Abbreviations used : HA, hemagglutinin ; 5-HT, serotonin.
Structure—Function Studies of the Eighth Hydrophobic Domain of a Serotonin Receptor
Article first published online: 18 JAN 2002
Journal of Neurochemistry
Volume 72, Issue 1, pages 413–421, January 1999
How to Cite
Hurley, J. H., Bloem, L. J., Pavalko, F., Liu, J., Tian, M., Simon, J. R. and Yu, L. (1999), Structure—Function Studies of the Eighth Hydrophobic Domain of a Serotonin Receptor. Journal of Neurochemistry, 72: 413–421. doi: 10.1046/j.1471-4159.1999.0720413.x
- Issue published online: 18 JAN 2002
- Article first published online: 18 JAN 2002
- α helix;
- G protein-coupled receptor;
- Hydrophobic domain;
Abstract : The most prominent structural feature of the G protein-coupled receptor superfamily is their seven hydrophobic domains, which are postulated to form membrane-spanning α helices. Some members of the G protein-coupled receptor family, specifically several serotonin (5-HT) receptors, possess eight hydrophobic domains. The importance of this extra hydrophobic domain, located at the N terminus of the receptor, is unknown. This question was addressed by deleting the extra hydrophobic region from the 5-HT2C receptor and comparing its function and topology with those of the wild-type receptor. Immunofluorescence microscopy was used to determine the location of the N terminus of the epitope-tagged wild-type and mutant receptors. The N terminus of both receptors was extracellular, suggesting that the extra hydrophobic domain does not change the topology of this receptor and is unlikely to be a membrane-spanning α helix. Radioligand-binding studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hydrophobic domains were sufficient for normal function in these assays. Interestingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild-type receptor containing eight hydrophobic domains, suggesting that the extra hydrophobic domain does impact the activity of this receptor by regulating its expression.
The members of the superfamily of G protein-coupled receptors share several structural and functional features, the most prominent being the presence of seven hydrophobic domains, postulated to form transmembrane α helices (Lefkowitz and Caron, 1988 ; O'Dowd et al., 1989 ; Raymond et al., 1990). This hypothesis was based largely on the known structure of bacteriorhodopsin (Unwin and Henderson, 1975 ; Ovchinnikov et al., 1979 ; Engelman et al., 1980 ; Pebay-Peyroula et al., 1997), an integral membrane protein that also contains seven hydrophobic regions. Structural and biochemical evidence from members of the G protein-coupled receptor family provides strong support for a seven-transmembrane domain structure, with the amino terminus located extracellularly and the carboxyl terminus in the cytoplasm (Dixon et al., 1987a,b ; Dohlman et al., 1987 ; Frielle et al., 1988). Although it is believed that most members of the G protein-coupled receptor family possess similar topologies, the actual prevalence of the seven-transmembrane domain structure within this receptor family is unknown.
The serotonin (5-HT) receptor family is the largest (over 30 receptors have been cloned) and the oldest biogenic amine receptor family. The primordial 5-HT receptor is estimated to be over 750 million years old (Peroutka and Howell, 1994). Several of its members possess an unusual feature : an eighth hydrophobic domain. Based on sequence alignment of this receptor with other G protein-coupled receptors, the hydrophobic domain at the N terminus is the “extra” hydrophobic domain (Fig. 1). This extra hydrophobic domain (domain E) is seen in the mouse (Lübbert et al., 1987 ; Yu et al., 1991), rat (Julius et al., 1988), and human (Saltzman et al., 1991) homologues of the 5-HT2C receptor, two Drosophila 5-HT receptors (Witz et al., 1990 ; Saudou et al., 1992), and the rat and mouse 5-HT7 receptor (Meyerhof et al., 1993 ; Plassat et al., 1993 ; Ruat et al., 1993). In contrast, domain E is not seen in the other 5-HT receptors, including the 5-HT2A receptor (Pritchett et al., 1988) (see Fig. 1), which is most closely related to the 5-HT2C receptor in both protein sequence and cellular function. Neither is the extra hydrophobic region (domain E) present in the human β2-adrenergic receptor (Kobilka et al., 1987), a prototypic G protein-coupled receptor, or in bacteriorhodopsin, the first protein determined to have seven membrane-spanning α helices (Unwin and Henderson, 1975 ; Ovchinnikov et al., 1979 ; Engelman et al., 1980). Nor has it been described in any other G protein-coupled receptors cloned to date.
