Abstract
Discovering the molecular and atomic mechanism(s) by which G-protein-coupled receptors (GPCRs) are activated by agonists remains an elusive goal. Recently, studies examining two representative GPCRs (rhodopsin and α1b-adrenergic receptors) have suggested that the disruption of a putative “salt-bridge” between highly conserved residues in transmembrane (TM) helix III, involving aspartate or glutamate, and helix VII, involving a basic residue, results in receptor activation. We have tested whether this is a general mechanism for GPCR activation by constructing a model of the 5-hydroxytryptamine (5-HT)2A receptor and characterizing several mutations at the homologous residues (Asp-155 and Asn-363) of the 5-HT2Aserotonin receptor. All of the mutants (D155A, D155N, D155E, D155Q, and S363A) resulted in receptors with reduced basal activity; in no case was evidence for constitutive activity revealed. Structure-function studies with tryptamine analogs and various Asp-155 mutants demonstrated that Asp-155 interacts with the terminal, and not indole, amine moiety of 5-HT2A agonists. Interestingly, the D155E mutation interfered with the membrane targeting of the 5-HT2A receptor, and an inverse relationship was discovered when comparing receptor activation and targeting for a series of Asp-155 mutants. This represents the first known instance in which a charged residue located in a putative TM helix alters the membrane targeting of a GPCR. Thus, for 5-HT2A receptors, the TMIII aspartic acid (Asp-155) is involved in anchoring the terminal amine moiety of indole agonists and in membrane targeting and not in receptor activation by salt-bridge disruption.
The precise mechanism by which G-protein-coupled receptors (GPCRs) are activated is currently unknown, although much current evidence suggests that conformational changes in transmembrane (TM) helices are required for G-protein activation (Farrens et al., 1996; Gether et al., 1997;Roth et al., 1997b; Gether and Kobilka, 1998). At least two different models have been proposed to account for some of the intramolecular rearrangements thought to result in receptor activation. The first model, derived from studies of rhodopsin, opsin, and α1b-adrenergic receptors (Cohen et al., 1992;Robinson et al., 1992; Porter et al., 1996; Porter and Perez, 1999) suggests that breaking a “salt-bridge” or a hydrogen-bonding interaction between a highly conserved polar residue in TMIII (Asp or Gln) and a positively charged/polar residue in TMVII (Lys or Asn, respectively) initiates receptor activation. The second model, derived from studies involving reciprocal mutations of gonadotropin-releasing hormone receptors (GnRHs; Ballesteros et al., 1998) suggests, instead, that a different highly conserved motif (D/ERY) at the TMIII/i2 interface is involved in receptor activation. In the second model, Ballesteros et al. (1998) propose that the arginine at position 139 of the GnRH receptor is constrained by the aspartic acid at position 137. This constraining of Arg-139 allows for the stabilization of the inactive state of the GnRH receptor (Ballesteros et al., 1998). A similar role for the D/ERY motif in the β2-adrenergic receptor was recently proposed based on mutagenesis and cysteine-accessibility studies (Rasmussen et al., 1999).
The 5-hydroxytryptamine (5-HT)2A serotonin receptor represents a convenient receptor in which to test the salt-bridge model because it could contain a hypothetical salt-bridge between Asp-155 and Asn-363. In this article, we show, first, by molecular modeling experiments that Asp-155 and Asn-363 are likely to be widely separated in three-dimensional space and that a salt-bridge between them is unlikely. We also report that a series of mutations of the Asp-155 locus (D155A, -E, -Q, and -N) all result in receptors with decreased constitutive activity, a result that contradicts the salt-bridge hypothesis. Also, by testing 5-HT analogs, we found that the main role of Asp-155 in the 5-HT2A receptor is to anchor the charged terminal amine group and that doing so facilitates interactions of the aromatic moiety of indoles with aromatic residues in TMVI, leading to receptor activation. Finally, we report a novel role for Asp-155: membrane targeting. These results indicate that in addition to anchoring charged residues, Asp-155 plays a prominent role in membrane targeting of 5-HT2A receptors.
