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CELLULAR AND MOLECULAR

Domain Swapping in the Human Histamine H₁ Receptor

Leiden/Amsterdam Center for Drug Research, Faculty of Sciences, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (R.A.B., G.D., R.L.); Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, United Kingdom (J.J.C., J.F.L.-G., G.M.); Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (R.G.B.); and School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, United Kingdom (P.G.S.)

Received February 24, 2004; accepted May 24, 2004.

	Abstract

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G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H₁ receptor. We also show the presence of domain-swapped H₁ receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate¹⁰⁷ in transmembrane (TM) 3 or phenylalanine⁴³² in TM6 to alanine results in two radioligand-binding-deficient mutant H₁ receptors. Coexpression of H₁D¹⁰⁷ A and H₁F⁴³²A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wild-type H₁ receptor. Interestingly, the H₁ receptor radioligands [³H]mepyramine and [³H]-(–)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B_max) for wild-type H₁ receptors but not for the radioligand binding site that is formed upon coexpression of H₁ D¹⁰⁷A and H₁ F⁴³²A receptors, suggesting the presence of different H₁ receptor populations.

Despite the important role of GPCRs in physiology and their position as important drug targets, detailed insight regarding molecular mechanisms of drug-GPCR interaction are still lacking. This is well illustrated by the fact that until recently GPCRs were thought to act as monomeric transmembrane entities (Devi, 2001). Accumulating evidence, using a variety of techniques, now suggests that various GPCRs exist in monomeric, dimeric, and various oligomeric forms (Maggio et al., 1999; Gomes et al., 2000; Dean et al., 2001). The existence of homodimers has been shown for e.g., ₂-adrenergic receptors (Hebert et al., 1996; Angers et al., 2000; Jordan et al., 2001), - and -opioid receptors (Cvejic and Devi, 1997; Gomes et al., 2000), metabotropic glutamate receptor 5 (Robbins et al., 1999), calcium-sensing receptor (Bai et al., 1998; Pace et al., 1999; Zhang et al., 2001), M₃ muscarinic receptor (Maggio et al., 1993a; Zeng and Wess, 1999), vasopressin V2 receptor (Zhu and Wess, 1998) and various somatostatin receptors (Rocheville et al., 2000; Pfeiffer et al., 2002), and dopamine receptor subtypes (Nimchinsky et al., 1997; Zawarynski et al., 1998; Gines et al., 2000).

The human histamine H₁ receptor (H₁R) is a prototypic GPCR that is an important target for pharmaceutical drug development. For many years H₁R antagonists (also often referred to as "antihistamines") have been successfully used for the treatment of a variety of allergic conditions (Woosley, 1996). We previously have successfully identified the H₁R binding site for prototypic H₁R antagonists (Wieland et al., 1999) and have reclassified various clinically used H₁R antagonists as inverse H₁R agonists on the basis of their ability to inhibit spontaneous H₁R activity (Bakker et al., 2000). In view of the emerging concept of GPCR dimerization we have now investigated the potential oligomerization of the H₁Rby utilizing three techniques: immunoprecipitation of N-terminally epitope-tagged receptors followed by immunodetection of the tagged receptors, time-resolved FRET, using a combination of fluorescently labeled antibodies recognizing N-terminally epitope-tagged receptors, as well as the complementation of radioligand binding sites upon coexpression of two distinct mutant receptors for which the individual mutations render the receptors radioligand insensitive. Our studies show that the histamine H₁ receptor is constitutively expressed as both monomeric and multimeric entities. Moreover, as previously suggested (Booth et al., 1999, 2002; Bucholtz et al., 1999; Choksi et al., 2000), our studies indicate that the recently introduced H₁ receptor radioligand [³H]-(–)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene ([³H]-(–)-trans-H₂-PAT) specifically binds to only a subpopulation of multimeric H₁Rs. These data shed a new light on the mechanism of drug receptor interaction at this therapeutically relevant target.