Computer-assisted analysis of the amino acid sequence of the 5-HT2C receptor by both the Eisenberg method (Eisenberg et al., 1984) and the Kyte and Doolittle algorithm (Kyte and Doolittle, 1982) predicts that this protein has eight hydrophobic regions capable of forming α-helical structures and traversing the lipid bilayer. Because the minimum number of amino acid residues in an α helix thought to be sufficient to cross the plasma membrane is 18-20, domain E with 21 amino acid residues (Table 1) is theoretically capable of spanning the lipid bilayer. In addition, as shown in Table 1, the average hydrophobic value for domain E is higher than that for the putative transmembrane domains III and VII but lower than that calculated for the other hydrophobic domains. Based on these considerations, it is possible that domain E may constitute a membrane-spanning α helix.
If domain E were to form a transmembrane domain, then it would predict a change in the topology of the 5-HT2C receptor compared with other G protein-coupled receptors. Two hypothetical models are depicted in Fig. 2. If domain E crosses the plasma membrane (Fig. 2A), then the amino terminus of the wild-type 5-HT2C receptor will be intracellular in contrast to the extracellular location of the N terminus of the G protein-coupled receptors characterized to date (O'Dowd et al., 1989). If domain E does not cross the plasma membrane (Fig. 2B), then the amino terminus of the wild-type 5-HT2C receptor will be extracellular, as is predicted for most G protein-coupled receptors. In the models in Fig. 2, we make the assumption that the seven transmembrane domains I-VII, the G protein loop, and the C terminus are arranged as described for other receptors (O'Dowd et al., 1989).
There are a number of possibilities for the functional significance of the extra hydrophobic domain in the 5-HT2C receptor. Domain E could be involved in ligand binding as the ligands for catecholamine receptors are known to bind within the hydrophobic domains (Strader et al., 1987, 1988). Another possibility is that domain E may have a role in G protein coupling. This region may be directly or indirectly involved in the conformational changes that occur after ligand binding and that are responsible for G protein activation. To address these questions, we studied the topology and function of the mouse 5-HT2C receptor and a mutant 5-HT2C receptor lacking domain E. Here we report the results.
MATERIALS AND METHODS
Construction of Del E mutant receptor
The cDNA clone of the mouse 5-HT2C receptor has been previously described (Yu et al., 1991). The Del E mutant was created using the full-length clone of the 5-HT2C cDNA in the pGEM-7 (Promega) vector and oligonucleotide-directed mutagenesis. This method was used to delete a 72-bp cDNA fragment encoding amino acids 11-34 of the 5-HT2C receptor (see Fig. 1, top). A 36-bp mutagenic oligonucleotide (5′ GGC ACT GCG GTG CGC TCA ACT GAC ACT TTT AAT TCC 3′) corresponding to 18 bases on either side of the region to delete was used in the method of Olsen and Eckstein (1990) with the Amersham mutagenesis kit. The mutagenesis product was confirmed with DNA sequencing.
