimeric receptors D/BV and B/DV, very similar values were obtained compared with the WT h5-HT_1B and h5-HT_1D receptors. The K_D values for [³H]GR 125743 ranged between 0.54 and 2.38 nM, in agreement with those obtained for the WT h5-HT_1B (0.65 nM) and h5-HT_1D (1.22 nM) receptors (Fig. 1). Maximal expression levels of [³H]5-CT and [³H]GR 125743 binding for each of the chimeric receptors were in the same range as for the WT h5-HT_1B and h5-HT_1D receptors: 0.51-2.17 and 0.22-2.67 pmol/mg protein, respectively.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the WT h5-HT1D, WT h5-HT1B, and chimeric 5-HT1D/1B receptors. Shaded rectangles, TMDs derived from the h5-HT1D receptor. Open rectangles, TMDs derived from the h5-HT1B receptor. Extramembrane regions are drawn as either a thin (h 5-HT1D) or a thick (h5-HT1B) line. N-term. and C-term., amino- and carboxyl-terminal ends of the receptor, respectively. The nomenclature of each chimeric receptor is explained in the text. The equilibrium dissociation constants (KD, nM) were determined for each WT and chimeric receptor as described in the text and are expressed as mean ± standard error values of two to four independent experiments.

Inhibition of [³H]5-CT and [³H]GR 125743 binding to WT h5-HT_1D and h5-HT_1B receptors and chimeric 5-HT_1D/1B receptors by 5-HT and ketanserin. Table 2 summarizes the affinity constants of 5-HT and ketanserin for WT h5-HT_1D and h5-HT_1B receptors and eight chimeric 5-HT_1D/1B receptors. 5-HT displaced [³H]5-CT binding with a similar affinity for both WT h5-HT_1D and 5-HT_1B receptors. 5-HT exhibited an 8-fold selectivity for the WT h5-HT_1D over the WT h5-HT_1B receptor when [³H]GR 125743 was used as a radioligand. The 5-HT_2A receptor antagonist ketanserin yielded an affinity of 24-27 nM for the WT h5-HT_1D receptor and a low binding affinity (K_i = 2193 to 2902 nM) for the WT h5-HT_1B receptor, regardless of whether [³H]5-CT or [³H]GR 125743 was used as a radioligand. Replacement of TMDs I-IV of the WT h5-HT_1D receptor by the equivalent amino acid region of the WT h5-HT_1B receptor (chimera D/BI-IV, Fig. 1) did not modify (p > 0.05) the binding affinity of ketanserin compared with the WT h5-HT_1D receptor. The 5-HT binding affinity was not modified for the D/BI-IV chimera when [³H]5-CT was used as a radioligand, but a 4-fold decrease (p < 0.001) in binding affinity was measured with [³H]GR 125743. The addition of TMD V (chimera D/BI-V) promoted a low affinity for ketanserin (K_i = 3,428->10,000 nM), being statistically similar to that of the WT h5-HT_1B receptor when [³H]GR 125743 was used as a radioligand. Otherwise, the binding affinity of 5-HT was similar to the WT h5-HT_1D receptor when measured with [³H]5-CT but significantly decreased (2.5-fold, p < 0.01) when analyzed with [³H]GR 125743. Exchange of TMDs VI and VII of the h5-HT_1B receptor into the h5-HT_1D receptor (chimera D/BVI-VII) did not modify (p > 0.05 versus the WT h5-HT_1D receptor) the binding affinities of ketanserin and 5-HT. Ketanserin showed a significantly lower binding affinity (15-46-fold, p < 0.001 versus the WT h5-HT_1D receptor) in the chimera D/B2ECL+V when only the second ECL and the fifth TMD of the h5-HT_1D receptor were exchanged by the equivalent amino acid domain of the h5-HT_1B receptor. The affinity of 5-HT remained unmodified (p > 0.05) compared with the WT h5-HT_1D receptor by using both radioligands. The reverse chimera (B/D2ECL+V) promoted a 25-34-fold higher affinity (p < 0.01 versus the WT h5-HT_1B receptor) for ketanserin but still differed (2-5-fold, p < 0.01) from that of the WT h5-HT_1D receptor. The affinity of the chimeric receptor B/D2ECL+V for 5-HT was not affected (p > 0.05 versus the WT h5-HT_1B receptor).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Schematic representation of point mutations and chimeric receptors created within the second ECL and the fifth TMD of the h5-HT1D receptor. Bold amino acid residues, diverge between the h5-HT1D and h5-HT1B receptors. The point mutations (PM) that have been constructed are shown (*), and their position in the h5-HT1D receptor sequence is indicated. Arrows, constructed chimeric receptors. Left and right arrowheads, the first and last amino acid of the h5-HT1D receptor replaced by the homologous region of the h5-HT1B receptor, respectively. Each chimeric receptor and point mutation was constructed by the modified overlap extension PCR-based technique using appropriate complementary mutagenic primers as described in the text. The corresponding IC50 values for ketanserin are summarized in Table 3.