	Materials and Methods

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Materials. Cell culture media, LipofectAMINE, penicillin, and streptomycin were obtained from Invitrogen (Carlsbad, CA), and calf serum was obtained from Integro BV (Dieren, the Netherlands). [³H]mepyramine (20 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Bovine serum albumin, chloroquine diphosphate, DEAE-dextran (chloride form), histamine (2-[4-imidazolyl]ethylamine hydrochloride), mepyramine (pyrilamine maleate), and polyethyleneimine were purchased from Sigma/RBI (Natick, MA). The H₁ receptor radioligand [³H]-(–)-trans-H₂-PAT (85 Ci/mmol) was synthesized as described (Wyrick et al., 1994; Booth et al., 2002). Gifts of mianserin hydrochloride (Organon NV, Oss, The Netherlands), (R)- and (S)-cetirizine hydrochloride (UCB Pharma, Brussels, Belgium), the expression vector pcDEF₃ (Goldman et al., 1996) (Dr. J. Langer), the cDNAs encoding the human H₁R (Fujimoto et al., 1999) (Dr. H. Fukui), the mutant human H₁R Asp¹⁰⁷Ala (Ohta et al., 1994; Moguilevsky et al., 1998) (UCB Pharma), and the mutant human H₃R Asp¹¹⁴Glu (Uveges et al., 2002) (Wyeth-Ayerst, Princeton, NJ) are greatly acknowledged.

Site-Directed Mutagenesis. The mutant human H₁ receptor Phe⁴³²Ala was created by Altered Sites' II (Promega, Madison, WI) according to the manufacturer's protocol. All mutant receptors were subcloned into the expression vector pcDEF₃ and verified by sequencing.

Cell Culture and Transfection. COS-7 African green monkey kidney cells were maintained at 37°C in a humidified 5% CO₂/95% air atmosphere in Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 5% (v/v) fetal calf serum. COS-7 cells were transiently transfected using the DEAE-dextran method as previously described (Wieland et al., 1999). The total amount of the transfected plasmid DNA was maintained constant by addition of pcDEF₃.

HEK293 cells were maintained at 37°C in a humidified 5% CO₂/95% air atmosphere in Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, supplemented with 10% (v/v) newborn calf serum. Cells were grown to 60 to 80% confluency before transient transfection in 100-mm dishes. Transfection was performed using LipofectAMINE reagent, according to the manufacturer's instructions.

Histamine H₁ Receptor Binding Studies. Cells used for radioligand binding studies were harvested 48 h after transfection and homogenized in ice-cold H₁R binding buffer (50 mM Na₂/K-phosphate buffer, pH 7.4). For saturation isotherms, cell membrane homogenates were incubated for 30 min at 30°C with 0.01 to 17.0 nM [³H]mepyramine or for 60 min at 30°C with 0.01 to 5.8 nM [³H]-(–)-trans-H₂-PAT, in a total assay volume of either 400 or 100 µl or buffer, respectively. For competition binding assays, the cell homogenates were incubated for 30 min at 30°C with 0.1 to 10,000 nM test ligand in the presence of 7 nM [³H]mepyramine or for 60 min at 30°C with 1 nM [³H]-(–)-trans-H₂-PAT in a total volume of 400 or 100 µl buffer, respectively. The nonspecific binding was determined in the presence of 1 µM mianserin for both radioligands. The incubations were stopped by rapid dilution with 3 ml ice-cold H₁R binding buffer. The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml of buffer, and radioactivity retained on the filters was measured by liquid scintillation counting. Binding data were evaluated by a nonlinear, least-squares curve-fitting procedure using Graphpad Prism (GraphPad Software Inc., San Diego, CA). Protein concentrations were determined according to Bradford (1976), using bovine serum albumin as a standard. All data are represented as mean ± S.E.M from at least three independent experiments in triplicate. Statistical significance was determined by Student's unpaired t test (p < 0.05 was considered statistically significant).