Materials and Methods
Site-Directed Mutagenesis and Plasmid Construction.
5-HT2A receptor mutants were constructed with the Quick-Change kit (Stratagene, San Diego, CA) with mutants expressed in pSVK3, as described previously (Choudhary et al., 1993, 1995; Roth et al., 1997b), and verified by dideoxy sequencing of the entire insert. Each mutant was then excised by EcoRI digestion, blunt-ended, gel purified, and subcloned into the eurkaryotic expression vector pIRESNEO with phosphorylated NotI linkers. The orientation of the insert was verified by restriction digestion and sequencing.
Cell Culture.
COS-7 cells were grown as previously detailed (Roth et al., 1997b) and transiently transfected with various receptor mutants with Fugene6 (Boehringer-Mannheim, Mannheim, Germany) in 100-mm dishes by scaling up the recommended procedure. Stably expressing cells lines were constructed in HEK-293 cells by transfecting with Fugene6 and selecting with 1 mg/ml G418-containing growth medium as previously detailed (Roth et al., 1995, 1997b).
Binding Assays.
Stably or transiently transfected cells were switched to serum-free medium for 24 h before harvest to remove serotonin and then harvested with a cell scraper as previously described (Roth et al., 1995; 1997b). Binding assays were performed with membrane preparations in a total volume of 0.5 ml with [3H]ketanserin or [3H]spiperone (for the D155E mutants) as the labeled ligand. For competition binding assays, 6 to 10 concentrations of unlabeled ligand spanning a range of 10,000-fold (typically 1–10,000 nM) were used. Agonist and antagonist competition binding assays were performed in a buffer of the following composition: 50 mM Tris-HCl, 10 mM Mg2+, 0.5 mM EDTA, 0.1% ascorbic acid, and 10 μM pargylline, pH 7.4 (Roth et al., 1995, 1997a). Typically, specific binding (defined by 1 μM spiperone) represented 90% of total binding with no more than 10% of the total counts bound. Data were analyzed with the LIGAND program (Munson and Rodbard, 1980) as previously detailed (Roth et al., 1995, 1997b) with differences in binding parameters analyzed with the F test. Protein was determined with the Bio-Rad procedure with BSA as standard.
Phosphoinositide Hydrolysis Assays.
For measurements of [3H]inositol monophosphate (IP) release, cells were loaded for 18 to 24 h with 1 μCi/ml [3H]inositol in serum-free and inositol-free medium as previously detailed (Roth et al., 1995, 1997b). Measurements of phosphoinositide (PI) hydrolysis were performed as previously detailed (Roth et al., 1995, 1997a).Kact andVmax values were determined with a nonlinear curve-fitting routine as previously described (Roth et al., 1995, 1997b).
Confocal Microscopy.
For investigation of receptor expression in COS-7 cells, cells were transfected in six-well plates with Fungene6 exactly as described by the manufacturer with native and mutant 5-HT2A receptors subcloned into pSVK3 together with pEGFP-N2 (Clontech Laboratories, Palo Alto, CA), which encodes for green fluorescent protein (GFP). GFP fluorescence was used as a control to assess transfection efficiency. At 24 h after transfection, cells were split into 24-well plates and grown on glass coverslips as previously described (Berry et al., 1996); 24 h later, the medium was changed to serum-free medium. After an overnight incubation, the medium was removed and cells were fixed and prepared for confocal microscopy as previously detailed with a polyclonal 5-HT2A receptor antibody (Berry et al., 1996). Microscopy was done in an identical manner with stable cell lines.
Molecular Modeling of 5-HT2A Receptors.