Binding profile of the WT h5-HT_1D and h5-HT_1B receptors and chimeric receptors D/B2ECL+V and B/D2ECL+V. A series of 5-HT receptor ligands were tested on the chimeric receptors D/B2ECL+V and B/D2ECL+V for inhibition of [³H]GR 125743 binding and compared with the binding profile obtained with the WT h5-HT_1B and h5-HT_1D receptors (Table 4). Representative binding curves are illustrated in Fig. 3. The mixed 5-HT_1A/1B/1D agonist L 694247 yielded a slight increase (1.6-fold, p < 0.01) in its binding affinity for the chimeric receptor B/D2ECL+V compared with the WT h5-HT_1B receptor, although no difference was apparent between D/B2ECL+V and the WT h5-HT_1D receptor. The agonists zolmitriptan and 5-HT displayed 8- and 25-fold selectivity for the WT h5-HT_1D over h5-HT_1B receptor. The chimeric receptors are not significantly different (p > 0.05) from their parental receptors in regard to these agonists; 8- and 33-fold selectivity was observed. The chimera B/D2ECL+V yielded a 4-fold decreased binding affinity (p < 0.01) for the agonist sumatriptan compared with the WT h5-HT_1B receptor, whereas the K_i values remained unmodified for D/B2ECL+V and WT h5-HT_1D receptors. The mixed 5-HT_1B/1D antagonist GR 127935 yielded similar binding affinities for the chimeric receptors compared with their respective WT receptors. The 5-HT_1B inverse agonist SB 224289 showed 35-fold selectivity for the WT h5-HT_1B receptor; 11-fold selectivity was conserved between the chimeric receptors B/D2ECL+V and D/B2ECL+V. The 5-HT₂ antagonist ritanserin displayed 8-fold selectivity for the WT h5-HT_1D over h5-HT_1B receptor. The chimeric receptors B/D2ECL+V and D/B2ECL+V yielded similar binding affinities for ritanserin as for the WT h5-HT_1D and 5-HT_1B receptors, respectively. Experiments conducted with [³H]5-CT yielded similar results for the comparison between the chimeric receptor B/D2ECL+V and the WT h5-HT_1B receptor (Table 5). Otherwise, slight variations were observed with L 694247, sumatriptan, GR 127935, and SB 224289 when comparing the chimeric receptor D/B2ECL+V with the WT h5-HT_1D receptor.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Inhibition curves of 5-HT receptor ligands for [3H]GR 125743 binding to a membrane preparation of Cos-7 cells expressing either WT h5-HT1B (), WT h5-HT1D (), chimeric D/B2ECL+V (), or B/D2ECL+V () receptors. Radioligand binding was performed as described in Table 3. Curves were constructed using the mean values from one representative experiment of three to six independent experiments. Mean Ki values are summarized in Table 3.

	Discussion

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The major finding of this study concerns the involvement of the second ECL and the fifth TMD in the high affinity binding of ketanserin and ritanserin to the h5-HT_1D receptor. This effect seems to be specific because the other ligands, including 5-HT, various 5-HT_1B/1D agonists, the mixed 5-HT_1B/1D antagonist GR 125743, and the selective 5-HT_1B inverse agonist SB 224289, displayed either no or only slight modifications in their ligand binding affinities. Moreover, these minor modifications were, in contrast to those of ritanserin and ketanserin, not observed by analysis of both [³H]5-CT and [³H]GR 125743 binding. These results point to a specific binding site for ketanserin and ritanserin, distinct from that for the diverse agonists, and also from the inverse agonist SB 224289. The observed changes in the binding profile of the chimeric receptors are likely to result from differences in certain amino acids that interact directly with ketanserin and ritanserin. It is unlikely to be a consequence of a global change in the receptor conformation because the binding affinities of the other ligands were either not or only slightly modified. Although ketanserin is a key compound that differentiates between recombinant 5-HT_1D versus 5-HT_1B receptors of human (Pauwels et al., 1996), guinea pig (Wurch et al., 1997b; Pauwels et al., 1998), and rat (Bach et al., 1993), displaying >100-fold selectivity for the 5-HT_1D receptor subtype, the affinity of ketanserin for the 5-HT_1D receptor is species dependent (Table 6). The antagonist is weakly selective (11-fold) at the rabbit 5-HT_1D receptor relative to the rabbit 5-HT_1B receptor (Wurch et al., 1997a) and apparently recognizes neither the canine 5-HT_1D nor the canine 5-HT_1B receptor subtypes (Zgombick et al., 1997). Amino acid alignment of a domain encompassing the second ECL and the fifth TMD of human, rat, guinea pig, rabbit, and dog 5-HT_1B and 5-HT_1D receptors and the h5-HT_2A receptor does not immediately point to amino acids that may be specifically involved in this differential ketanserin-binding profile (Table 6). The lack of amino acid homology between receptors with either a high or a low affinity for ketanserin, especially between the canine 5-HT_1D versus the 5-HT_1B receptors and the h5-HT_2A versus the 5-HT_1D receptors, suggests a complex ligand/receptor interaction.

Segregation between agonist and antagonist binding sites in G protein-coupled receptors. Although the h5-HT_1D receptor, like other G protein-coupled receptors (Strader et al., 1988), binds agonists and antagonists in a competitive manner (Pauwels et al., 1996), a structural overlap of agonist and antagonist binding sites has not been clearly demonstrated. Alternatively, distinct binding sites for agonists and antagonists may be involved, but they influence the receptor configuration in such a way that agonist and antagonist cannot be bound at the same time. Our results with the chimeric receptors D/BI-V, D/B2ECL+V, and B/D2ECL+V could differentiate between a 5-HT- and a ketanserin-binding site. The data suggest that the binding sites of ketanserin and ritanserin and of the agonists for the h5-HT_1D receptor are not identical. Thus, the competitive antagonism by ketanserin and ritanserin does not imply that they share the same molecular interaction points with the receptor as the agonist. A similar hypothesis has been put forward for the competitive antagonism of substance P by several nonpeptide antagonists at the neurokinin NK1 receptor (Cascieri et al., 1995). Site-directed mutagenesis studies of -adrenergic receptors (Chung et al., 1988; Strader et al., 1988), dopamine D₂ receptors (Mansour et al., 1992), and chimeric 5-HT_2A/5-HT_2C receptors (Roth et al., 1993) also have demonstrated a differential implication of receptor domains in agonist compared with antagonist binding.

Importance of the second ECL and fifth TMD in ligand binding to G protein-coupled receptors. TMD V of several G protein-coupled receptors is likely to play an important role in agonist and antagonist binding, especially via the ability of its hydroxylated residues to form hydrogen bonds. TMD V of the h5-HT_1B receptor previously has been reported to participate in the binding of the agonist sumatriptan; it may form a hydrogen bond with a Ser507 residue of the WT h5-HT_1B receptor (Table 6; Granäs et al., 1995). Replacement of Thr508 (Table 6) by a serine residue in the h5-HT_1B receptor induced a 7.5- and 13-fold decrease in 5-HT affinity and potency, respectively (Veldman and Bienkowski, 1994). This Thr508 residue (or Ser508 in h5-HT_2A) also may be involved in hydrogen bonds to the hydroxyl group of the aromatic ring of 5-HT (Trumpp-Kallmeyer et al., 1992). Similar hydroxylated amino acid residues are found at the same location in TMD V of other serotonergic, -adrenergic, and -adrenergic receptors; in particular, the same amino acid residues (Ser507-Thr508; Table 6) are present in the h5-HT_1D receptor. A hydroxylated threonine residue (Thr196, position 511 in Table 6) in TMD V of the rat 5-ht₆ receptor has been shown to interact with the N1 position of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond with the hydroxyl moiety of threonine (Boess et al., 1997). Johnson et al. (1994) reported that Ala242 (position 511 in Table 6) in TMD V of the rat 5-HT_2A receptor plays an important role in the structure-activity relationship for ergolines and tryptamines. This single amino acid is implied in the high affinity of the antagonist mesulergine, a N1-methylergoline, for the rat 5-HT_2A receptor (Kao et al., 1992). The h5-HT_2A receptor, for which this amino acid is replaced by a serine residue (Ser511; Table 6), is unable to bind mesulergine. The alanine residue, present at the position 511 (Table 6) in TMD V of both the h5-HT_1B and h5-HT_1D receptor subtypes, seems not to be critical for ketanserin binding.