Time-Resolved FRET. The experiments were conducted as described previously (McVey et al., 2001; Carrillo et al., 2003). Briefly, HEK293 cells were transfected with cDNA encoding the human H₁R-FLAG or H₁R-c-myc or cotransfected with both constructs together. Time-resolved FRET was assessed in whole cells expressing the aforementioned receptors or in membrane homogenates from these cells. The final amount of sample per assay was 250 µg of protein in the case of cell membranes and 1.2 to 1.5 x 10⁶ cells when using whole cells. Samples were incubated for 2 h at room temperature in phosphate-buffered saline (16 mM Na₂HPO₄, 5 mM NaH₂PO₄, and 150 mM NaCl) containing 50% (v/v) newborn calf serum, 5 nM Eu³⁺-labeled anti-c-myc (PerkinElmer Life and Analytical Sciences), and 15 nM allophycocyanin-labeled anti-FLAG (Cis Bio International, Gif, Yvette Cedex, France) antibodies. After the incubation samples were washed twice with phosphate-buffered saline, the final pellet was resuspended in 50 µl of the same buffer and transferred to a 384-well microtiter plate. Energy transfer was measured by exciting the Eu³⁺ at 320 nm and monitoring the allophycocyanin emission for 1000 µs at 665 nm using a Victor² 1420 Multilabel Counter (PerkinElmer Life and Analytical Sciences) configured for time-resolved fluorescence after a 50-µs delay.

GPCR Coimmunoprecipitation Studies. Coimmunoprecipitation studies using FLAG- and c-myc-tagged forms of the human H₁R were performed as in (McVey et al., 2001; Ramsay et al., 2002), except that 30 U/ml endoglycosidase F was added to deglycosylate the receptor.

	Results

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Coimmunoprecipitation of Human H₁ Receptors. The human H₁R was modified at the N terminus to include c-myc or FLAG epitope tags immediately after the N-terminal methionine. Following transient coexpression of both tagged forms of the receptor and immunoprecipitation with anti-FLAG antibodies, the samples were resolved by SDS-PAGE and immunoblotted to detect anti-c-myc immunoreactivity. Two bands of an apparent molecular mass of 48 and 100 kDa were observed, which would be consistent with the detection of both monomeric and dimeric forms of the H₁ receptor (Fig. 1A). Other bands of higher molecular mass were also detected, which could be due to either the presence of higher order oligomers or receptor aggregation during the denaturalization of the samples prior to SDS-PAGE. Furthermore, the anti-c-myc immunoreactivity was not detected when the c-myc- and FLAG-tagged forms of the receptor were expressed in separate cell populations nor when these two forms of the receptor were expressed independently and mixed before immunoprecipitation. Thus, the formation of aggregates during the solubilization process can be ruled out. To confirm appropriate expression of both tagged forms of the receptor, samples of the cell lysates were also immunoblotted with anti-c-myc and anti-FLAG antibodies (Fig. 1B).

View larger version (37K): [in this window] [in a new window]   Fig. 1. Coimmunoprecipitation of differentially epitope-tagged forms of the human H1-receptor: evidence for constitutive homo-oligomerization. A, HEK293 cells were transiently transfected with empty vector (lane 1), the cDNA encoding the FLAG-H1R (lane 2), the cDNA encoding the c-myc-H1R (lane 3), or both cDNAs for the FLAG-H1R and c-myc-H1R (lane 4). Prior to immunoprecipitation, cell lysates from the separately expressed FLAG-H1R and the c-myc-H1R were physically mixed (lane 5). Cell lysates were immunoprecipitated with anti-FLAG (lanes 1–5) antibodies, the samples resolved by SDS-PAGE, and then immunoblotted with anti-c-myc. B, cell lysates were prepared from transiently transfected HEK293 cells and immunoblotted with anti-FLAG antibodies or anti-c-myc antibodies.