Addition of epitope tag
A highly antigenic nine-amino acid sequence (YPYDVP-DYA) from the hemagglutinin (HA) protein of the human influenza virus (Wilson et al., 1984), the HA epitope, was added to the N terminus of both the wild-type and the Del E mutant receptors immediately after the initial methionine. The sequence encoding the initial methionine and the HA epitope tag was subcloned into the KpnI and XhoI sites in the polylinker of pcDNA3 (referred to hereafter as pcHA). Two oligonucleotides were designed that could amplify both receptor cDNAs and add restriction sites for subcloning. The oligonucleotide, Mou5HT2C-1 (5′ CCG CTC GAC GTG AAC CTG GGC ACT GCG GTG 3′) anneals at the 5′ end of the cDNA at the second codon (resulting in the deletion of the original methionine) and contains an XhoI site (underlined). The oligonucleotide, Mou-5HT2C-2 (5′ CCG GGG CCC TTA CAC ACT ACT AAT CCT CTC 3′), anneals to the stop codon and contains an ApaI site (underlined). Polymerase chain reaction products were isolated and digested with ApaI and XhoI. These fragments were cloned into pcHA that had been digested with ApaI and XhoI. The resulting plasmids were verified by sequencing and are referred to as HA-5-HT2C for the epitope-tagged wild-type 5-HT2C receptor and HA-Del E for the epitope-tagged Del E receptor, respectively.
Xenopus oocyte expression and electrophysiology
Receptor proteins were expressed in Xenopus laevis oocytes after injection of in vitro transcribed RNA. Transcripts were synthesized from the T7 RNA polymerase promoter using an in vitro transcription kit (Ambion). Oocytes were harvested from mature female X. laevis frogs and separated from follicle cells with collagenase using standard protocols. Oocytes were injected with 1 ng of RNA in 50 nl of H2O with a Drummond automatic microinjector. Three days after injection, oocytes were voltage-clamped at -60 mV with two glass electrodes (filled with 3 M KCl and having a resistance of 0.5-3 MΩ) using an Axoclamp 2A (Axon Instruments). Oocytes were superfused with ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 5 mM HEPES, pH 7.5) containing the desired concentration of serotonin. Membrane currents were recorded on a Gould chart recorder and on computer using pCLAMP software (Axon Instruments).
Cell culture and transfection
For ligand-binding studies, the cDNA clones for the 5-HT2C and Del E receptors were subcloned into a mammalian expression vector, SVpoly. For immunofluorescence, the epitopetagged clones, HA-5-HT2C and HA-Del E, in pcDNA3 were used. COS-7 (ATCC) and AV12 cells were used for transient transfections. All cells were grown in Dulbecco's modified Eagle's medium and 10% fetal calf serum supplemented with gentamicin and Fungizone. Transfections were done with a modified (Ca)3(PO4)2 protocol (Chen and Okayama, 1987). This procedure uses an N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid buffer at low pH (6.95) and allows the DNA-(Ca)3(PO4)2 precipitate to form gradually in the medium in an incubator with lowered CO2 levels (3%). Cells were harvested 2-3 days after transfection.
Membrane preparation and radioligand binding
Membrane proteins were prepared as previously described (Fargin et al., 1988). In brief, cells were lysed in hypotonic buffer (20 mM Tris-Cl, pH 7.4, 5 mM EDTA) by Dounce homogenization. Unlysed cells and nuclear debris were removed by low-speed centrifugation (3,000 g), and the supernatant was centrifuged at 30,000 g. Proteolytic degradation was minimized by keeping all samples and solutions on ice and by the addition of a protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 μg/μl leupeptin, 1 μg/μl antipain, 1 μg/μl aprotinin, 1 μg/μl pepstatin). The pelleted membrane proteins were resuspended in binding buffer (50 mM Tris-Cl, pH 7.4, 5 mM EDTA) with the addition of 10 μM pargyline and 1 mg/ml ascorbate for agonist-binding studies. The radioligand for binding studies was [3H]mesulergine (77-84 Ci/mmol ; Amersham), a serotonergic antagonist. Saturation binding of [3H]mesulergine (0.1-5 nM) was done with 5 μM mianserin to define nonspecific binding. For inhibition binding experiments, 1 nM [3H]mesulergine was used with varying concentrations of unlabeled competitors. Each concentration was assayed in triplicate. Binding reactions were incubated at 30°C for 1 h, and the bound and free ligand were separated by vacuum filtration onto GF/B filters presoaked in 3% polyethylenimine using a Brandel cell harvester. Filters were rapidly washed three times with 5 ml of cold 50 mM Tris-Cl (pH 7.4). The filters were counted by liquid scintillation. Computer-assisted nonlinear regression analysis was used to determine the binding parameters.
AV12 cells were plated onto coverslips at low density and transiently transfected with the HA-5-HT2C or the HA-Del E plasmid as described above. Three days later, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 20 min on ice. Some coverslips were permeabilized with 0.2% Triton X-100 in Tris-buffered saline for 2 min at room temperature and rinsed in fresh Tris-buffered saline for 5 min before staining. The cells were incubated with either the 12CA5 monoclonal antibody for the HA epitope tag at 20 μg/ml (Boehringer-Mannheim) or with a rabbit polyclonal antiserum against α-actinin (diluted 1 : 20) (Pavalko and LaRoche, 1993) for 60 min at 4°C. The coverslips were washed extensively (three times for 30 min each) and stained with secondary antibody, anti-mouse IgG coupled to fluorescein (Jackson Immunoresearch Laboratories) or anti-rabbit IgG coupled to rhodamine (Jackson Immunoresearch Laboratories) for HA or α-actinin visualization, respectively. The coverslips were washed as above and fixed to microscope slides with Permount. The slides were examined with a Nikon microscope equipped with epifluorescence optics.
Effect of domain E on topology of 5-HT2C receptor
To characterize the topological arrangement of domain E of the 5-HT2C receptor, we sought to localize the amino terminus. We first generated a mutant receptor lacking the 24-amino acid domain E (Fig. 1). This mutant receptor was designated “Del E” receptor. Then, the HA epitope tag was added to the amino terminus of both the wild-type 5-HT2C and the Del E receptors. The addition of the HA epitope tag did not affect either the receptor's ligand-binding characteristics or its ability to activate G proteins (data not shown). Epitope tagging has been used extensively to determine the location of a particular protein or a protein domain and to study protein trafficking during synthesis or internalization (von Zastrow and Kobilka, 1992 ; Chen et al., 1993 ; von Zastrow et al., 1993). If domain E traverses the plasma membrane, then the N terminus should be located on the intracellular side and removing domain E should switch the location of the N terminus to the extracellular side. If domain E does not cross the membrane, then its deletion will have no effect on the location of the N terminus.
Immunofluorescence was utilized to localize the epitope-tagged N terminus of the 5-HT2C and Del E receptors, using cultured cells transiently transfected with either the HA—5-HT2C or the HA—Del E receptor. Figure 3 shows the results of a representative experiment. Binding of the anti-HA antibody was performed both in nonpermeabilized cells and in Triton X-100-permeabilized cells. In addition, as a negative control to demonstrate that antibodies do not have access to intracellular proteins in nonpermeabilized cells, antibody binding to a known intracellular antigen, α-actinin, was performed in permeabilized and nonpermeabilized cells. The anti-HA antibody reacts strongly in both the permeabilized and the nonpermeabilized cells transfected with the HA—5-HT2C receptor (Fig. 3a and c), suggesting that the N terminus of this receptor is extracellular. In addition, a marked staining around the cell periphery indicates a plasma membrane localization of the receptor. The binding of α-actinin antibody is strong in all permeabilized cells (Fig. 3b and f) but almost completely absent in nonpermeabilized cells (Fig. 3d and h), demonstrating that antibodies did not have access to intracellular antigens in nonpermeabilized cells. The binding of the anti-HA antibody to cells transfected with the HA-Del E receptor showed a pattern similar to that of the HA—5-HT2C receptor. Both the permeabilized and the nonpermeabilized cells are labeled with the anti-HA antibody (Fig. 3e and g), indicating that the N terminus of both receptors is also extracellular. Based on these results, we conclude that deletion of domain E did not reorient the N terminus, suggesting that in the wild-type 5-HT2C receptor, domain E does not form a membrane-spanning domain.