A model of the TM domain in the rat 5HT2A receptor was constructed by using computer graphics, molecular mechanics, and molecule dynamics. The MIDASPLUS computer program was used for computer graphics, and the AMBER 4.1 all atom force field was used for energy minimizations and molecular dynamics simulations. Models of TMI–VII with standard α-helical geometries (φ = −65° and ψ = −40°) were constructed. Each helix was capped by acetamide at its N terminus and N-methyl-amide at its C terminus. A Pro kink in TMVII that includes both a bend and a twisting of the exposed faces of the helical segments before and after the pro kink (Perlman et al., 1997) was introduced in the present model. Each of the seven helices was then energy minimized. The resultant energy minimized structures were assembled into a 7TM bundle according to the projection map of the frog rhodopsin structure (Baldwin et al., 1997). Information about interhelical interactions proposed according to data obtained in site-directed mutagenesis experiments with various GPCRs also was used in the model building.
The Gaussian94 programs (Gaussian94, revision D.4) were used for single point calculations of electrostatic potentials around 4-methyl-2,5-dimethoxyphenylisopropylamine (DOM),N,N-demethyl 5-HT, and gramine at the HF/6–31G* level. Atomic point charges were projected from these potentials by using the RESP program of the AMBER program package. Several different DOM/receptor complexes, oneN,N-demethyl 5-HT/receptor complex and one gramine/receptor complex were constructed by using interactive computer graphics. All ligand/receptor complexes were refined by energy minimization, followed by 100-ps molecular dynamics simulation in which position restraints on Cα-atoms were applied, and energy minimization. During the DOM/receptor simulations, distance restraints were applied to restrain specific DOM-receptor hydrogen bonds.
Results
Molecular Modeling Implies No Interaction between Asp-155 and Asn-363.
A new molecular model for agonist binding to the 5-HT2A receptor was constructed that incorporates the following features: 1) the most recent data in the form of an α-carbon template from Joyce Baldwin (Baldwin et al., 1997); 2) data derived from mutagenesis studies of the 5-HT2Areceptor (Choudhary et al., 1993, 1995; Roth et al., 1997b; Sealfon et al., 1995); and 3) cysteine substitution/accessibility studies of the closely related D2-dopamine receptor (Fu et al., 1996). The model implies that the prototypic agonist DOM binds via charge-charge (via Asp-155), aromatic-aromatic (via Trp-336, Phe-339, and Phe-340) and hydrogen bond-like interactions with Ser-159, Ser-239, and Asn-343 (Fig. 1A). In the model shown in Fig. 1B, intramolecular interactions also are highlighted with a focus on a highly conserved aspartic acid (Asp-155) in TMIII. As shown in Fig. 1B, the Asp-155 side chain interacts with Asn-343 in TMVI, and the Asn-363 side chain interacts with Thr-134 in TMII and Trp-151 in TMIII.
Because of recent competing models that imply distinct roles for intramolecular charge-charge interactions, we also examined the relative distances in our model between Asp-155 and Asn-363. As seen in Fig. 1B, Asp-155 and Asn-363, according to our model, are some distance from each other (distance between Cα-atoms is 10.9 Å) and not likely to interact. Our modeling results did, however, predict the following: 1) Asp-155 is probably involved via a polar interaction with agonists, 2) Asp-155-Asn-363 do not form a constraining salt-bridge in the 5-HT2A receptor, and 3) that Asp-155 interacts with other polar residues to facilitate helical-helical interactions.
We next modeled the interactions of two test compoundsN,N-dimethyl 5-HT and gramine, only one of which,N,N-dimethyl 5-HT, is predicted to interact with Asp-155. Gramine is not predicted to interact with Asp-155 because it is one carbon shorter than N,N-dimethyl 5-HT (Fig. 2). Figure 2 shows energy minimized ligand-receptor complexes after 100 ps of molecular dynamics simulation. The protonated amine group of the agonist forms a strong salt bridge with Asp-155 (TMIII) in theN,N-dimethyl 5-HT/receptor complex, but not in the gramine-receptor complex (N-O distances: 3.6 and 4.0 Å in the gramine/receptor complex and 2.7 and 2.9 Å in theN,N-dimethyl 5-HT/receptor complex). Interestingly, the model of the gramine/receptor complex predicts a weak hydrogen-bonding interaction between the protonated amine group of gramine and Ser-159 (TMIII). These modeling results predict that the terminal amine moiety, and not the indole nitrogen, ofN,N-dimethyl 5-HT and related indoles interacts with Asp-155.
