Abstract
Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study G␣ i signaling in vivo (1, 2) . Also, G␣ s -coupled G protein-coupled receptors have been engineered to generate G␣ s -coupled RASSLs (3, 4). In this study, we exploited the differences in binding pockets between different classes of H 1 receptor agonists and identified the first G␣ q/11 -coupled RASSL. The mutant human H 1 receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H 1 receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H 1 receptors.

Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study G␣ i signaling in vivo (1, 2). Also, G␣ s -coupled G protein-coupled receptors have been engineered to generate G␣ s -coupled RASSLs (3, 4) . In this study, we exploited the differences in binding pockets between different classes of H 1 receptor agonists and identified the first G␣ q/11 -coupled RASSL. The mutant human H 1 receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H 1 receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H 1 receptors.

Receptors activated solely by synthetic ligands (RASSLs) 2 have recently been developed as tools to control G protein signaling in vivo (5) . RASSLs are G protein-coupled receptors (GPCRs) that no longer respond to their endogenous ligands but can still be activated by synthetic ligands (5) . Given the biological importance of GPCRs, the ability to stimulate a single G protein pathway in a tissue of choice in vivo is useful for the understanding of subsequent changes in downstream signaling. The use of RASSLs has already provided valuable insight into the effect of G␣ i signaling in the heart. Stimulation of a G␣ i -coupled RASSL selectively expressed in the murine heart resulted in a strong (3-fold) and rapid (within 1 min after drug administration) decrease in heart rate (1) . In the future, RASSLs may further be applied, e.g. to create reversible models of disease states, or combined with microarrays, to result in gene expression fingerprints of specific G protein pathways (for review, see Ref. 6 ).

There are four major classes of G proteins, named after their respective ␣-subunit (G␣ s , G␣ i , G␣ q/11 , and G␣ 12 ). Each class couples to specific signal transduction pathways, which are well characterized in vitro (for a review, see Ref. 7). The first described RASSL couples through G␣ i proteins (5) and thus far is the only RASSL being applied in vivo (1, 2, 8) . Also, more recently, several G␣ s -coupled RASSLs have been developed (3, 4) . Thus far, there have been no reports about either G␣ q/11 -or G␣ 12 -coupled RASSLs.

The human histamine H 1 receptor (H 1 R) couples through G␣ q/11 proteins and thereby activates phospholipase C, resulting in, for example, inositol phosphate hydrolysis and increased concentrations of intracellular Ca 2ϩ (9, 10) . We previously demonstrated that the H 1 receptor also activates NF-B, both in a ligand-induced as well as in a constitutive manner, via G␣ q/11 proteins (11). Although several G␣ qcoupled GPCRs also activate G␣ 12 (for a review, see Ref. 12) , the H 1 R does not seem to couple to G␣ 12 (10, 11, 13) .

By mutational analysis, we previously characterized several amino acids that are involved in the binding of histamine and several synthetic agonists, such as the histaprodifens (a new class of specific H 1 R agonists) (14) . Mutation of Phe 435 (6.55) to Ala in TM6 (transmembrane domain 6) resulted in a strong decrease in affinity and potency of histamine, whereas only marginally affecting affinities and potencies of histaprodifens. In this study, we showed that for another class of H 1 R agonists, the substituted 2-phenyl-histamines (PheHAs, Ref. 15), mutation of F435A results in strongly increased affinities and potencies, thereby identifying the first G␣ q/11 -coupled RASSL.
EXPERIMENTAL PROCEDURES
Materials-pNF-B-Luc was obtained from Stratagene (La Jolla, CA). ATP disodium salt, bovine serum albumin, chloroquine diphosphate, DEAE-dextran (chloride form), histamine dihydrochloride, mepyramine (pyrilamine maleate), glycerol, Triton X-100, and polyethyleneimine were purchased from Sigma. D-Luciferin was obtained from Duchefa Biochemie BV (Haarlem, The Netherlands). Cell culture media, penicillin, and streptomycin were obtained from Invitrogen. Cell Culture and Transfection-COS-7 African green monkey kidney cells were maintained at 37°C in a humidified 5% CO 2 , 95% air atmosphere in Dulbecco's modified Eagle's medium containing 50 IU/ml penicillin, 50 g/ml streptomycin, and 5% (v/v) fetal bovine serum. COS-7 cells were transiently transfected using the DEAE-dextran method as described previously (11) .
EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-The cDNA encoding the human H 1 R (17) was subcloned in the pAlter plasmid (Promega), and point mutations were created according to the manufacturer's protocol (Altered Sites II, Promega). cDNA of all mutant and wild-type receptors were subcloned into the expression plasmid pcDEF3 (16 3 H]mepyramine. The nonspecific binding was determined in the presence of 1 M mianserin. The incubations were stopped by rapid dilution with 3 ml of ice-cold H 1 R binding buffer. The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethylenimine. Filters were washed twice with 3 ml of H 1 R binding buffer, and radioactivity retained on the filters was measured by liquid scintillation counting. Binding data were evaluated by a non-linear, least squares curve-fitting procedure using GraphPad Prism ® (GraphPad Software, Inc., San Diego, CA).
EXPERIMENTAL PROCEDURES
Analytical Methods-Protein concentrations were determined according to Bradford (18) , using bovine serum albumin as a standard. All data shown are expressed as means Ϯ S.E. Statistical analyses were carried out by non-paired Student's t test. p values Ͻ 0.05 were considered to indicate a significant difference (a, p Ͻ 0.05; b, p Ͻ 0.01; c, p Ͻ 0.001).
EXPERIMENTAL PROCEDURES
Molecular Modeling-Our H 1 R homology model was obtained using the bovine rhodopsin crystal structure (Protein Data Bank entry 1L9H, Ref. 19 ) as the template. The third intracellular loop that connects transmembranes 5 and 6 was omitted due to its large size. Side chains were added using the homology module of InsightII (20) . Water molecules present in the crystal structure were not incorporated. The initial model was refined by a steepest descent energy minimization. The minimized model was placed in a dodecahedral box filled with simple point charge water (21) , and a second minimization step using steepest descent was performed. Hereafter, a 20-ps molecular dynamics (MD) run was performed with positions restraints (1000 kJ ⅐ mol Ϫ1 nm Ϫ2 ) on all heavy protein atoms. Finally, the model was refined by a 100-ps MD run. In this structure, Phe 435 was changed to Ala with InsightII, thus creating a model of the F435A H 1 R.
EXPERIMENTAL PROCEDURES
Histamine and 2-(3-chlorophenyl)histamine (ClPheHA) were docked in the WT and F435A models using AutoDock 3.0.0 (22) applying default parameters. The monocationic ligands were assigned Löw-din atomic charges obtained after a single point ab initio restricted Hartree-Fock calculation using 6-31Gr* with the GAMESS US package (23) . The protein was assigned with KOLLUA partial charges using the SYBYL program (version 6, Tripos, St. Louis). Only essential hydrogens were taken into account.
EXPERIMENTAL PROCEDURES
The obtained ligand-receptor complexes were minimized using the steepest descent method. These ligand-receptor complexes were placed in a dodecahedral box, with a minimum distance of 7 Å between protein and the box. The system was solvated with simple point charge water, and the protein partial charges were assigned by GROMACS.
EXPERIMENTAL PROCEDURES
Again, a steepest descent procedure was performed on the system. Subsequently, a set of MD runs with position restraints (1000 kJ⅐ mol Ϫ1 nm Ϫ2 ) was applied to the system in which a controlled release of the restraints was performed. A run for 50 ps with position restraints on all heavy protein atoms and all ligand atoms was performed, with the protein hydrogens and all water molecules unrestrained. Consecutively, a run was performed for 20 ps with position restraints on all C␣-atoms, all heavy atoms of residues Asp 107 and Asn 198 , and all ligand atoms. Finally, a MD run was performed for 1 ns with only position restraints on C␣-atoms. During the last 500 ps of this run, the presence of hydrogen bonds was analyzed using cutoff distance between heavy atoms of 3.5 Å and a cutoff angle between acceptor donor and hydrogen of 60°.
EXPERIMENTAL PROCEDURES
All minimizations and MD simulations were performed using the GROMACS software package and the GROMOS 43a1 force field (24, 25) and LINCS constraints (26) on all bonds. An NPT ensemble was generated using both the Berendsen thermostat and the barostat with default settings for 300 K and 1 bar (25) .