Time-Resolved Fluorescent Resonance Energy Transfer. Time-resolved FRET fluorescence results (665-nm emission after excitation at 320 nm) obtained with the different samples are shown in Fig. 2. In both cases, whole and cell membranes, there is a clear specific signal when comparing the results observed for cells coexpressing both epitope-tagged receptors in relation to the ones resulting from a mixture of membranes or cells individually expressing each of those receptors. This FRET signal can only be explained due to the resonance energy transfer from anti-c-myc-Eu³⁺ antibodies bound to H₁R-c-myc receptors to anti-FLAG-allophycocyanin antibodies bound to H₁R-FLAG receptors. Since this resonance energy transfer can only take place within 10 nm, these date indicate the formation of H₁R multimers in living cells.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Time-resolved FRET. Upon measuring fluorescence emission at 665 nm, after excitation at 320 nm, tr-FRET signal (filled bars) is seen using either membranes or cells coexpressing H1R-FLAG and H1 R-c-myc, due to the resonance energy transfer of specific H1R-c-myc bound to anti-c-myc-Eu3+ antibody to the specific H1 R-FLAG-bound anti-FLAG-allophycocyanin antibody. Mixing of two populations of cells independently expressing each epitope-tagged receptor resulted in a reduced tr-FRET signal (open bars).

Similar results were obtained for FLAG-tagged gpH₁ receptors expressed in CHO cells using the experimental assay conditions according to Gazi et al. (2003), indicating also that gpH₁ receptors form multimers in living cells.

[³H]Mepyramine Binding Studies to Mutant H₁ Receptors. In our study using human H₁R mutants in which either Asp¹⁰⁷ or Phe⁴³² is changed into alanine, we confirm (Fig. 3A) previous studies reporting the loss of [³H]mepyramine binding to guinea pig H₁Rs in which the corresponding amino acids (Asp¹¹⁶ and Phe⁴³²) were changed to alanine (Wieland et al., 1999). Moreover, we observed that transient coexpression of both H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³² Ala mutants resulted in reconstitution of [³H]mepyramine binding sites. Furthermore, the reconstitution of [³H]mepyramine binding was only demonstrated when the H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³²Ala mutants were cotransfected. Physical mixing of the membranes, expressing H₁ Asp¹⁰⁷ Ala, or H₁ Phe⁴³² Ala receptors, prior to [³H]mepyramine binding, did not lead to significant binding (Fig. 3A). In parallel, cotransfection of a mutant human histamine H₃ receptor, H₃ Asp¹¹⁴Glu, in combination with the H₁ Phe⁴³² Ala mutant, did not result in significant [³H]mepyramine binding (Fig. 3A). A detailed characterization of the [³H]mepyramine binding sites that are formed upon coexpression of H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³²Ala receptors by saturation analysis revealed a K_d for [³H]mepyramine of 1.8 ± 0.1 nM (Fig. 3B). Although the K_d value of [³H]mepyramine for the reconstituted binding site is in agreement with its K_d value for the wild-type human H₁ receptor (K_d = 1.2 ± 0.1 nM), the number of binding sites is greatly reduced (see Table 1). A more detailed investigation of the pharmacological profiles of the [³H]mepyramine binding sites that are formed upon coexpression of both H₁ Asp¹⁰⁷Ala and H₁ Phe⁴³² Ala mutant receptors revealed a clear human H₁R profile of these reconstituted [³H]mepyramine binding sites (Fig. 3C; Table 2), including the known stereoselectivity for the enantiomers of cetirizine (Bakker et al., 2000). The difference in affinity of mepyramine for the wild-type H₁R and for the reconstituted [³H]mepyramine binding site that is formed upon coexpression of both H₁ Asp¹⁰⁷Ala and H₁ Phe⁴³² Ala mutant receptors may result from Hill coefficients (n_H) that deviate from unity (Lazareno and Birdsall, 1993). However, the Hill coefficients for mepyramine displacing [³H]mepyramine from either wild-type H₁Rs or from the reconstituted binding sites do not deviate from unity (n_H = –0.9 ± 0.1 and –1.1 ± 0.1, respectively; see also Table 2).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Reconstitution of [3H]mepyramine binding. A, [3H]mepyramine binding to COS-7 cells membranes of cells transiently transfected with cDNA encoding the empty expression vector pcDEF3 (mock), the cDNA encoding the H1R Asp107Ala (D107A), the cDNA encoding for the H1R Phe432Ala (F432A), or equal amounts of the cDNAs encoding either H1R Asp107Ala and H1R Phe432Ala receptors (D107A + F432A) or H3R Asp114Glu and H1R Phe432Ala receptors (D114E + F432A). Also shown is the binding of [3H]mepyramine to a 1:1 mix of membranes of transiently transfected cells with cDNA encoding either H1R Asp107Ala or H1R Phe432Ala (D107A/F432A). B, representative saturation isotherm of [3H]mepyramine binding to COS-7 membranes, transiently cotransfected with both H1R Asp107Ala and H1R Phe432Ala (Kd = 1.8 ± 0.1 nM, Bmax = 0.34 ± 0.1 pmol/mg protein; see also Table 1). C, representative radioligand displacement curves on COS-7 membranes, transiently coexpressing both H1R Asp107Ala and H1R Phe432Ala, for histamine (), R- () and S- () cetirizine, (–)-trans-H2-PAT (), and mepyramine (). See also Table 2.