Effect of Asp-155 and Asn-363 Mutations on Agonist-Mediated PI Hydrolysis.
We next examined the ability of various Asp-155 mutants and a single Asn-363 mutant to activate PI hydrolysis, to determine whether the predictions from the molecular modeling studies were correct. Two hypotheses were tested: 1) if the Asp-155 and Asn-363 loci are essential for stabilizing the receptor in an inactive conformation via an interhelical hydrogen bond, then disrupting this hydrogen bond should induce constitutive activity; and 2) if the Asp-155 and Asn-363 loci are essential for anchoring the terminal amine moieties of serotonergic agonists, then structurally modified ligands should activate the receptor in a predictable manner. To test the first hypothesis, we measured the basal PI hydrolysis in transiently and stably transfected cells expressing the various Asp-155 mutants.
As can be seen from Figs. 3 and4, stably transfected HEK-293 cells expressing the native 5-HT2A receptor had higher basal activities than cells expressing the D155A, D155E, D155N, D155Q (Fig. 3), or N363A (Fig. 4) mutants. In every instance, the basal PI hydrolysis was attenuated in cells expressing the D155X or N363A mutants, despite the fact that at least some of these mutants (D155E and N363A) were expressed at equivalent levels based on radioligand binding (Table 1 and Fig. 4). Because no constitutive activity was measured, the results do not support the hypothesis that a hydrogen bond between Asp-155 and Asn-363 stabilizes the receptor in an inactive conformation.
Asp-155 Anchors the Terminal Amine Moiety of Tryptamine Analogs.
We next examined two tryptamine analogs to identify the likely role of the Asp-155 locus in ligand recognition with stable cell lines for the native receptor and the D155E mutant. Several molecular models (Almaula et al., 1996) and some limited mutagenesis-based findings (Wang et al., 1993) have suggested that Asp-155 anchors the terminal amine of indoles to the 5-HT2Areceptor, although this assumption has not been previously tested. To test these models, we evaluated the abilitiesN,N′-dimethyltryptamine (DMT) and gramine to activate PI hydrolysis at the native and D155E mutant.
In initial studies, we examined the ability of various Asp-155 mutants to bind [3H]ketanserin and [3H]spiperone. As shown in Table 1, only the D155E mutant expressed [3H]spiperone, binding whereas the D155A, D155N, and D155Q mutants were all unable to specifically bind either of the radioligands tested. As is seen in Table 1, the D155E mutation decreased the affinity of the 5-HT2A receptor for a number of ligands. Our finding that the D155A, -N, and -Q mutants were all unable to bind any ligand, coupled with the discovery that the D155E mutant had decreased affinity for a number of 5-HT2A agonists and antagonists (Table 1), suggests that the charge as well as relative orientation of the carboxylate group is essential for high-affinity binding.
We reasoned that if the Asp-155 locus binds the terminal nitrogen of tryptamines, and not the N-1 nitrogen, than gramine should be more active at the D155E mutant than at the native receptor. As shown in Fig. 5 and Table2, gramine was active at D155E receptors and inactive at native receptors. In contrast,N,N′-DMT was much more active at native receptors compared with the D155E mutant. These results are in accord with our model that predicts that the terminal amine moiety of indole agonists interacts with Asp-155.
D155E Mutation Does Not Affect Receptor Expression But Does Affect Receptor Transport to Plasma Membrane.