RESULTS

Mutation F435A Differentially Modulates Affinities of Several
Classes of H 1 R Agonists-At the H 1 R, several H 1 R agonists show a higher affinity than the natural agonist histamine. The histamine analogs 2-(3-trifluoromethyl-phenyl)histamine (CF 3 PheHA), histaprodifen, suprahistaprodifen, and the newly characterized H 1 R agonist 8R-lisuride bind 9 -1000-fold better to the H 1 R as compared with histamine (TABLE  ONE) . Previously, we characterized Phe 435 (6.55), as a specific interaction point for histamine (14) . Mutation of this residue into alanine resulted in a 25-fold decrease in histamine affinity without drastic alterations in the affinity of the agonists histaprodifen and suprahistapodifen (27) and is implicated in ligand binding in both human and guinea pig H 1 Rs (14, 28). The F435A mutation removes an aromatic ring structure from the binding pocket, and therefore, potential interactions with either ligands or other amino acids in the H 1 R-binding pocket may be lost. However, exchanging the bulky Phe 435 (6.52) with the smaller alanine may also increase the size of the ligand-binding pocket and thus create space for the trifluoromethyl-phenyl moiety of CF 3 PheHA, resulting in an increased affinity for this ligand. To address the latter possibility, we created additional mutant H 1 Rs (F435V and F435L) that vary in the size of the side chain at position 6.55.
Mutation F435A Differentially Modulates Affinities of Several
The generated mutant receptors are expressed at comparable levels (B max ϭ 13.6 -20.1 pmol/mg of protein) in transiently transfected COS-7 cells and bind the H 1 R radioligand [
Mutation F435A Differentially Modulates Affinities of Several
3 H]mepyramine with virtually unchanged affinity (K d ϭ 1.5-5.7 nM), as determined by radioligand saturation binding experiments (TABLE TWO) . Although the expression levels are similar, there is a clear difference in constitutive GPCR activities of the tested H 1 R mutants as measured by an NF-B-driven reporter gene assay (Fig. 2, white bars) . Although the basal activity of H 1 R F435L H 1 R is comparable with the WT H 1 R, the level of constitutive NF-B activation of H 1 R F435V is twice as high, whereas for mutant H 1 R, F435A constitutive activity is hardly detectable. Although the levels of constitutive NF-B activation vary between the mutant H 1 Rs, their agonist-induced responses are comparable (Fig. 2, black bars) , indicating that G protein-coupling of the mutant H 1 Rs is not impaired.
Mutation F435A Differentially Modulates Affinities of Several
Binding Analysis of WT and Phe 435 Mutant H 1 Rs-Similar to the mutant F435A H 1 R (pK i ϭ 2.7), the mutant H 1 R F435V (pK i ϭ 2.9) and F435L (pK i ϭ 2.9) exhibit a strong decrease in their affinity for histamine as compared with the WT receptor (pK i ϭ 4.1) (TABLE THREE) . Again, these mutant receptors exhibit an increased affinity for CF 3 PheHA. The exchange of Phe 435 for leucine, valine, or alanine results in mutant H 1 Rs with a 47-, 124-, and 54-fold increased affinity for CF 3 PheHA respectively, as compared with the WT receptor (pK i ϭ 5.0) (TABLE THREE) . For these mutant H 1 Rs, the affinity of CF 3 PheHA greatly exceeds their affinity of the endogenous ligand histamine, the mutant F435V H 1 R exhibiting the greatest selectivity for the synthetic agonist CF 3 PheHA over histamine (17,000-fold).
Mutation F435A Differentially Modulates Affinities of Several
We tested two other substituted PheHAs, ClPheHA and 2-(3,4-dichlorophenyl)histamine (Cl 2 PheHA), on the mutant H 1 Rs as well. For the WT H 1 R, the affinity of ClPheHA (pK i ϭ 5.3) is slightly higher then than that of CF 3 PheHA (pK i ϭ 5.0). An (additional) para-chloro substituent is not favorable for H 1 R affinity (pK i ϭ 4.6), fitting previous findings for both human and rodent H 1 Rs (29) . Similar to CF 3 PheHA, the affinity of ClPheHA is increased upon mutation of Phe 435 into leucine (pK i ϭ 6.8), valine (pK i ϭ 7.3), or alanine (pK i ϭ 6.7). Although for Cl 2 PheHA, H 1 R affinities are lower and increases in affinity are smaller, the same trend upon mutation is observed (pK i values, 4.6, 5.3, 5.8, and 5.4, respectively). Similar to CF 3 PheHA, for ClPheHA and Cl 2 PheHA, mutation F435V also results in the largest increase in affinity, as compared with the WT receptor.