Effect of domain E on Cl- currents in Xenopus oocytes
To explore the potential functional effect of domain E, we examined the Del E receptor's ability to activate a G protein-mediated signal transduction pathway. In Xenopus oocytes, stimulation of phospholipase C-coupled receptors activates an endogenous Ca2+ -dependent Cl- channel (Gundersen et al., 1983 ; Dascal et al., 1987). The mutant receptor Del E was tested for its ability to activate the Ca2+ -dependent Cl- current in Xenopus oocytes. In vitro transcribed RNA for the 5-HT2C receptor or the Del E receptor was injected into mature oocytes. After 3 days, oocytes were stimulated with a single concentration of 5-HT and the Cl- current was measured. In RNA-injected oocytes, 5-HT evoked a transient depolarizing Cl- current both in oocytes expressing the 5-HT2C receptor and in those expressing the Del E receptor (Fig. 4A and B). Both receptors were capable of activating the Cl- current in a concentration-dependent manner (Fig. 4C) with EC50 values that were similar for the 5-HT2C (96 nM, 95% confidence interval 42-219 nM) and the Del E (46 nM, 95% confidence interval 21-92 nM) receptors. In addition, the maximal responses did not differ for the 5-HT2C (2,319 nA, 95% confidence interval 1,861-2,778 nA) and the Del E (1,700 nA, 95% confidence interval 1,251-2,149 nA) receptors. These data suggest that the structural core of the receptor for activating the G protein-mediated signaling pathway is preserved in the Del E receptor.
Effect of domain E on ligand-binding affinities and on receptor expression
Since both the wild-type 5-HT2C and the Del E receptors could elicit Cl- currents in Xenopus oocytes, we sought to characterize the binding affinities of these receptors in a more quantitative manner. Saturation binding with [3H]mesulergine, a high-affinity 5-HT antagonist (Pazos et al., 1984), was carried out on membrane proteins from COS-7 cells that were transiently transfected with either the 5-HT2C or the Del E receptor clone. Both receptors displayed specific and saturable binding of mesulergine with almost identical dissociation constants (Fig. 5). The KD values for mesulergine derived from five experiments were 0.57 ± 0.12 nM for the 5-HT2C receptor and 0.64 ± 0.16 nM for the Del E receptor.
We also tested the ability of several compounds to compete for [3H]mesulergine binding to the two receptors. The apparent inhibition constants (Ki) for three antagonists (mesulergine, mianserin, and LY53857) and two agonists (5-HT and m-chlorophenylpiperazine) were not different for the 5-HT2C and Del E receptors (Table 2). These values are very comparable with other published results for both the antagonists and the agonists (Hoyer et al., 1985 ; Hoyer, 1988). The lack of noticeable differences in ligand-binding affinities suggests that the structural core of the receptor for ligand binding is preserved in the Del E receptor.
|Mesulergine||0.63 ± 0.12||0.55 ± 0.13|
|Mianserin||1.07 ± 0.24||1.11 ± 0.34|
|LY53857||1.09 ± 0.23||0.99 ± 0.22|
|5-HT||127 ± 24||158 ± 24|
|m-CPP||289 ± 120||276 ± 128|
Although no binding affinity differences were evident between the two receptors, one differences became apparent during the course of our studies : The Del E receptor was consistently expressed at a higher level than the 5-HT2C receptor in transfected cells. Receptor expression is defined here as the maximum number of binding sites in the membrane fraction (Bmax/mg of protein). Because the efficiency of transient transfections is variable from one experiment to the next, we always conducted parallel transfections with the two receptors in each experiment, thus providing a basis for correlating their relative expression. In 12 of 13 experiments where the wild-type 5-HT2C and Del E receptors were transfected side by side, the Del E receptor was expressed at about twofold higher levels, and across the multiple experiments, a good correlation is seen for the twofold higher expression of the Del E receptor (Fig. 6). When the ratio of Del E to 5-HT2C expression was calculated for each experiment (Fig. 6, inset), the average ratio for the 13 experiments was 1.92 ± 0.19, which is significantly different from 1 (p < 0.01). A ratio of 1 would be expected if the two receptors were processed and inserted into the membrane at comparable levels. In addition, based on observation of the immunofluorescence studies with AV12 cells, it appears that approximately equal numbers of cells were transfected with each receptor. Thus, these data suggest that the extra hydrophobic domain E may impact the activity of this receptor by regulating its expression level.