It is conceivable that the D155A, -E, -N, and -Q mutants did not display constitutive activity because of problems with protein expression and/or transport to the cell surface. To investigate these possibilities, we initially examined the effects of the Asp-155 mutations on the surface expression of the native and mutant receptors in transiently transfected COS-7 cells by immunofluorescent confocal microscopy.
As is shown in Fig. 6E, the transfection efficiency of the native and D155A, -E, -N, and -Q mutants were similar compared with the transfection efficiency of GFP. These results imply that the D155A, -E, -N, and -Q mutations do not alter the ability of the receptor protein to be synthesized and expressed in COS-7 cells. Figure 6, A through D, shows that all constructs tested were expressed primarily intracellularly in COS-7 cells, as has been frequently seen when exogenous proteins are expressed in COS-7 cells.
To assess the ability of the mutant and native receptors to be expressed on the cell surface, we examined stably transfected HEK-293 cells (Fig. 7). As can be seen, the D155A and D155N mutations had no effect on targeting of the 5-HT2A receptor to the cell surface, whereas the D155E mutation resulted in an accumulation of receptors in an intracellular compartment. Dual-label studies with specific markers for the endoplasmic reticulum demonstrated that the D155E mutation accumulates in the endoplasmic reticulum (Fig.8).
Discussion
The major findings of this study are that the Asp-155 locus of the 5-HT2A receptor, which is conserved in all biogenic amine GPRCs, is 1) involved in membrane targeting, 2) anchoring the terminal amine of indole ligands, and 3) is not involved in a putative hydrogen bonding interaction with Asn-363 (Helix VII) to constrain the receptor in an inactive conformation. The second and third findings were based on three independent lines of investigation, all of which yielded equivalent conclusions: site-directed mutagenesis studies, molecular modeling results, and studies with indole analogs. Collectively, these results demonstrate that the Asp-155 locus is the likely anchoring site of the terminal amine of indoles that bind to 5-HT receptors. Additionally, our data demonstrate that this locus plays a previously unsuspected role in membrane targeting.
Prior studies with rhodopsin (Robinson et al., 1992) and α1b-adrenergic receptors (Porter et al., 1996;Porter and Perez, 1999) have suggested a common activation mechanism for both TMVII proteins. This activation mechanism involves, in part, the disruption of a strong interaction, called a salt-bridge, between residues in TMIII and TMVII. In rhodopsin, the putative interaction is between Glu-113 and Lys-296, whereas in the α1b-adrenergic receptor the interaction is between Asp-125 and Lys-331. The Lys-296-rhodopsin and Lys-331-α1b-loci are not precisely homologous, although it is conceivable that strong interactions between Lys-331 and Asp-125 in the α1b-adrenergic receptor exist. According to one current model, the Asp-125-Lys-331 interaction (or Glu-113-Lys-296 for rhodopsin) constrains the receptor in an inactive conformation (Porter et al., 1996; Porter and Perez, 1999). Disruption of this interaction by the insertion of the terminal amine of catecholamines in the α1b-adrenergic receptor relieves this constraint and allows for conformational changes that lead to receptor activation. Interestingly, constitutively active mutants can be constructed for both rhodopsin and α1b-adrenergic receptors in which one of the members of the salt-bridge is mutated to disrupt the interaction (Robinson et al., 1992; Porter et al., 1996; Porter and Perez, 1999).
The 5-HT2A receptor contains a homologous motif with an aspartic acid in TMIII (Asp-155) and an asparagine in TMVII (Asn-363). Molecular modeling studies, however, showed that Asp-155 and Asn-363 are not likely to be involved in an intramolecular interaction of the type that would constrain the 5-HT2Areceptor in an inactive conformation. In support of the modeling results, site-directed mutagenesis studies at the Asp-155 and Asn-363 locus yielded, in every case, receptors with lower levels of constitutive activity. These results support the hypothesis that the Asp-155 locus, at least for the 5-HT2A receptor, is not involved in receptor activation via a mechanism similar to that proposed for rhodopsin and α1b-adrenergic receptors.