Mutation F435A Differentially Modulates Affinities of Several
Additionally, we tested a series of 2-cyclohexyl-histamines (CxHAs), varying in spacer length between the imidazole ring and the cyclohexyl moieties: CxHA, 2-(methyl-cyclohexyl)-histamine (MeCxHA), and 2-(ethyl-cyclohexyl)-histamine (EthCxHA). The affinities of the CxHAs are approximately equal to the affinity of histamine (TABLE THREE) . Again, mutation of Phe 435 into alanine, valine, or leucine results in increased affinities as compared with the WT H 1 R (TABLE THREE) . The increases in affinity, however, are smaller (maximum 10-fold) than those observed for the PheHAs (maximum 120-fold). Although changes in affinity are less prominent, for EthCxHA, we observe a correlation between the space available in the binding pocket (Ala 3 Val 3 Leu 3 Phe) and the affinities for the mutant receptors (F435A, pK i 4.9; F435V, pK i ϭ 4.6; F435L, pK i ϭ 4.3; and WT, pK i ϭ 4.1, TABLE THREE). For both CxHA and MeCxHA, the highest affinities are observed for the F435V mutant H 1 R, following the trend observed for the PheHAs.
Mutation F435A Differentially Modulates Affinities of Several
Functional Analysis of WT and Phe 435 Mutant H 1 Rs-Using an NF-B-driven reporter gene assay, we evaluated the potencies of several 2-phenylsubstituted histamine analogs for mutant and WT H 1 Rs. For the WT H 1 R, these analogs are less potent than the endogenous ligand histamine (pEC 50 ϭ 6.9) (TABLE THREE). Fig. 3 clearly illustrates that as compared with the WT H 1 R, the potency of HA at the H 1 R F435A is decreased 200-fold, whereas the potency of ClPheHA is increased 2,600-fold. Also, for CF 3 PheHA and Cl 2 PheHA, potencies were increased upon the mutation F435A; again ClPheHA (pEC 50 ϭ 9.4) and CF 3 PheHA (pEC 50 ϭ 9.2) are equipotent, and Cl 2 PheHA is clearly less potent (pEC 50 ϭ 7.9) (TABLE THREE). For mutant H 1 Rs F435L and F435V, similar trends are observed, with PheHA potencies for H 1 R F435V being slightly higher than for F435A and F435L H 1 Rs. For mutant H 1 Rs, F435A, F435V, and F435L ClPheHA and CF 3 PheHA are agonists with subnanomolar potencies, which exceed the potency of histamine 70,000 -280,000-fold.
Mutation F435A Differentially Modulates Affinities of Several
Molecular Modeling of WT and F435A Mutant H 1 Rs-Our model of the H 1 R, based on the bovine rhodopsin crystal structure, was used to visualize the interactions of either histamine or ClPheHA with the WT and F435A mutant H 1 R. Models of both WT and F435A H 1 Rs were optimized by molecular dynamics simulations. Hereafter, histamine and ClPheHA were automatically docked in both WT and F435A mutant H 1 R models. Finally, a second round of molecular dynamics was used to optimize the receptor-ligand complexes. In the WT H 1 R, both histamine and ClPheHA show an ionic interaction between the conserved Asp 107 (3.32) in TM3 (transmembrane domain 3) and their ethylamine group over distances of 2.6 and 3.0 Å, respectively (Fig. 4, A and  C) . As expected, differences between histamine and ClPheHA can be observed for interaction with Phe 435 . In binding histamine, Phe 435 has an indirect role, keeping Phe 432 in an edge-to-face orientation toward the imidazole moiety of histamine (Fig. 4A) . In this orientation, a hydrogen bond can be formed between the distal nitrogen of the imidazole ring of histamine and Asn 198 at a distance of 2.7 Å and is observed during 47% of the MD simulation. In binding ClPheHA, Phe 435 directly interacts with the ligand by -stacking with the phenyl moiety of ClPheHA (Fig. 4B) . By this interaction, Phe 435 prevents hydrogen bonding of the imidazole ring with Asn 198 since the distance exceeds 5 Å. The difference in interaction with Phe 435 between histamine and ClPheHA is further illustrated by observed changes in receptor-ligand interactions upon the mutation F435A. For histamine, the mutation F435A releases Phe 432 , thereby allowing histamine to shift toward Asp 107 , resulting in an increased distance between the agonist and Asn 198 (7.0 Å) and the loss of the hydrogen bond between Asn 198 and the imidazole ring of histamine (Fig. 4C) . Upon the mutation F435A, we observed an opposite effect for ClPheHA; removal of the -stacking with Phe 435 allows the ligand to move deeper in the binding pocket, enabling hydrogen bonding of the imidazole ring with Asn 198 at a distance of 2. 4 Å. This hydrogen bond is observed during 78% of the MD simulation (Fig. 4D) .