[³H]-(–)-trans-H₂-PAT Binding Studies to Mutant H₁ Receptors. Previous findings suggests that the recently described H₁R radioligand [³H]-(–)-trans-H₂-PAT, might selectively label H₁R dimers (Booth et al., 2002). In agreement with the previously reported binding-characteristics of [³H]-(–)-trans-H₂-PAT to rat and guinea pig H₁Rs (Booth et al., 1999, 2002; Bucholtz et al., 1999; Choksi et al., 2000), we observed a high-affinity [³H]-(–)-trans-H₂-PAT binding site in COS-7 cells expressing the wild-type human H₁R (K_d = 1.2 ± 0.4 nM) and a significant lower number of [³H]-(–)-trans-H₂-PAT binding sites (B_max = 3.4 ± 1.0 pmol/mg protein) in comparison with the number of [³H]mepyramine binding sites that can be detected in the same preparation (Table 1).

Measuring [³H]-(–)-trans-H₂-PAT binding to COS-7 cells membranes expressing the mutant human H₁ Asp¹⁰⁷Ala or H₁ Phe⁴³² Ala receptors individually, did not result in any significant specific binding (<25 dpm, see also Fig. 4A). However, upon coexpression of both mutant receptors, we observed the specific reconstitution of [³H]-(–)-trans-H₂-PAT binding sites (Fig. 4). The reconstitution of [³H]-(–)-trans-H₂-PAT binding sites was only observed upon coexpression of H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³² Ala mutant H₁Rs, physical mixing of membranes of cells individually expressing either H₁ Asp¹⁰⁷ Ala or H₁ Phe⁴³² Ala receptors, prior to radioligand binding experiments, did not lead to the reconstitution of [³H]-(–)-trans-H₂-PAT binding sites. A detailed characterization of the [³H]-(–)-trans-H₂-PAT binding sites that are formed upon coexpression of H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³² Ala receptors by saturation analysis revealed a K_d for [³H]-(–)-trans-H₂-PAT of 3.0 ± 0.6 nM (Fig. 4B; Table 1). Although the K_d value of [³H]-(–)-trans-H₂-PAT for the reconstituted binding sites is in agreement with its K_d value for the wild-type H₁R (K_d = 1.2 ± 0.4 nM), the number of binding sites is greatly reduced (see Table 1). A more detailed investigation of the pharmacological profiles of the [³H]-(–)-trans-H₂-PAT binding sites that are formed upon coexpression of both H₁ Asp¹⁰⁷Ala and H₁ Phe⁴³² Ala mutant receptors revealed a clear human H₁-receptor profile of these reconstituted [³H]-(–)-trans-H₂-PAT binding sites (Table 2), including the known stereoselectivity for the enantiomers of cetirizine (Bakker et al., 2000). However, the pharmacological profile is not identical to that of the wild-type H₁R, the difference in affinity of mepyramine for the wild-type H₁R and for the reconstituted [³H]-(–)-trans-H₂-PAT binding site that is formed upon coexpression of both H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³² Ala mutant receptors may in part result from the Hill coefficient (n_H) that deviate from unity, which may result in affinity values that differ from their actual K_d values (Lazareno and Birdsall, 1993). Although the Hill coefficient for mepyramine displacing [³H]-(–)-trans-H₂-PAT from wild-type H₁Rs is close to unity (n_H = –1.1 ± 0.1), the Hill coefficient for mepyramine displacing [³H]-(–)-trans-H₂-PAT from the reconstituted binding sites deviates from unity (n_H = –0.5 ± 0.1; see also Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Reconstitution of [3H]-(–)-trans-H2-PAT binding sites upon coexpression of H1 Asp107 Ala and H1 Phe432Ala receptors. A, specific [3H]mepyramine (open bars) [3H]-(–)-trans-H2-PAT (filled bars) binding, to COS-7 cells membranes, expressing either H1 Asp107Ala (D107A) or H1 Phe432Ala receptors (F432A), or coexpressing H1 Asp107Ala and H1 Phe432Ala receptors (D107A + F432A). B, representative saturation isotherm of [3H]-(–)-trans-H2-PAT binding to COS-7 membranes coexpressing H1 Asp107Ala and H1 Phe432 Ala receptors (Kd = 3.0 ± 0.6 nM, B = 0.32 ± 0.1 pmol/mg protein; see also Table 1).

