Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

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 _1b-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 ₂-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-HT_2A 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-HT_2A receptors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Site-Directed Mutagenesis and Plasmid Construction. 5-HT_2A 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). K_act and V_max 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-HT_2A 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-HT_2A receptor antibody (Berry et al., 1996). Microscopy was done in an identical manner with stable cell lines.

Molecular Modeling of 5-HT_2A Receptors. A model of the TM domain in the rat 5HT_2A 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 -helical geometries ( = 65° and  = 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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Molecular Modeling Implies No Interaction between Asp-155 and Asn-363. A new molecular model for agonist binding to the 5-HT_2A receptor was constructed that incorporates the following features: 1) the most recent data in the form of an -carbon template from Joyce Baldwin (Baldwin et al., 1997); 2) data derived from mutagenesis studies of the 5-HT_2A receptor (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.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Molecular-modeling studies of the 5-HT2A receptor reveal that Asp-155 (TMIII) and Asn-363 (TMVII) do not interact. View of the C-chains of TM helices, DOM, and aromatic and polar residues within a 3.5-Å sphere radius of the DOM molecule (top) in an energy minimized complex after 100-ps molecular dynamic simulation, viewed from the extracellular side. Bottom, Asp-155 (TMIII) interacts strongly with both the protonated amine group of DOM and Asn-343 (TMVI), whereas Asn-366 (TMVII) interacts strongly with Trp-151 (TMIII) and Thr-134 (TMII). Color coding of ligand atoms: white, C; red, O; green, N; and cyan, H.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Molecular modeling reveals that indole-ligands interact via the terminal and not indole amine moiety. View of the C-chains of TM helices, the ligand and aromatic and polar residues within a 3.5-Å sphere radius of the ligand molecule in energy-minimized complexes after 100-ps molecular dynamics simulations, viewed from the extracellular side. Top, model of the gramine/receptor complex. Bottom, model of the N,N-dimethyl 5-HT/receptor complex. Color coding of the receptor: cyan, Asp-155 (TMIII); green, Trp-336, Phe-339, Phe-340 (TMVI), and Phe-365 (TMVII); and orange, Ser-159, Thr-160 (TMIII), Ser-207 (TMIV), Ser-239 (TMV), and Asn-343 (TMVI). Color coding of ligand atoms: white, C; red, O; green, N; and cyan, H.

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.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Mutations at the D155X locus result in lower constitutive activity. Shown are the mean ± S.E. for n = 4 separate determinations in which basal PI hydrolysis was measured as described in Materials and Methods for D155A, -E, -N, and -Q, and native 5-HT2A receptors. *P < .01 versus native.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   The Asn-363A receptor does not show constitutive activity. Shown are dose-response studies for 5-HT at native and N363A receptors. Data represent mean ± S.E. for triplicate determinations of a typical experiment that has been replicated three times. For the native receptor, the Kact and Vmax values were 31 ± 5 nM and 3377 ± 119 cpm, whereas for the N363A receptor, the following Kact and Vmax values were obtained: 460 ± 38 nM and 1271 ± 79 cpm (both significantly lower at the P < .01 level than native receptors). The max for the N128A receptor was 1.4 ± 0.3 pmol/mg, which was not different from the max for the native receptor (1.8 ± 0.6 pmol/mg).

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-HT_2A receptor, although this assumption has not been previously tested. To test these models, we evaluated the abilities N,N'-dimethyltryptamine (DMT) and gramine to activate PI hydrolysis at the native and D155E mutant.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Gramine selectively activates the D155E receptor. Shown are dose-response studies for gramine and DMT at D155E (top) and native (bottom) receptors stably expressed in HEK-293 cells. Data represent mean ± S.E. for triplicate determinations of a typical experiment that was replicated three times.

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.

View larger version (73K): [in this window] [in a new window]   Fig. 6.   The D155X mutants are expressed at equivalent levels in COS-7 cells. Shown are representative immunofluorescent confocal micrographs in which native (A), D155A (B), D155E (C), and D155Q (D) receptors were transiently expressed in COS-7 cells and then fixed for immunofluorescent analysis. E shows the mean transfection ratio ± S.E. of the native and D155X mutant 5-HT2A receptors. For the experiments in E, COS-7 cells were transiently cotransfected with equivalent amounts of D155A, -E, -N, and -Q or native receptors and a eukaryotic expression vector for GFP (pEGFP). At 48 h after transfection, cells were switched to regular medium containing 5% dialyzed serum (to remove serotonin) and 18 h later were fixed and prepared for confocal microscopy as previously detailed (Berry et al., 1996). Cells were photographed at 400× with rhodamine and fluoresceine filters for Texas Red-labeled 5-HT2A receptor and GFP fluorescence, respectively. The total number of fluorescent cells/field was then counted in the red and green channels separately. Transfection ratio was calculated as follows:

View larger version (101K): [in this window] [in a new window]   Fig. 7.   The D155E mutant is selectively impaired in plasma membrane targeting. A through D show representative confocal micrographs for native 5-HT2A receptors (A), D155A receptors (B), D155N receptors (C), and D155E receptors (D) stably expressed in HEK-293 cells. For all constructs with the exception of the D155E mutant, plasma membrane immunofluorescence was prominent (large arrows). For the D155E mutant, the majority of 5-HT2A-like immunofluorescence was intracellular (small arrows).

View larger version (30K): [in this window] [in a new window]   Fig. 8.   D155E receptors are trapped in the endoplasmic reticulum. Figure 4 shows representative dual-label confocal micrographs in which 5-HT2A-like immunoreactivity (red channel) and endoplasmic reticulum-like immunoreactivity (green channel) were simultaneously measured in stably transfected HEK-293 cells. The left-hand column shows results for native receptors, whereas the right-hand column shows results with the D155E mutant. A small rim of plasma membrane 5-HT2A-like immunoreactivity is seen for the D155E mutant (large arrows).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The major findings of this study are that the Asp-155 locus of the 5-HT_2A 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 _1b-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 _1b-adrenergic receptor the interaction is between Asp-125 and Lys-331. The Lys-296-rhodopsin and Lys-331-_1b-loci are not precisely homologous, although it is conceivable that strong interactions between Lys-331 and Asp-125 in the _1b-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 _1b-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 _1b-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-HT_2A 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-HT_2A receptor 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-HT_2A receptor, is not involved in receptor activation via a mechanism similar to that proposed for rhodopsin and _1b-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-HT_2A 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-HT_2A 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.

View larger version (69K): [in this window] [in a new window]   Fig. 9.   Molecular modeling predicts that gramine but not DMT will successfully interact with the D155E mutant receptor. Top, results of docking studies in which DMT was docked to the native 5-HT2A receptor. A strong interaction with Asp-155 in TMIII and aromatic residues in TMVI (Trp-336, Phe-340) are predicted. Middle, mutating Asp-155  Glu-155 (shown in red) results in hindrance of DMT binding. Bottom, results of docking studies in which gramine, which is one carbon shorter than DMT, successfully interacts with Glu-155, leading to a favorable orientation to interact with aromatic residues in helix VI to cause receptor activation.

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.