DISCUSSION
Most H 1 R agonists are structurally closely related to histamine and have the imidazole ring either replaced by other heterocycles (pyridylethylamine, thiozolylethylamine) or substituted at the 2-position (histaprodifens, PheHAs) (30) . Already in 1994, we demonstrated that H 1 R agonists, although structurally similar, appear to have different binding modes (31) . Recently, we extended this conclusion to histaprodifens. In contrast to histamine, for this class of compounds, interactions with either Asn 198 or Phe 435 are no longer required for high affinity H 1 R binding (14) . Asn 198 , however, still appeared crucial for receptor activation. To establish whether the interaction with Phe 435 is specific to histamine alone, we tested several other H 1 R agonists for interaction with Phe 435 . Comparable with histaprodifen, the affinities of suprahistaprodifen, pyridylethlamine, and 8R-lisuride were not altered by the mutation F435A. For CF 3 PheHA however, the affinity for H 1 R F435A was 54-fold higher than for the WT H 1 R. Studying receptor-mediated NF-B activation, the differences in potency of CF 3 PheHA between WT and F435A mutant receptors (1,500-fold) even exceeded the observed differences in affinity. Similar trends are observed for the 3-chlorosubstituted PheHA (ClPheHA) and to a lesser extent for 3,4-dichlorosubstituted PheHA (dClPheHA). These observations strongly suggested that despite structural (histamine-like moiety) and/or functional similarity (agonism), the precise orientation of H 1 R agonists within the H 1 Rbinding pocket differs.
DISCUSSION
We used computer modeling to examine the different H 1 R binding modes for histamine and the synthetic agonist ClPheHA. For histamine, we observed hydrogen bonding between one of the imidazole nitrogens and Asn 198 , whereas for ClPheHA, this interaction is not observed in our molecular dynamics simulations. We previously demonstrated that interaction with Asn 198 appears to be a prerequisite for H 1 R activation (14) . This explains why although the affinity of histamine for the WT H 1 R is lower than that of ClPheHA, histamine is more efficacious as an H 1 R agonist.
DISCUSSION
Our computational studies suggested that upon mutation of Phe 435 to alanine, the aromatic interaction of Phe 432 with the imidazole ring of histamine is lost. Histamine gained in conformational freedom (entropy) and the distance between Asn 198 , and the imidazole nitrogen of histamine increased to 7 Å, suggesting the loss of hydrogen bonding. The loss of this interaction and the increase in entropy most likely accounted for the observed decreased in affinity and potency of histamine (as compared with the WT H 1 R). In contrast, for ClPheHA, the distance between its imidazole moiety and Asn 198 was decreased in the F435A H 1 R, enabling hydrogen bonding and thus facilitating both high affinity binding and potent activation of the F435A H 1 R.
DISCUSSION
To delineate the effect of mutating the bulky, aromatic Phe 435 to a small, aliphatic alanine, mutant H 1 Rs were created in which the side chain at this position have intermediate sizes: Phe 435 3 Leu 3 Val 3 Ala. For none of the PheHAs did we observe gradual size-dependent changes in affinity or potency. Instead, increased affinities and potencies appeared an all-or-nothing phenomenon, correlating with the presence or absence of the aromatic Phe 435 . The various mutations also affected affinities of MeCxHA and CxHA, in a similar but attenuated fashion. Since we did observe a small but clear correlation between affinity and side chain bulk at position 6.55 for EthCxHA, having an ethyl chain as a spacer between histamine and cyclohexyl moieties, we speculated that only for this ligand, the space in binding pocket is limiting over the full array of receptors. Potentially, increasing the spacer length between histamine moiety and (substituted) phenyl ring would also result in PheHAs that can discriminate between the various mutant H 1 Rs.