These data indicate that reconstitution of radioligand binding sites upon the coexpression H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³²Ala mutant H₁Rs is accompanied by the detection of a considerable lower number of radioligand binding sites, when compared with the number of radioligand binding sites upon expression of wild-type H₁ receptors. Moreover, although both [³H]mepyramine and [³H]-(–)-trans-H₂-PAT identify a significantly different number of binding sites for the wild-type H₁R (Booth et al., 2002), they identify an identical number of reconstituted radioligand binding sites that are formed upon coexpression of the two radioligand binding defective mutant human H₁Rs: H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³²Ala (Table 1).

	Discussion

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Recently, compelling evidence has emerged that GPCRs may be present as oligomers in the plasma membrane. Various receptors belonging to different GPCR subfamilies have been shown to form dimeric and/or oligomeric (Milligan, 2001; Rios et al., 2001; Gazi et al., 2002; Agnati et al., 2003). The occurrence of dimeric GPCRs has been shown using a variety of techniques, which has also resulted in the identification of several mechanisms of GPCR dimerization. Two structural models for dimer formation have been suggested (Hebert et al., 1996): contact dimers and domain-swapped dimers (Dean et al., 2001; Filizola et al., 2002; George et al., 2002). In contact dimers, certain domains of individual receptors interact, most likely through hydrophobic interactions, while maintaining their respective ligand binding domains. The domain-swapped dimers are thought to form two ligand binding domains that are created upon the mutual exchange of transmembrane domains from both receptors (Maggio et al., 1993a; Gouldson et al., 2000) (see also Fig. 5).

View larger version (37K): [in this window] [in a new window]   Fig. 5. Models of human H1 receptor dimerization. GPCRs were thought to function as monomers (A); however, several lines of evidence suggest GPCRs can form oligomeric structures. Dimeric forms of GPCRs may consist of contact dimers between receptor monomers (B) or of domain-swapped dimers involving the reciprocal exchange of TM domains between receptors within the dimer resulting in trans-complementation (C). Different TM domains have been reported to be involved in the formation and stabilization of contact dimers, such as an interface involving TM4–5 (Guo et al., 2003) or an interface involving TM5–6 (as indicated here) (Maggio et al., 1993a; Gouldson et al., 2000; Liang et al., 2003) or involving TM1 (Gouldson et al., 2000; Overton and Blumer, 2002; Carrillo et al., 2003; Liang et al., 2003). The TM domains harboring the mutation in mutant H1Rs are indicated in gray (TM3 for H1 D107A and TM6 for H1 F432A); functional ligand binding sites (L) and nonfunctional ligand binding sites (X) are indicated.

In the present study, we have investigated the potential dimerization of the human histamine H₁R. We demonstrate that immunoprecipitation of epitope-tagged H₁Rs with specific antibodies results in the detection of bands of apparent size corresponding to the estimated molecular weight of monomeric, dimeric, as well as higher order oligomeric human H₁Rs. Although these studies clearly indicate the possible formation of dimeric H₁Rs, several issues have been raised about the use of coimmunoprecipitation studies for demonstrating dimerization of GPCRs (Milligan, 2001). Further studies will need to be performed to verify the functional expression of the higher order oligomeric forms of the H₁R; however, we have obtained evidence for dimerization of epitope-tagged H₁Rs using time-resolved fluorescence resonance energy transfer (tr-FRET) experiments. Although these data indicate the presence of dimeric H₁Rs at the cell surface of living cells and in membrane preparations derived from these cells, the H₁R dimers detected by these techniques may represent a heterogeneous population of homodimers formed by contact dimers as well as domain-swapped dimers. Receptor mutagenesis approaches have been used to demonstrate the formation of domain-swapped dimers of _2C-adrenergic receptors as well as of the M₂ and M₃ muscarinic receptors and type 1 angiotensin II receptors when they were coexpressed (Maggio et al., 1993a,b; Monnot et al., 1996). In these studies, coexpression of two radioligand-binding defective mutant receptors reconstituted a functional radioligand binding site, suggesting the molecular association of complementary transmembrane domains of two different defective mutant receptors. Several studies have identified critical amino acid residues in the H₁R antagonist binding pocket (Ohta et al., 1994; Moguilevsky et al., 1998; Wieland et al., 1999). Some of the mutant H₁Rs from these studies were defective in the binding of the H₁R radioligand [³H]mepyramine. Since the mutations were located in different domains of the H₁R, we investigated whether the mechanism of intermolecular complementation could be observed for this bioaminergic receptor. Thus, we have coexpressed two radioligand binding defective mutant human H₁Rs, H₁ Asp¹⁰⁷ Ala (harboring a mutation in TM3) and H₁ Phe⁴³²Ala (harboring a mutation in TM6) and monitored the formation of potential domain-swapped H₁R dimers by radioligand binding assays. Coexpression of H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³²Ala receptors, but not physical mixing of membranes of cells individually expressing either human H₁ Asp¹⁰⁷ Ala or H₁ Phe⁴³² Ala receptors, reconstituted a [³H]mepyramine binding site exhibiting a pharmacological profile closely resembling that of the wild-type H₁R, including the affinities for [³H]mepyramine and the stereospecificity toward the enantiomers of cetirizine (Bakker et al., 2000). Thus, both H₁ Asp¹⁰⁷Ala and H₁ Phe⁴³² Ala receptors can adopt a conformation allowing them to interact with H₁R ligands. These data clearly illustrate the formation of domain-swapped H₁R dimers in which there is the reciprocal exchange of TM domains 6 and 7 between the receptors present in the dimer. The expression level of the reconstituted binding site is considerably lower than that of the wild-type receptor. However, one should consider that the H₁R dimers that are formed by domain swapping upon coexpression of the two binding-defective mutant H₁Rs is expected to yield dimeric H₁Rs of three different compositions and only one of these will contain a single complete binding pocket and not two as expected for the H₁R dimer that is formed by domain swapping of wild-type H₁Rs (Fig. 5). Moreover, the two binding defective mutant H₁Rs may also form both monomeric as well as contact dimeric H₁Rs, which will not bind [³H]mepyramine due to their respective mutations (see also Fig. 5).

In previous studies, we have used [³H]-(–)-trans-H₂-PAT as an alternative high-affinity radioligand to label H₁Rs in guinea pig brain, rat brain, and human H₁Rs expressed in CHO cells (Booth et al., 1999, 2002; Choksi et al., 2000). The use of [³H]-(–)-trans-H₂-PAT or [³H]mepyramine as an H₁R radiotracer in displacement studies yields comparable H₁R affinities of a variety of H₁R ligands; however, [³H]-(–)-trans-H₂-PAT consistently labels a lower number of H₁Rs than [³H]mepyramine, indicating the presence of H₁R subpopulations that may be selectively recognized by certain ligands (Booth et al., 2002). Different levels of receptor expression have also been detected for muscarinic (Lee and el-Fakahany, 1985; el-Fakahany et al., 1986; Wreggett and Wells, 1995; Park et al., 2002) and dopamine (see Armstrong and Strange, 2001, and refs. cited therein; Logan et al., 2001; Seeman et al., 2003) receptor binding sites when using different radioligands. These findings have been explained by the formation of receptor dimers or oligomers (Wreggett and Wells, 1995; Armstrong and Strange, 2001; Logan et al., 2001; Park et al., 2002; Seeman et al., 2003). We have suggested that the H₁R radioligand [³H]-(–)-trans-H₂-PAT selectively labels H₁R dimers, whereas [³H]mepyramine labels both mono- and multivalent forms of the H₁R (Booth et al., 2002). Consistent with these suggestions, we observe a [³H]-(–)-trans-H₂-PAT binding site in COS-7 cells coexpressing H₁ Asp¹⁰⁷ Ala and H₁ Phe⁴³² Ala receptors. Expression of H₁ Asp¹⁰⁷Ala or H₁ Phe⁴³² Ala receptors or physical mixing of membranes of cells individually expressing either H₁ Asp¹⁰⁷Ala or H₁ Phe⁴³² Ala receptors did not lead to the formation of [³H]-(–)-trans-H₂-PAT binding sites. Therefore, our data indicate that both [³H]mepyramine and [³H]-(–)-trans-H₂-PAT recognize a multimeric H₁R that is formed by domain swapping. Moreover, Asp¹⁰⁷ and Phe⁴³² are crucial residues in the H₁R for the interaction with either mepyramine (Ohta et al., 1994; Moguilevsky et al., 1998; Wieland et al., 1999) or (–)-trans-H₂-PAT.

Our studies with H₁ Rs indicate that although [³H]mepyramine and [³H]-(–)-trans-H₂-PAT binding to wild-type receptors results in a discrepancy in the observed H₁ receptor expression level, a nearly identical number of H₁ receptors is detected using either radioligand when binding is selectively performed on domain-swapped dimeric H₁ receptors. These data demonstrate that when radioligand binding experiments are performed on one presumably independent population of H₁ receptors, there is no discrepancy in the number of ligand binding sites. Therefore, these data confirm our previous suggestion on the notion of distinct wild-type H₁ receptor subpopulations (Booth et al., 2002).

In conclusion, we demonstrate the existence of dimeric H₁Rs; in particular, domain-swapped H₁R dimers. These domain-swapped dimers form a subpopulation of H₁Rs exhibiting a classical H₁R pharmacological profile when profiled using diverse radioligands. The current results are in accord with previous findings on the binding of [³H]-(–)-trans-H₂-PAT in mammalian brain and suggest dimeric H₁R subpopulations may also occur in the CNS. The significance of these findings for H₁ receptor function and ligand responses needs to be further evaluated.

	Footnotes

 
This study was supported in part by UCB Pharma (Brussels, Belgium) and by the European Union BIOMED 2 program "Inverse Agonism. Implications for Drug Research" (to R.A.B., G.D., and R.L.).

This work was presented at the XXXIII Annual Meeting of the European Histamine Research Society which was held from April 28 to May 2, 2004, Düsseldorf/Köln, Germany.

doi:10.1124/jpet.104.067041.

ABBREVIATIONS: GPCR, G-protein-coupled receptor; H₁R, human histamine H₁ receptor; FRET, fluorescence resonance energy transfer; [³H]-(–)-trans-H₂-PAT, [³H]-(–)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene; PAGE, polyacrylamide gel electrophoresis; n_H, Hill coefficient; tr, time resolved; TM, transmembrane.

¹ These authors contributed equally to this article.

Address correspondence to: Dr. Rob Leurs, Leiden/Amsterdam Center for Drug Research, Faculty of Sciences, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands. E-mail: leurs{at}few.vu.nl

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