QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Im, W. B. Articles by Dinh, D. M. Search for Related Content PubMed PubMed Citation Articles by Im, W. B. Articles by Dinh, D. M.

Positive Allosteric Modulator of the Human 5-HT_2C Receptor

Biology II/Neurobiology, Pharmacia, Kalamazoo, Michigan (W.B.I., C.L.C., G.L.A.) and Research and Development Discovery Technologies (D.M.D.), Pharmacia, Kalamazoo, Michigan

Received December 2, 2002; accepted April 1, 2003

	Abstract

Top Abstract Methods and Materials Results Discussion References

 
The human 5-hydroxytryptamine-2C (5-HT_2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be L-threo--D-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 µM) markedly enhanced [³H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([³H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT–activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [³H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT_2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT_2A, 5-HT_2B, 5-HT₆, 5-HT₇, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the -D-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [³H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.

	Methods and Materials

Top Abstract Methods and Materials Results Discussion References

 
Cloning of the cDNA for the human 5-HT_2C receptor into a mammalian expression vector, PCI-Neo (Promega, Madison, WI), has been described elsewhere (Alberts et al., 1999) and was cloned into the PCRscript vector via a blunt end ligation. The recombinant vector was used to transfect human embryonic kidney 293 cells (HEK293), NIH3T3, and a human epithelial cell line (SH-EP) using Ca²⁺ phosphate precipitation techniques. Stably transfected cells were selected in the presence of G418 (400 µg/ml). Cell membranes expressing the 5-HT_2C receptor were prepared with the use of procedures described elsewhere (Alberts et al., 1999).

For [³H]5-HT binding, scintillation proximity binding assays were initially carried out using wheat germ agglutinin-coated beads saturated with membranes from HEK293-A cell line expressing human 5-HT_2C receptors. Assay mixtures contained [³H]5-HT at 4 nM and test ligands from Pharmacia (Peapack, NJ) chemical library at 10 µM in medium that contained 100 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, and 20 mM HEPES/Tris, pH 7.4. Nonspecific binding was measured in the presence of mianserin at 5 µM. Hits from the high-throughput screening were further examined using regular filtration-binding techniques as described elsewhere (Alberts et al., 1999). Briefly, binding of [³H]5-HT or [³H]mesulergine to 5-HT_2C membranes was measured in the above-described medium with use of the radioactive ligand at varying concentrations (0.1 to 20 nM for typical binding profiles) and 5 to 20 µg membrane protein in a total volume of 500 µl. Reaction mixtures were incubated at 23°C for 60 min and filtered over Whatman GF/B filters under vacuum (Whatman, Clifton, NJ), which were then washed three times with 4 ml of an ice-cold 50 mM Tris/HCl buffer, pH 7.4. Nonspecific binding was estimated in the presence of excess unlabeled clozapine (100 µM). Ligand stock solutions were prepared in 0.1% ascorbic acid. Displacement of [³H]mesulergine (2 nM) by test compounds at various concentrations (competition assay) was carried out in the same manner.

[³⁵S]GTPS binding was measured by following the procedure reported earlier (Chabert et al., 1994) in medium that contained 25 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 0.5 mM dithiothreitol, 0.003% mM digitonin, 2 nM [³⁵S]GTPS (3 to 5 x 10⁵ cpm /assay), and approximately 10 µg of membrane protein in a volume of 120 µl. Membranes were preincubated with 100 µM 5'-adenylylimidodiphosphate for 30 min at room temperature and subsequently with 10 µM GDP for 10 min on ice. Test ligands were included at 10 µM, unless indicated otherwise. Reaction mixtures were incubated for 45 min at 30°C and were filtered over a Whatman GF/B filter under vacuum. Filters were washed three times with 4 ml of an ice-cold buffer that contained 100 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 25 mM MgCl₂. Agonist-induced [³⁵S]GTPS binding was obtained by subtracting that which was observed without agonists. Binding data were analyzed using a nonlinear regression method (Sigma plot).

The agonist-induced IP₃ release in intact cells was measured using the inositol-1,4,5-trisphophate ³H radioreceptor assay kit from PerkinElmer Life Sciences (Boston, MA). Briefly, cells were grown in a 24-well plate to approximately 80% confluence and were treated with 5-HT or test ligands at indicated concentrations for 45 s (initially a time course from 0 to 1200 s). Each reaction was stopped with trichloroacetic acid (20% final concentration), and each was then extracted with 1,1,2-trichloro-1,2,2-trifluoroethane and trioctyl amine. An aliquot (300 µl) was analyzed for IP₃ using [³H]IP₃/IP₃ receptor preparations from calf cerebellum by following the protocols provided by the vender. For each experiment, a dose-response profile for IP₃ was constructed by adding known amounts of exogenous IP₃ to trichloroacetic acid extracts of untreated cells.

5-HT– or PNU-69176E–induced [³H]inositol phosphate (IP) accumulation was measured in cells that had been labeled with [myo-³H]inositol for 24 h. Briefly, semiconfluent cell cultures in 24-well plates were washed and incubated with 0.5 µCi of [myo-³H]inositol in 0.5 ml of Dulbecco's modified Eagle's medium without inositol for 24 h. After washing the cells, they were treated with 5-HT or PNU-69176E at 10 µM in the presence of Li (10 mM) and pargyline (10 µM) for 30 min. IPs were extracted from cells and isolated using an anion exchanger, AG1-X8 (formate form; Bio-Rad, Hercules, CA) column chromatography as described elsewhere (Berridge et al., 1982).

	Results

Top Abstract Methods and Materials Results Discussion References

 
To search for positive allosteric modulators, the Pharmacia chemical library was screened with use of the assay of [³H]5-HT binding to the human 5-HT_2C receptor. The receptor was heterologously expressed in HEK293 cells at a density of 45 ± 3 pmol/mg of protein (HEK293-A). From the screening, we discovered PNU-69176E (Fig. 1), which at 10 µM markedly stimulated [³H]5-HT (2 nM) binding (300%). The concentration-response profile for PNU-69176E was biphasic (Fig. 2A). At concentrations of less than 25 µM, the drug enhanced [³H]5-HT binding, but at higher concentrations it decreased binding. Peak stimulation of [³H]5-HT binding by PNU-69176E was observed at the drug concentration of 25 µM, with a net increase of 355 ± 37%, as normalized to that in the absence of the drug. As the drug concentration increased to levels greater than 25 µM, [³H]5-HT binding decreased gradually and disappeared at the drug concentration of 200 µM. The latter phase is probably caused by disturbances of membrane structures by the amphipathic PNU-69176E, which contains both a hydrophobic long alkyl chain and a polar head group (Fig. 1). The biphasic profiles fitted to a two-site logistic equation. The stimulatory phase for PNU-69176E showed an EC₅₀ value of 6.3 ± 1 µM and a slope of 2.3 ± 0.5, and the inhibitory phase showed an IC₅₀ value of 61 ± 5 µM and a slope of 3.6 ± 0.6 (Table 1; Fig. 2A). Also, saturation binding assays for [³H]5-HT at concentrations from 0.09 to 48 nM were carried out with or without PNU-69176E at 10 µM (Fig. 2B). Without the drug, [³H]5-HT binding linearly increased and showed no sign of saturation, even at 48 nM [³H]5-HT. In contrast, in the presence of PNU-69176E (10 µM), 5-HT binding data fitted to one site-binding model with a K_D of 17 ± 0.8 nM and maximal binding of 32 ± 0.8 pmol/mg of protein, which accounts for nearly 80% of the total binding site, as estimated from [³H]mesulergine binding.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical structure of PNU-69176E.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effects of PNU-69176E on [3H]5-HT binding to 5-HT2C and to analogous 5-HT receptors and [3H]quinpirole binding to dopamine receptors as expressed in HEK293 cells. A, profile for [3H]5-HT (2 nM) binding with PNU-69176E at various concentrations. Binding experiments were carried out at 23°C for 1 h in HEK293 cell membranes expressing the human 5-HT2C receptor. Nonspecific binding was estimated in the presence of clozapine at 20 µM. The solid line in the plot represents the data fitting to a two-site logistic equation (Table 1). B, binding profiles for [3H]5-HT at various concentrations with or without 10 µM PNU-69176E. The binding data in the absence of PNU-69176E showed no sign of saturation up to the concentration of [3H]5-HT of 48 nM, but in the presence of PNU-69176E, it fitted to one-site binding model with a KD of 17 ± 0.8 nM and maximal binding of 32 ± 0.8 pmol/mg of protein. C, effects of PNU-69176E on [3H]5-HT (2 nM) binding to 5-HT2A, 5-HT2B, 5-HT2C, 5-HT6 and 5-HT7 receptors and [3H]quinpirole (5 nM) binding to human dopamine D2-long and D3 receptors expressed in HEK293 cells. Nonspecific binding was obtained in the presence of excess antagonist for each receptor. The data represent a composite of three duplicate concentration-response profiles (A and C) and two duplicate profiles (B).

View this table: [in this window] [in a new window]   TABLE 1 Analysis of biphasic concentration-response profiles for the actions of PNU-69176E on [3H]5-HT binding (Fig. 2) and GTP [35S] binding (Fig. 4) to the human 5-HT2C receptor in HEK293 cell membranes using a two-site logistic equation Effects of PNU-69176E at various concentrations on [3H]5-HT (2 nM) binding to the 5-HT2C receptor were measured in HEK293 cell membranes expressing the receptor using filtration techniques. Their effects on GTP [35S] binding were measured in the same membrane preparation and as a function of drug concentrations with or without mesulergine in excess (100 µM), and they were normalized to the level observed with 5-HT at a saturating concentration (10 µM). Mesulergine by itself showed no appreciable effects on GTP [35S] binding, but it blocked PNU-69176E-induced GTP [35S] binding.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effects of PNU-69176E on [35S]GTPS binding in membranes from HEK293 cells expressing the human 5-HT2C receptor. A, [35S]GTPS binding was measured as a function of drug concentrations with or without mesulergine in excess (100 µM) and was normalized to the level observed with 5-HT at a saturating concentration (10 µM). Metergoline by itself showed no appreciable effects on [35S]GTPS binding but blocked PNU-69176E–induced [35S]GTPS binding. Amounts of mesulergine-sensitive [35S]GTPS binding were plotted as a function of drug concentrations, and fitted to a two-site logistic equation (Table 1 and text). B, 5-HT concentration-response profiles for [35S]GTPS binding in the presence or absence of PNU-69176E at 10 µM. To determine potential interactions of 5-HT and PNU-69176E, the amount of [35S]GTPS binding induced by PNU-69176E (10 µM) alone was subtracted from the 5-HT dose-response profiles with the drug. The data thus obtained fitted to a single rectangular hyperbolic equation (dashed line). PNU-69176E increased Emax to 120 ± 5% compared with that obtained with 5-HT alone and decreased the EC50 value from 27 ± 4 to 10 ± 2 nM.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effects of PNU-69176E on [3H]mesulergine (antagonist) binding to 5-HT2C receptors and on displacement of [3H]mesulergine binding by 5-HT (low-affinity sites). A, binding data for [3H]mesulergine at various concentrations with or without PNU-69176E at 10 µM were analyzed using Scatchard analysis. PNU-69176E (10 µM) produced no appreciable effects on KD (2.0 ± 0.2 nM) and maximal binding (45 ± 3 pmol/mg of protein) for [3H]mesulergine. B, similar analysis of [3H]mesulergine binding as a function of PNU-69176E concentrations from 3 to 25 µM. No changes in KD () and Bmax () were noted, except for a 45% reduction of Bmax with 25 µM PNU-69176E, probably because of its amphipathic property. C, 5-HT monophasically displaced [3H]mesulergine (3 nM) binding, and the displacement data fitted to a model for a single binding site. The 5-HT Ki decreased from 159 ± 12 to 86 ± 10, 36 ± 3, 10 ± 1, and 6.4 ± 0.9 nM, when the concentration of PNU-69176E was progressively increased to 2.5, 5, 10, and 20 nM, respectively. The plots in A represent two triplicate ([3H]mesulergine) or one triplicate ([3H]mesulergine + PNU-69176E 10 µM) concentration-response profiles. The plots in B represent two duplicate concentration-response profiles.

Typically, G protein-coupled receptors interact with agonists via high- and low-affinity sites, and their relative affinities can be examined with competition experiments using a radioactive antagonist. Displacement of [³H]mesulergine by 5-HT at 5-HT_2C receptors, however, fitted well to a single site-binding model with a K_i of 159 ± 12 nM (Fig. 2). This monophasic profile indicates more than 90% of receptors existing in low-affinity states for 5-HT, leaving only a negligible receptor population in high-affinity states, probably caused by high-receptor density of the cloned cells (Alberts et al., 1999). PNU-69176E concentration-dependently shifted the displacement curve to the left (Fig. 3C). The K_i for 5-HT decreased from 159 ± 12 to 86 ± 10, 36 ± 3, 10 ± 1, and 6.4 ± 0.9 nM in the presence of PNU-69176E at 2.5, 5, 10, and 20 µM, respectively. Such parallel shifts of the displacement curve may indicate that the whole receptor population undergoes gradual and uniform conformational changes in the presence of the drug. In the presence of PNU-69176E at 20 µM, the affinity of 5-HT (6.4 ± 0.9 nM) approached that of high-affinity sites as measured with [³H]5-HT (K_D = 5 nM) (Julius et al., 1988; Alberts et al., 1999). Moreover, the ability of PNU-69176E to enhance 5-HT affinity was reversible. We treated 5-HT_2C membranes with PNU-69176E at 10 µM for 30 min and then washed out upon dilution (4-fold) and ultra-centrifugation. In such treated membranes, 5-HT replaced [³H]mesulergine with a K_i of 128 ± 33 nM, which was not appreciably different from that of membranes which were not exposed to PNU-69176E at all.

Not only 5-HT but also other agonists improved their affinity to the 5-HT_2C receptor in the presence of PNU-69176E. From competition experiments with [³H]mesulergine in the presence of PNU-69176E at 10 µM, we found that the K_i values of Org 37684, 1-(3-chlorophenyl)piperazine hydrochloride, 2,5-dimethoxy-4-iodoamphetamine, and 5-carboxamidotryptamine decreased by 12-, 8-, 7, and 3.4-fold, respectively: from 342 ± 28 to 29 ± 4 nM for Org 37684, from 228 ± 12 to 32 ± 2 nM for 2,5-dimethoxy-4-iodoamphetamine, from 523 ± 81 to 64 ± 12 nM for 1-(3-chlorophenyl)piperazine hydrochloride, and from 4081 ± 612 to 1210 ± 185 nM for 5-carboxamidotryptamine. In the same experiments, the K_i for 5-HT decreased from 167 ± 15 to 14 ± 3 nM, approximately 12-fold. This indicates the universal action of PNU-69176E on 5-HT_2C agonists, albeit to somewhat differential degrees.

Agonist binding to analogous 5-HT and dopamine receptors was not affected by PNU-69176E at 10 µM. The drug showed no appreciable effect on [³H]5-HT (2 nM) binding to 5-HT_2A, 5-HT_2B, 5-HT₆, and 5-HT_7A receptors and also no effect on [³H]quinpirole binding to dopamine D2-long and D3 receptors (Fig. 2C).

Functionally, cloned 5-HT_2C receptors in HEK293 cells couple to both pertussis toxin-insensitive G_q/11 (Julius et al., 1988) and pertussis toxin-sensitive G_i subtypes of G proteins (Alberts et al., 1999). GDP/GTP exchange at G subunits is an early step for G protein activation and could be monitored with [³⁵S]GTPS (a slowly hydrolyzable analog) binding to G_i. The exchange at G_q/11 is not considerable because of their much slower turnover rates in isolated states (Pang and Sternweis, 1990; Smrcka et al., 1991). In HEK293-A cells, 5-HT concentration-dependently enhanced mesulergine-sensitive [³⁵S]GTPS binding, whereas mesulergine by itself showed no appreciable effects on the basal [³⁵S]GTPS binding (Alberts et al., 1999). The basal, mesulergine-sensitive [³⁵S]GTPS binding, however, increased as a function of PNU-69176E concentrations (Fig. 4), and its concentration-response profile was biphasic and similar to that observed with [³H]5-HT binding. At concentrations less than 25 µM, the drug progressively increased mesulergine-sensitive [³⁵S]GTPS binding, but at concentrations greater than 25 µM, it gradually decreased the [³⁵S]GTPS binding, and at 200 µM the drug largely abolished it. Peak stimulation by PNU-69176E was observed at 25 µM, with a net increase of 71 ± 5% (500 fmol [³⁵S]GTPS binding per mg of protein), as normalized to maximal stimulation by 5-HT (10 µM). The concentration profile fitted again to a two-site logistic equation (Fig. 4). The stimulatory phase for PNU-69176E displayed an EC₅₀ value of 7.7 ± 0.6 µM and a slope of 2.5 ± 0.3, and its inhibitory phase exhibited an IC₅₀ value of 49 ± 2 µM and a slope of 3.4 ± 0.9 (Table 1). These parameters are very similar to those obtained from similar analysis of PNU-69176E–stimulated [³H]5-HT binding data (see above). This indicates a common, underlying mechanism(s) by which PNU-69176E affects [³H]5-HT and [³⁵S]GTPS binding to 5-HT_2C receptors. It should be noted that in HEK293 cell membranes without heterologous expression of 5-HT_2C receptors, no mesulergine-sensitive [³⁵S]GTPS binding was induced by PNU-69176E at concentrations ranging from 2.5 to 20 µM.