DISCUSSION
RASSLs are GPCRs that no longer respond to endogenous ligands but can still be activated by synthetic ligands (5) . Previously, such mutant GPCRs have been introduced as new tools to selectively study specific G protein pathways in vivo (1) . So far, RASSLs for both G i and G s pathways have been described (3) (4) (5) . For mutant receptors F435A, F435V, and F435L, the affinity and potency of histamine are strongly decreased as compared with the WT H 1 R, whereas the affinities and potencies of PheHAs are strongly increased (TABLE THREE) . These mutant receptors can therefore be classified as the first G␣ q/11 -coupled RASSLs. It is noteworthy that these mutant H 1 Rs differ in their level of constitutive NF-B activation. The constitutive activity of H 1 R F435L was similar to that of the WT H 1 R, whereas it is hardly detectable for H 1 R F435A and increased for H 1 R F435V. Since mutant receptors F435L and F435V displayed basal NF-B activation, they are not "solely activated by synthetic ligands," and therefore, only H 1 R F435A fits the criteria of RASSL.
DISCUSSION
It is important to notice that ligand-independent constitutive activity of RASSLS can be apparent in vivo. Chronic overexpression of Ro1, a G␣ i -coupled RASSL, in transgenic mice caused mortality, independent of stimulation with the synthetic agonist (2). This mortality could be blocked by treatment with either the specific G i -blocker pertussis toxin or the Ro1 antagonist nor-binaltorphimine (2). Apparently, constitutive Ro1-signaling was accountable for this mortality, and this receptor was not a real RASSL. For the F435A-RASSL, we believe that no constitutive activity-related side effects can be expected since, even at receptor expression levels exceeding 10 pmol/mg, no basal NF-B signaling is observed using a sensitive reporter gene assay. Besides our G␣ q/11 -coupled RASSL (H 1 R F435A), H 1 R F435L may also be useful for future in vivo use. When replacing the endogenous H 1 R, it would create a knockout system, displaying the H 1 R-like constitutive signaling but lacking histamine-induced responses. One would, however, still be able to induce H 1 R responses by administration of synthetic agonists.
DISCUSSION
The in vivo use of the RASSL Ro1 has generated considerable knowledge on the effect of G␣ i signaling on heart rate (1) and cardiomyopathy (2) . Currently, the Ro1 has also been expressed in other tissues of transgenic mice, including several brain areas, to further address the effects of G i signaling (6) . At this moment, much less is known about in vivo effects of G␣ q/11 stimulation. Our current G␣ q/11 -coupled RASSL may be employed to fill that gap of in vivo knowledge. Although the H 1 R RASSL can still be stimulated by the endogenous ligand histamine at high concentrations, plasma levels of histamine in healthy humans, rats, or rabbits are well below that level (2-250 nM) (32) (33) (34) (35) . When in vivo application is limited to tissues in which histamine concentrations are not exceptionally high, potentially excluding, for example, brain (synaptic histamine) and immune system (mast cell contents), the H 1 R RASSL is expected to remain quiescent until stimulation with synthetic agonists. Future research should focus on further modifications of our RASSL to make it even less responsive to histamine.
DISCUSSION
In conclusion, we exploited subtle differences in the binding pockets for different classes of H 1 R agonists and showed that the precise orientation of histamine and 2-phenylhistamine in the binding pocket is quite different. As a result, we have thus identified H 1 R F435A (6.55) as the first G␣ q/11 -coupled RASSL. This mutant H 1 R combines strongly decreased affinities and potencies for the endogenous ligand histamine with improved affinities and potencies for PheHAs, a synthetic class of H 1 R agonists. In contrast to mutant H 1 Rs F435V and F435L, this receptor lacks constitutive activity, which is a characteristic, we believe, that should be emphasized more strongly in the development of future RASSLs.

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