A large number of molecular-modeling and site-directed mutagenesis studies with various 5-HT-family receptors have suggested that the Asp-155 locus is involved in anchoring amine-moieties of indole ligands (Ho et al., 1992; Branchek, 1993; Chanda et al., 1993; Choudhary et al., 1993, 1995; Wang et al., 1993; Kuipers et al., 1994; Albert et al., 1996; Almaula et al., 1996; Boess et al., 1998). None of these prior studies, however, was designed to answer which of the nitrogens on 5-HT and related ligands was involved in ligand recognition. It is possible, for instance, that the indole nitrogen (N-1) and not the terminal nitrogen is involved in anchoring indoles and related ligands.
To address this question, we evaluated reciprocal receptor and ligand “mutations”. In these experiments, we increased the length of the carboxylate side chain of aspartic acid by one carbon by making the D155E mutant. We then tested a 5-HT analog gramine, which is one carbon shorter than N,N′-DMT. We reasoned that if the terminal and not indole nitrogen was anchored by Asp-155 and D155E then gramine should have greater efficacy at the D155E receptor than at the native receptor and that N,N′-DMT should have lower efficacy at D155E compared with the native receptor. As predicted, we discovered that gramine had negligible efficacy at the native receptor, whereas gramine was a partial agonist at the D155E receptor Additionally, N,N′-DMT was less potent at the D155E compared with the native receptor.
Interestingly, the ability of gramine to activate the D155E receptor occurred despite the fact that the Asp-155E mutant was expressed predominantly intracellularly. It is likely that because of the high levels of expression (1–2 pmol/mg protein), even a small percentage of receptors that are expressed on the cell surface are sufficient to maximally activate PI hydrolysis. Thus, the small rim of 5-HT2A receptors expressed on the cell surface (Figs. 7 and 8) is sufficient for maximal activation of PI hydrolysis.
The final test was to examine molecular-modeling results. Our prior studies have implied that Phe-340 and Trp-336, in TMVI, are involved with anchoring 5-HT and other agonists (Choudhary et al., 1993, 1995). Additionally, recent findings (D. A. Shapiro, K. R. Kristiansen, W. K. Kroeze, and B. L. Roth, unpublished data) suggest that phenylalanine and serine residues in TMV also are involved in anchoring 5-HT and related ligands to the 5-HT2A receptor. Docking gramine and DMT to these residues allows only for a favorable interaction between the terminal amine of gramine and D155E and DMT and Asp-155 (Fig. 9). Collectively, all of these results are consistent with our current model that stipulates that the terminal amine moiety of 5-HT and related ligands interacts with the Asp-155 locus.
Novel Role of Conserved Charged Residue in Membrane Targeting Suggests an Inverse Relationship between Altered Membrane Targeting and Receptor Activation for Asp-155 Mutants.
We also found that Asp-155 plays an unpredicted role in membrane targeting. Prior studies of rhodopsin and other GPCRs have implicated residues located in the amino and carboxy terminus being involved in the membrane targeting of receptors (Rodriguez et al., 1992; Schulein et al., 1996a, 1998;Heymann and Subramanian, 1997). To our knowledge, no prior studies have implicated charged residues found in the TM helices in membrane targeting of GPCRs. It should be noted, however, that relatively few studies have addressed the membrane localization (or lack thereof) of mutant receptors, despite the fact that large numbers of studies have been done on these mutant receptors. It is also important to realize that using COS-7 cells alone would have led to the erroneous conclusion that none of the receptors was correctly targeted.