Signaling responses for 5-HT_2C receptors could also be examined with transient IP₃ release in intact cells during a short incubation period (e.g., 1 min) or [³H]IP accumulation during a longer incubation period (e.g., 30 min) in the presence of lithium and pargeline. In this study, 5-HT (10 µM) transiently increased IP₃ releases in HEK293 cells, reaching a peak at the incubation time of 45 s. PNU-69176E (10 µM) by itself also increased IP₃ releases with the same time course and a peak that reached 71% of the maximal 5-HT (10 µM) response (2.4 ± 0.4 pmol/well) (Fig. 5). Similar results were obtained with [³H]IP accumulation. Both 5-HT and PNU-69176E increased [³H]IP accumulation as a function of time in cells labeled with [myo-³H]inositol (Fig. 5). Maximal accumulation of [³H]IP was observed at the incubation time of 30 min, and PNU-68176E (10 µM) alone induced [³H]IP accumulation up to 83% of that observed with 5-HT (10 µM) (Fig. 5). The drug responses were blocked by mesulergine (10 µM), an antagonist to 5-HT_2C. These results are consistent with the view that the drug renders 5-HT_2C receptors constitutively active.

View larger version (15K): [in this window] [in a new window]   Fig. 5. IP3 release or [3H]IP accumulation induced by PNU-69176E alone in HEK293 cells heterologously expressing the human 5-HT2C receptors. A, IP3 release was measured using a FlashPlate assay kit (PerkinElmer Life Sciences) upon treatment of cells with 10 µM 5-HT or 10 µM PNU-69176E for indicated durations (from 10 to 300 s) at room temperature. A transient increase of IP3 release was observed, reaching a peak in 45 s and then returning to the basal level in 5 min. B, [3H]IP accumulation was measured in cells prelabeled with [myo-3H]inositol upon exposure to PNU-69176E at 10 µM or 5-HT 10 µM for 30 min, following a procedure described elsewhere (Berridge et al., 1982). The basal level of [3H]IP accumulation was negligible during a 30-min incubation period and was not reduced in the presence of mesulergine (antagonist) at 100 µM. The data in A represent the mean ± S.E. from three triplicate concentration-response profiles, and those in B represent the mean ± S.E. from two quadruplicate concentration-response profiles.

To investigate potential functional interactions between PNU-690176E and 5-HT, we examined [³⁵S]GTPS binding as a function of 5-HT concentrations with or without PNU-69176E (10 µM) (Fig. 4B). 5-HT concentration-dependently increased [³⁵S]GTPS binding with an EC₅₀ value of 27 ± 4 nM. PNU-69176E at 10 µM visibly shifted the 5-HT profile upward. After subtraction of the portion induced by PNU-69176E alone, the 5-HT profile fitted to a single rectangular hyperbola with an EC₅₀ value of 10 ± 1.2 nM for 5-HT and a maximal level of 120 ± 5%, as normalized to that of 5-HT alone (Fig. 4B). This indicates that PNU-69176E (10 µM) potentiates the 5-HT action by decreasing the EC₅₀ value from 27 to 10 nM and increasing maximal stimulation by 20%.

We also examined the effect of 5-HT at submaximal (5 nM) and saturating concentrations on IP₃ production with or without PNU-69176E. Peak IP₃ level was measured with a 45-s exposure to 5-HT. At a submaximal concentration of 5 nM, 5-HT induced an IP₃ release of 0.6 ± 0.1 pmol/well, and PNU-69176E (10 µM) alone induced an IP₃ release of 1.3 ± 0.2 pmol/well. In combination of the two, the peak IP₃ level reached a nearly maximal level of 1.8 ± 0.2 pmol/well. At a saturating concentration of 200 nM, 5-HT increased IP₃ release by 2.0 ± 0.2 pmol/mg of protein, and its action was not augmented by PNU-69175E at 10 µM (2 ± 0.1 pmol/mg of protein). This suggests that PNU-69176E and 5-HT share the same IP₃ signaling pathways, but their functional potentiation was not as evident as with [³⁵S]GTPS binding, probably because of involvements of downstream amplification and threshold steps for IP₃ release.

In the course of studying the pharmacology of the 5-HT_2C receptor, we obtained several mammalian cell lines, HEK293, SH-EP, and NIH3T3, stably expressing the receptor at various receptor densities, as estimated from maximal binding of [³H]mesulergine (antagonist) (Table 2). The highest receptor density was 45 ± 3 pmol/mg of protein for the HEK293 cell line we studied (HEK293-A), followed by SH-EP-A, NIH3T3, and HEK293-B at receptor densities of 12.4 ± 2, 11.9 ± 0.6, and 6.6 ± 0.1 pmol/mg of protein, respectively. Despite widely variable receptor densities, all of these cell lines showed robust agonist-induced [³⁵S]GTPS binding, which was blocked by N-ethylmaleimide (100 µM), an inhibitor of G_i and G_o subtypes of G proteins (data not shown). Also, PNU-69176E enhanced the affinity of 5-HT to low-affinity sites and increased the basal, mesulergine-sensitive [³⁵S]GTPS binding. The K_i value for 5-HT low-affinity sites ranged from 159 to 223 nM in these cell lines and decreased in the presence of PNU-69176E (10 µM) to10 to 32 nM (Table 2). The drug (10 µM) increased mesulergine-sensitive [³⁵S]GTPS binding by 23 to 50% as normalized to that of 5-HT at 10 µM (Table 1). We conclude that the positive allosteric modulation of 5-HT_2C receptors by PNU-69176E was not dependent on receptor density or on specific cell lines.

View this table: [in this window] [in a new window]   TABLE 2 Effect of PNU-69176E (10 µM) on 5-HT binding (low-affinity sites) in competition with [3H]mesulergine and 5-HT-induced GTP [35S] binding in several mammalian cell lines expressing human or rat 5-HT2C receptors at various densities data represent the mean ± S.E. from two duplicate concentration-response profiles. Receptor density was estimated from maximal binding of [3H]mesulergine to 5-HT2C.

Structurally, PNU-69176E consists of two moieties, a long alkyl chain (undecyl) and a polar moiety including the -D-galactopyranoside (Fig. 1). Analogs of PHA-69176E with a shorter alkyl chain (methyl to hexyl) showed no effect on [³H]5-HT binding to 5-HT_2C receptors (Table 3). PNU-68607E (methyl), PNU-65287E (ethyl), PNU-63502E (propyl), PNU-61734E (n-butyl), PNU-62804E (t-butyl), PNU-67220E (pentanoyl), and PNU-62344E (n-hexyl) at 10 µM did not stimulate [³H]5-HT binding to 5-HT_2C receptors. Also long alkyl amides, PNU-8750 (N-(4-acetoamido-1-naphthylsulfonyl), PNU-33078 [(N-[2-(dimethylamino)ethyl]-N-methyl)], PNU-43240 (N,N-diethyldodecanamide), and PNU-170158 (N-phenyl dodecanamide) at 10 µM failed to stimulate [³H]5-HT binding to 5-HT_2C receptors (Table 3). These results indicate that the undecyl chain and the specific polar group seem to be essential for PNU-68176E to exert positive allosteric modulation on 5-HT_2C receptors.

	Discussion

Top Abstract Methods and Materials Results Discussion References

 
Allosteric modulations have been reported for several types of G protein-coupled receptors. Muscarinic M₁ and M₂ receptors were allosterically modulated by gallamine, pancuronium, and alcuronium, affecting local ligand binding sites (Tuek and Prota, 1995). Various biogenic amine receptors interact with Na⁺ and Zn²⁺ via the aspartate residue at their second transmembrane segment, leading to changes in the binding properties of some ligands (Schetz and Sibley, 2000). Various serotonergic receptors, 5-HT_1A, 5-HT_2A, and 5-HT_2C, as expressed in Xenopus laevis oocytes, have been reported to show oleamide-sensitive receptor signals (Thomas et al., 1997), although this was not reproducible in our hands in mammalian expression systems (W. B. Im, C.L. Chio, G. L. Alberts, unpublished observations). At PGE₂ receptors, L-171837 reportedly enhanced [³H]PGE₂ binding but blocked PGE₂-induced [³⁵S]GTPS binding (Carriere et al., 2000), probably indicating complex conformational perturbations via multiple interaction sites. Overall, these earlier studies have indicated the presence of allosteric modulatory sites at G protein-coupled receptors, but modulatory actions through these sites were limited to local impacts on certain ligand-binding sites, species-dependent modulations, or nonspecific functional perturbations. In this study, we discovered a positive allosteric modulator that was highly selective for 5-HT_2C receptors. PNU-69176E profoundly enhanced [³H]5-HT binding to the human 5-HT_2C receptor by selectively increasing the 5-HT affinity to its low-affinity sites (more than 20-fold), with no effect on antagonist binding, and it also rendered the receptor to be constitutively active, as measured with mesulergine-sensitive [³⁵S]GTPS binding, transient IP₃ release, and [³H]IP accumulation. These actions of PNU-69176E were not dependent on receptor density or specific cell lines, as shown with several mammalian cell lines (HEK293, NIH3T3, and SH-EP) at various receptor densities (6 to 45 pmol/mg of protein).

Interestingly, the modes of action for PNU-69176E are considerably different from those for conventional allosteric modulators of membrane receptors interacting with a single class of high-affinity sites (e.g., benzodiazepines). First, concentration-response profiles for PNU-69176E showed a Hill coefficient of nearly 3, indicating multiple cooperative binding sites. Second, the drug induced gradual and uniform conformational changes in the receptor population instead of converting a fractional population to high-affinity states, probably reflecting gradual occupancy of its multiple binding sites. Finally, structurally, PNU-69176E resembles amphipathic lipid metabolites with a long alkyl chain and a polar head group, both of which seem to be essential for its modulatory actions on 5-HT_2C. Thus, various amphipathic lipid metabolites could have modulatory action on 5-HT_2C. In this respect, it is noteworthy that cloned 5-HT_2C receptors expressed in mammalian cells, e.g., NIH3T3 cells, reportedly display some constitutive activity, as monitored with clozapine- or mesulergine-sensitive basal inositol accumulation in intact cells (Barker et al., 1994). However, no constitutive activity of the 5-HT_2C receptor was detected in isolated membranes from NIH3T3 or HEK293 cells, as measured by [³⁵S]GTPS binding and [³H]IP accumulation. It is conceivable that such a constitutive activity could be induced by specific lipid metabolites of relatively short half-lives, thus detectable only in intact cells.

Constitutive activation of G protein-coupled receptors has been frequently reported on mutations at various regions of receptors. This study shows another route of constitutive activation of G protein-coupled receptors, namely allosteric modulation by specific amphipathic compounds and perhaps certain lipid metabolites.

	Footnotes

 
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; IP₃, inositol 1,4,5-triphosphate; PNU-69176E,L-threo--D-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thio-monohydrochloride (2S-cis); HEK, human embryonic kidney; IP, inositol phosphate; [³⁵S]GTPS, guanosine-5'-O-(3-[³⁵S]thio)triphosphate; Org 37684, (S)-3-[(2,3-dihydro-5-methoxy-1H-inden-4-yl)oxy]-pyrollidine HCl; PGE₂, prostaglandin E₂.

Address correspondence to: Dr. Wha Bin Im, BiologyII/Neurobiology, 0216-209-512, Pharmacia, 301 Henrietta Street, Kalamazoo, MI 49007. E-mail: wbim{at}am.pnu.com

	References

Top Abstract Methods and Materials Results Discussion References

 
Alberts GL, Pregenzer JF, Im WB, Zaworski PG, and Gill GS (1999) Agonist-induced GTP³⁵S binding mediated by human 5-HT_2C receptors expressed in human embryonic kidney 293 cells. Eur J Pharmacol 383: 311-319.[CrossRef][Medline]

Barker EL, Westphal RS, Schmidt D, and Sanders-Bush E (1994) Constitutively active 5-hydroxytryptamine 2C receptors reveal novel inverse agonist activity of receptor ligands. J Biol Chem 269: 11687-11690.[Abstract/Free Full Text]

Berridge MJ, Downes PC, and Henry MR (1982) Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem J 206: 587-595.[Medline]

Canton H, Verriele L, and Colpaert FC (1990) Binding of typical and atypical antipsychotics to 5-HT1C and 5-HT2 sites: clozapine potently interacts with 5-HT1C sites. Eur J Pharmacol 191: 93-96.[CrossRef][Medline]

Carriere MC, de Lean A, Gareau Y, and Metters KM (2000) A specific allosteric modulator of prostaglandin E₂ binding to the prostanoid EP₁ receptor, in Proceedings of the Annual Meeting for American Society of Biochemistry and Molecular Biology and American Society of Pharmacology and Experimental Therapeutics; 2000 June 4–8; Boston, MA. FASEB J 14: A203.

Chabert C, Cavegn C, Bernard A, and Mills A (1994) Characterization of the functional activity of dopamine ligands at human recombinant dopamine D4 receptors. J Neurochem 63: 62-65.[Medline]

Conn PJ, Sanders-Bush E, Hoffman BJ, and Hartig PR (1979) A unique serotonin receptor in choroid plexus is linked to a phosphatidylinositol turnover. Proc Natl Acad Sci USA 76: 4350-4354.[Abstract/Free Full Text]

Cowen PJ, Clifford EM, Walsh AE, Williams C, and Fairburn CG (1996) Moderate dieting causes 5-HT_2C receptor supersensitivity. Psychol Med 26: 1155-1159.[Medline]

Dourish CT (1995) Multiple serotonin receptors: opportunities for new treatments for obesity? Obes Res 3 (Suppl 4): 449S-462S.[Medline]

Epstein RP, Segman R, Benjamin J, Osher Y, Nemanov L, and Belmaker RH (1997) 5-HT_2C serotonin receptor gene polymorphism associated with the human personality trait of reward dependence: Interaction with dopamine D4 receptor and dopamine D3 receptor polymorphisms. Am J Med Genet 74: 65-72.[CrossRef][Medline]

Julius D, MacDermott AB, Axel A, and Jessell TM (1988) Molecular characterization of a functional cDNA encoding the serotonin-1C receptor. Science (Wash DC) 241: 558-564.[Abstract/Free Full Text]

Kaufman MJ, Hartig PR, and Hoffman BJ (1995) Serotonin 5-HT_2C receptor stimulates cyclic GMP formation in choroid plexus. J Neurochem 64: 199-205.[Medline]

Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, Thomas D, Baxter GS, Forbes IT, Ham P, et al. (1996) In vivo and in vitro profile of SB206553, a potent 5-HT2C/5-HT2B receptor antagonist with anxiolytic-like properties. Br J Pharmacol 117: 427-434.[Medline]

Moreau JL, Jenck F, Martin JR, Perrin S, and Haefely WE (1993) Effects of repeated mild stress and two antidepressant treatments on the behavioral response to 5-HT_1C receptor activation in rats. Psychopharmacology (Berl) 110: 140-144.[CrossRef][Medline]

Pang I-H and Sternweis PC (1990) Purification of unique subunit of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized subunit. J Biol Chem 265: 18707-18712.[Abstract/Free Full Text]

Salzman AG, Morse B, Whitman MM, Ivanschenko Y, Jaye M, and Felder S (1991) Cloning of the human serotonin 5-HT₂ and 5-HT_1C receptor subtypes. Biochem Biophys Res Commun 181: 1469-1478.[CrossRef][Medline]

Sanders-Bush E and Breeding M (1991) Choroid plexus epithelial cells in primary culture: a model of 5-HT1C receptor activation by hallucinogenic drugs. Psychopharmacology (Berl) 105: 340-346.[CrossRef][Medline]

Schetz JA and Sibley DR (2000) Three distinct allosteric sites on a G protein coupled receptor modulate the pharmacology of the binding site crevice, in Proceedings of the Annual Meeting for American Society of Biochemistry and Molecular Biology and American society of Pharmacology and Experimental Therapeutics; 2000 June 4–8; Boston, MA. FASEB J 14: A180.

Smrcka AV, Helper JR, Brown KO, and Sternweis PC (1991) Regulation of polyphosphoinositide-specific phospholipase C activity by purified G_q. Science (Wash DC) 251: 804-807.[Abstract/Free Full Text]

Stam NJ, Vanderheyden P, van-Alebeek C, Klomp J, de-Boer T, van-Delft AM, and Olijve W (1994) Genomic organization and functional expression of the gene encoding the human serotonin 5-HT_2C receptor. Eur J Pharmacol 269: 339-348.[CrossRef][Medline]

Tecott LH, Sun LM, Akana SF, Strack AM, Lowenstein DH, Dallman MF, and Julius D (1995) Eating disorder and epilepsy in mice lacking 5-HT_2C serotonin receptors. Nature (Lond) 374: 542-546.[CrossRef][Medline]

Thomas EA, Carson MJ, Neal MJ, and Sutcliffe JG (1997) Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide. Proc Natl Acad Sci USA 94: 14115-14119.[Abstract/Free Full Text]

Tuek S and Proka J (1995) Allosteric modulation of muscarinic acetylcholine receptors. Trends Pharmacol Sci 16: 205-212.[CrossRef][Medline]

Xie E, Zhu L, and Chang LS (1996) The human serotonin 5-HT_2C receptor: complete cDNA, genomic structure and alternatively spliced variant. Genomics 35: 551-561.[CrossRef][Medline]

This article has been cited by other articles:

				V. Avlani, L. T. May, P. M. Sexton, and A. Christopoulos Application of a Kinetic Model to the Apparently Complex Behavior of Negative and Positive Allosteric Modulators of Muscarinic Acetylcholine Receptors J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 1062 - 1072. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Im, W. B. Articles by Dinh, D. M. Search for Related Content PubMed PubMed Citation Articles by Im, W. B. Articles by Dinh, D. M.