The small group of 5-HT receptors that have eight hydrophobic domains are an unusual variation of the more common seven-hydrophobic domain motif. The amino acid sequences of the extra hydrophobic domain in the mouse (Yu et al., 1991), rat (Julius et al., 1988), and human (Saltzman et al., 1991) 5-HT2C receptors are almost identical, which is not surprising considering the high overall homology these receptors share. In contrast, there is little homology between the extra hydrophobic domains of the mammalian and Drosophila receptors, between the 5-HT7 and 5-HT2C receptors, or even between the Drol and Dro2A receptors. The presence of this unusual feature in several distantly related serotonin receptors raises questions about its origin and functional significance. Furthermore, a naturally occurring receptor variant that has more than seven hydrophobic domains provides an opportunity to examine the “seven-transmembrane domain” rule, so as to obtain clues about the structure—function relationship of this motif in G protein-coupled receptors. To undertake such a study, we chose the mouse 5-HT2C receptor. Our approach was to remove the “extra” eighth hydrophobic domain in the 5-HT2C receptor to create the Del E receptor and to compare it with the wild-type 5-HT2C receptor that has eight hydrophobic domains.
Our first goal was to determine the location of the N terminus of the 5-HT2C receptor and whether or not domain E crossed the lipid bilayer. The results of our immunofluorescence study indicate that domain E does not traverse the plasma membrane and that the N terminus of the mouse 5-HT2C receptor is extracellular (Fig. 3). Additional evidence that the N terminus of the 5-HT2C receptor is extracellular comes from a study describing the production and characterization of polyclonal antiserum to the N terminus of the human 5-HT2C receptor (Sharma et al., 1997). HEK cells transfected with the human 5-HT2C receptor cDNA exhibited strong cell surface staining with this antiserum. Confirmation of this topology in native cells expressing the 5-HT2C receptor is still necessary, but it seems likely that the 5-HT2C receptor retains the seven-transmembrane motif despite the additional hydrophobic domain and that domain E does not traverse the lipid bilayer (Fig. 2B).
It has been hypothesized that this hydrophobic domain could be a signal peptide that is cleaved during processing (Abramowski and Staufenbiel, 1995). The data presented here as well as the results using the N terminal-directed antisera described above (Sharma et al., 1997) do not support this conclusion. If domain E is part of a signal sequence that is cleaved off, our epitope tag antibody would not detect cell surface staining of the 5-HT2C receptor. Moreover, we had previously created an additional receptor mutant (Hurley, 1995) with a deletion of the putative transmembrane domains I—VII and their connecting loops ; the resulting “receptor” (▵I—VII) contains only the epitope tag, N terminus, domain E, the loop between domain E and transmembrane domain I, and the C terminus of the 5-HT2C receptor. In vitro transcription and translation of ▵I—VII were done with and without the addition of microsomal membranes. The ▵I—VII protein was ~16 kDa (as predicted) in the absence and presence of microsomal membranes, suggesting that this protein was neither cleaved nor glycosylated at Asn39.
The effect of domain E on the ligand-binding properties of the 5-HT2C receptor was studied by comparing the 5-HT2C and Del E receptors. Our radioligand-binding studies did not detect any differences in the binding affinities of the agonists and the antagonists we tested (Fig. 5 ; Table 2), demonstrating that the structural core of the 5-HT2C receptor for ligand binding is preserved in the Del E receptor.
The potential effect of domain E on G protein-mediated signaling was examined by expressing the 5-HT2C and Del E receptors in Xenopus oocytes and testing their ability to activate the Ca2+ -dependent Cl- current via stimulation of phospholipase C. Our data demonstrate that domain E is not required for G protein-mediated activation of the phospholipase C pathway in oocytes. These data do not rule out the possibility of modulatory effects of domain E on this pathway, because small differences in activation of the phospholipase C pathway may not be detectable in the Xenopus oocyte expression system. The 5-HT2C receptors have been classically described as signaling through the phospholipase C pathway (Conn et al., 1986 ; Wolf and Schutz, 1997) ; however, several reports indicate that the 5-HT2C receptor can activate additional signal transduction pathways in vivo (Kaufman et al., 1995) as well as in heterologous expression systems (Backstrom and Sanders-Bush, 1997 ; for review, see Gerhardt and van Heerikhuizen, 1997). Although domain E is not necessary for activation of phospholipase C in oocytes, it will be of interest to determine whether domain E has any impact on other signal transduction pathways involving the 5-HT2C receptor.
A noteworthy observation made during the course of our studies is that the Del E receptor is expressed at about twice the level of the 5-HT2C receptor. In this regard, it is interesting to note that there are reports of low expression levels of other 5-HT receptors with eight hydrophobic domains (Witz et al., 1990 ; Saudou et al., 1992). For the two Drosophila serotonin receptors that contain an extra hydrophobic domain in their amino terminus, the amino terminus was truncated in an effort to obtain higher expression levels (Witz et al., 1990 ; Saudou et al., 1992). These observations do not necessarily imply a functional role of domain E in modulation of receptor expression, but it is interesting to speculate that this region may have a role in receptor regulation or localization.
The 5-HT2C receptors exhibit some unusual characteristics such as agonist-independent activation and/or atypical down-regulation caused by agonists as well as inverse agonists (Barker et al., 1994 ; Westphal and Sanders-Bush, 1994 ; Labrecque et al., 1995), RNA editing (Burns et al., 1997), and alternatively spliced mRNAs that code for truncated receptors (Canton et al., 1996), for which the mechanisms and/or function have not been completely described. One plausible, though yet-to-betested, hypothesis is that domain E could be a protein interaction or recognition site. This could constitute another mechanism by which multiple 5-HT2 receptors with similar signal transduction pathways and pharmacology profiles are able to subserve distinct functions in the nervous system and peripheral tissues, i.e., by targeting of this receptor to particular subcellular locations or particular G proteins, effectors, or other regulatory proteins.
Data from our study illustrate that domain E is not necessary for the 5-HT2C receptor to function in ligand binding and activating at least one G protein-coupled pathway. The “extra” hydrophobic domain in the mouse 5-HT2C receptor does not cross the plasma membrane ; therefore, the seven-transmembrane domain motif is conserved. These results support the idea that the seven-transmembrane domain motif in most G protein-coupled receptors is sufficient to constitute the structural core for receptor functions.
We thank Drs. M. Baez, M. Cohen, and D. Nelson (Lilly Research Laboratories) for gifts of AV12 cells and LY53857. We also thank Dr. Anton Mestek for constructing the pcDNA3 vector containing the HA epitope tag. This work was supported in part by NIH grants NS28190, DA09116, DA09444, and DA11891 to L. Y. J.H.H. and L.J.B. were supported by an NIH training grant (HD07373). J.L. was supported by a John B. Hickam Memorial postdoctoral fellowship from the American Heart Association, Indiana Affiliate, Inc. L.Y. was the recipient of an NIH Research Career Development Award (NS01557).
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