Prior studies examining the mechanisms by which GPCRs are targeting to plasma membranes have focused on intracellular and C terminus residues. Thus, Heymann and Subramanian (1997) showed that the C terminus was essential for appropriate membrane targeting of rhodopsin. Similar results were obtained by Rodriguez et al. (1992) for the luteinizing hormone receptor. Finally, recent studies by Schulein and colleagues (Schulein et al., 1996b, 1998) have suggested that a di-leucine motif in the C terminus is responsible for appropriate membrane targeting. To our knowledge, no prior studies have demonstrated a role for residues in TMIII (or for highly conserved charged residues) in membrane targeting.
How the D155E mutation affects membrane targeting is not clear, although it is likely that an alteration of intramolecular hydrogen-bonding interactions (e.g., with Asn-343 in TMVI; Fig. 1A) occurs. How this affects membrane targeting is unknown, although it is unlikely that it is simply due to “misfolding” of the receptor. If misfolding occurred, we would have predicted that the D155E receptor would be unable to bind radioligands and activate second messenger production. In fact, the D155E mutation was the only one of a series of four mutations at the Asp-155 locus that resulted in a functional receptor. Interestingly, even though the other Asp-155 mutants tested (D155A and D155N) were nonfunctional, they were correctly transported to the plasma membrane. These results imply that the ability of D155X mutants to bind ligand and induce receptor activation affects membrane targeting. Thus, the D155A, -N, and -Q mutants, which were all targeted correctly in HEK-293 cells, were all unable to bind radioligand with high affinity and be activated. By contrast, the D155E mutant bound radioligands such as spiperone with high affinity and was able to be activated by 5-HT, DMT, gramine, and other agonists. These results imply a reciprocal role between receptor activation and membrane targeting for mutants at the Asp-155 locus. A systematic study of the effects of other TM helix mutations on plasma membrane targeting would provide us with clues regarding the other rules that govern 5-HT2A receptor targeting in particular and GPCR targeting in general. To our knowledge, no prior studies with GPCR mutants have suggested this novel role of conserved TM domain residues in membrane targeting.
In conclusion, we have demonstrated by a combination of approaches (site-directed mutagenesis, reciprocal ligand alterations, and molecular modeling) that the terminal amine moiety of 5-HT and related ligands is anchored via Asp-155. Additionally, our studies suggest that a disruption of a strong association between a conserved motif in TMIII and TMVII is not a general mechanism of GPCR activation. Finally, our studies demonstrate that the D155E mutation has an unexpected and important role for membrane targeting of the 5-HT2A receptor. Because few prior studies have examined membrane targeting of GPCR mutants, it is likely that a systematic study of the role(s) TM residues have for membrane targeting will illuminate the processes involved in receptor assembly.
Footnotes
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Send reprint requests to: Bryan L. Roth, M.D., Ph.D., Department of Biochemistry; Room W438, Case Western Reserve University Medical School, 10900 Euclid Ave., Cleveland, OH 44106-4935. E-mail:roth{at}biocserver.cwru.edu
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↵1 This study was supported in part by National Institutes of Health Grants RO1MH57635, Research Scientist Development Award KO2MH01366, a gift from the Heffter Research Foundation, and a National Alliance for Research on Schizophrenia and Depression Independent Investigator Award (to B.L.R.). D.L.W. was supported in part by a National Alliance for Research on Schizophrenia and Depression Young Investigator Award.
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↵2 Current address: Institute of Molecular Pharmacology, Molecular Modeling Group, Alfred Kowalke Str. 4, D-10315 Berlin, Germany.
- Abbreviations:
- GPCR
- G-protein-coupled receptor
- TM
- transmembrane domain
- GnRH
- gonadotropin-releasing hormone receptor
- 5-HT
- 5-hydroxytryptamine
- IP
- inositol monophosphate
- PI
- phosphoinositide
- GFP
- green fluorescent protein
- DOM
- 4-methyl-2,5-dimethyoxyphenylisopropylamine
- DMT
- dimethyltryptamine
- Received December 3, 1999.
- Accepted March 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics