The histamine H₁ protein is a G protein-coupled receptor (GPCR) first cloned and characterized from bovine adrenal gland in 1991 (Yamashita et al., 1991). The cDNA genes encoding the H₁ receptor from other species were cloned soon after, including for human (De Backer et al., 1993). Southern blot analysis with H₁ receptor probes indicates that there are no related genes in various species, and there is no compelling evidence for H₁ receptor subtypes (Smit et al., 1999), although interspecies heterogeneity regarding H₁ pharmacology is known (Seifert et al., 2003). In most types of mammalian smooth muscle, endothelial, and brain tissue, histamine activation of H₁ receptors triggers Gα_q protein activation with subsequent stimulation of phospholipase C (PLC) and increased intracellular formation of inositol phosphates (IP) and diacylglycerol (Hill et al., 1997).

In mammalian brain and adrenal gland, activation of H₁ receptors also stimulates adenylyl cyclase (AC) and intracellular formation of cAMP. In rat brain, H₁-mediated stimulation of cAMP formation is enhanced by protein kinase C activation and is dependent on intra- and extracellular calcium (Garbarg and Schwartz, 1988). In bovine adrenal cells, H₁-mediated stimulation of cAMP formation also is dependent on extracellular calcium (Marley et al., 1991). In H₁-transfected CHO (CHO-H₁) cells, histamine H₁ activation augments forskolin-stimulated cAMP formation by a mechanism not sensitive to extracellular calcium, nor protein kinase C activation, and is pertussis toxin-insensitive (Leurs et al., 1994). Thus, although details such as G protein, kinase, and calcium involvement are not clear, H₁ receptors also can modulate AC/cAMP signaling in addition to PLC/IP signaling. This dual signaling mechanism now is thought to be common among GPCRs.

The phenomenon of multiple signaling pathways associated with a single GPCR can be described within the framework of the three-state model of GPCR activation (Leff et al., 1997), wherein GPCRs isomerize between inactive and constitutively active states (Kenakin, 2001). GPCR activation causes dissociation of heterotrimeric (α,β,γ) G protein subunits, the Gα subunit can then activate transducer protein (e.g., PLC and AC) to alter second messenger concentration. In addition to a role for G-βγ subunits in signal transduction (Clapham and Neer, 1997), it also is now realized the same GPCR can couple to different Gα proteins to result in “multifunctional signaling” (Milligan, 1993). A critical assumption of the GPCR multifunctional signaling theory is that a heterogeneity of active receptor conformations exists and that agonist ligands differ in their ability to induce, stabilize, or select among receptor conformations, as described in the “stimulus trafficking” hypothesis (Kenakin, 2001). It follows that, upon binding, agonist ligand chemical structural parameters are among the most important determinants of GPCR conformation that influences type of Gα protein and signaling pathway activated.

Previously, it was reported that the novel selective histamine H₁ ligand (-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (trans-PAT; Fig. 1) stimulates tyrosine hydroxylase activity and dopamine synthesis in rat and guinea pig forebrain in vitro and in vivo, by activating presynaptic H₁ receptors (Booth et al., 1999; Choksi et al., 2000). This effect of trans-PAT is similar to that observed for the endogenous agonist histamine (Fleckenstein et al., 1993), and effects of both ligands can be blocked specifically by typical H₁ antagonists such as triprolidine (Fig. 1). Based on these results, trans-PAT was proposed as a putative agonist at H₁ receptors linked to modulation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine neurotransmitter synthesis (Choksi et al., 2000). Subsequently, it was reported that trans-PAT also behaves as a potent antagonist (pA₂ = 9.2) regarding H₁-mediated contraction of guinea pig ileum, and trans-PAT fully blocks H₁ receptor activation of PLC/IP formation in CHO-H₁ cells (Booth et al., 2002). Radioreceptor competition binding results, using the standard H₁ antagonist radioligand [³H]mepyramine (Fig. 1), show the Hill coefficient (n_H) for the slope of the displacement curve by trans-PAT consistently is ∼0.9 using membranes prepared from rodent tissues and H₁-transfected clonal cell lines (Booth et al., 1999, 2002; Choksi et al., 2000), characteristic of agonist ligand binding at a GPCR, according to the ternary complex model with limiting availability of G protein (De Lean et al., 1980). The complex H₁ receptor activity shown by trans-PAT also has been observed in pilot studies for another PAT-type derivative, (±)-cis-5-phenyl-7-dimethylamino-5,6,7,8-tetrahydro-9H-benzocycloheptane (cis-PAB; Fig. 1), that behaves as an H₁ agonist or antagonist, depending on the functional assay (Moniri and Booth, 2004). The preliminary functional results reported for trans-PAT and cis-PAB are reminiscent of activities shown by certain dopamine D₂ receptor ligands that are agonists at postsynaptic D_2L receptors but antagonists at presynaptic D_2L receptors (Kilts et al., 2002; Mottola et al., 2002). The term “functional selectivity” was used to describe this phenomenon wherein a ligand that acts as an agonist at a GPCR linked to one particular signaling pathway may act as an antagonist at the same receptor linked to another signaling pathway. Functional selectivity can be exploited for drug design purposes as a practical application of the multifunctional signaling and stimulus-trafficking hypotheses.

In this article, we report the unique H₁ binding and selective functional activity (PLC/IP versus AC/cAMP signaling) of cis-PAB and trans-PAT, two novel H₁ ligands that are from the same chemical class but differ primarily with regard to stereochemistry (Fig. 1). Results of these studies suggest delineation of ligand molecular structural parameters that determine functionally selective binding should be an important consideration involved in designing GPCR-active drugs with predictable and selective pharmacotherapeutic effects versus untoward side effects.

Materials and Methods

Chemicals.trans-PAT (Fig. 1) was synthesized as described previously (Wyrick et al., 1993). Briefly, the benzylstyrylketone was cyclized to the tetralone intermediate and reduced to the (±)-cis- and (±)-trans-tetralols. This diastereomeric tetralol mixture was converted to the free amine, followed by dimethylation and fractional recrystallization, to isolate (±)-trans-PAT. The (±)-trans-PAT enantiomeric mixture was converted to the (-)-camphorsulfonic acid diastereomeric salt and resolved by fractional recrystallization to yield the pure (1R,3S)-(-)-trans-PAT isomer (Wyrick et al., 1993; Bucholtz et al., 1998, 1999). To synthesize cis-PAB (Fig. 1) (Wyrick et al., 1995), the corresponding diphenylpentenone was cyclized to the tetrahydrobenzocycloheptanone and reduced to the (±)-cis- and (±)-trans-tetrahydrobenzocycloheptanol diastereomers, which could be separated using silica gel column chromatography. Conversion of the (±)-cis-alcohols to the free amine, followed by dimethylation, gave (±)-cis-PAB as a solid that could be purified as the HCl salt; (±)-trans-PAB resisted crystallization and remained a gum. Resolution of the (5R,7S/5S,7R)-(±)-cis-PAB enantiomers currently is underway.

[³H]Mepyramine (specific activity 20 Ci/mmol), l-[1-¹⁴C]tyrosine (54 Ci/mmol), and [2-³H(N)]myoinositol (24 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Other compounds were obtained in highest purity from Sigma-Aldrich (St. Louis, MO).

CHO Cell Transfection and Culture. Chinese hamster ovary cells deficient in dihydrofolate reductase (CHO-K1) were stably transfected with the guinea pig histamine H₁ receptor cDNA (Traiffort et al., 1994). Clonal transfects expressing the H₁ receptor (CHO-H₁) were selected for in α-minimal essential media without ribo-nucleosides and supplemented with 10% fetal bovine serum and 2 mM l-glutamine. For binding and functional studies, CHO-H₁ cells were grown to 90% confluence in 75-cm² tissue culture flasks containing α-minimum essential medium, supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 0.1% penicillin-streptomycin (100 units/100 μg/ml), in a humidified atmosphere of air, CO₂ (95: 5%) at 37°C.

Null-transfected CHO-K1 cells were cultured as described above and used to verify that effects observed were H₁ receptor-dependent (selective H₁ antagonists also were used). Essentially, no H₁ radioligand specific binding was detected in membranes prepared from CHO-K1 cells as [³H]mepyramine total and nonspecific saturation binding was 480 ± 49 and 460 ± 27 fmol/mg protein (mean ± S.E.M.), respectively. Also, essentially no histamine receptor-mediated IP or cAMP second messenger formation was detected in lysates of CHO-K1 cells because second messenger levels were 99 ± 1.3% of basal control values (mean ± S.E.M.) after exposure to 10 μM histamine for 15 to 45 min.

Radioreceptor Assays. Radioligand competition and saturation binding assays were performed using membrane homogenate prepared from CHO-H₁ cells, as reported previously (Booth et al., 2002). For competition binding assays, membranes were incubated with ∼K_D concentration of the standard H₁ antagonist radioligand [³H]mepyramine (1.0 nM) plus test ligand (0.01–10,000 nM). For saturation isotherms, membranes were incubated in 0.01 to 10.0 nM [³H]mepyramine; some conditions included the H₁ antagonist triprolidine, trans-PAT, or cis-PAB, where possible, at ∼K_0.5, 10 times K_0.5, or 100 times K_0.5. concentration. Both assays used 50 mM Na⁺-K⁺ phosphate buffer (total assay volume was 0.4 ml), incubation was for 30 min at 25°C, and nonspecific binding was defined by triprolidine (10 μM). Inhibition data were analyzed by nonlinear regression using the sigmoidal curve-fitting algorithms in Prism 3.0 (GraphPad Software Inc., San Diego, CA) to determine IC₅₀ and Hill coefficient (n_H). In light of the incompletely characterized nature of the interaction between the H₁ receptor and the novel ligands used here, ligand affinity is expressed as an approximation of K_i values by converting IC₅₀ data to K_0.5 values using the equation K_0.5 = IC₅₀/1 + L/K_D, where L is the concentration of radioligand having affinity K_D (Cheng and Prusoff, 1973). Each experimental condition was run in triplicate and each experiment was performed a minimum of three times to determine S.E.M.

Measurement of [³H]Inositol Phosphate Formation in CHO-H₁ Cells. Formation of [³H]IP was measured in CHO-H₁ cells, as described previously (Booth et al., 2002). Briefly, CHO-H₁ cells were incubated overnight in 12-well culture plates (∼7.0 × 10⁴ cells/well) with [³H]myo-inositol (0.4 μCi), a precursor of the PLC-β substrate phosphatidylinositol. Aliquots of drug stocks were added in triplicate in the presence of 50 mM LiCl (total well volume 0.5 ml) and incubation continued at 37°C for 45 min. After aspiration of media, wells were placed on ice and lysed by incubation with 50 mM formic acid (15 min). Formic acid was neutralized with ammonium hydroxide and well contents were added to individual AG1-X8 200-400 formate resin anion exchange columns. Ammonium formate/formic acid (1.2 M/0.1 M) was used to elute [³H]IP directly into scintillation vials for counting of tritium. Resulting data were analyzed using the nonlinear regression algorithm in Prism 3.0 and are expressed as mean percentage of control [³H]IP formation, and potencies are expressed as concentrations required to produce 50% maximal [³H]IP formation (EC₅₀) ± S.E.M. (n ≥ 3).

Measurement of cAMP Formation in CHO-H₁ Cells. Formation of cAMP was measured in CHO-H₁ cells grown in 24-well plates, preincubated for 5 min in serum-free media in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1.0 mM), followed by addition of drug stocks in serum-free media. After incubation for 15 min at 37°C, plates were placed on ice, and the cells were lysed by addition of 0.1 M HCl followed by sonication. Well contents were centrifuged (700g; 10 min) individually, and aliquots of the supernatants were used in the direct-cAMP immunoassay kit (Assay Designs Inc., Ann Arbor, MI). Data were expressed as mean percentage of control cAMP formation as obtained by linear standard curve extrapolation, and potencies are expressed as concentrations required to produce 50% maximal cAMP formation (EC₅₀) ± S.E.M. (n ≥ 3).

Results

H₁ Radioreceptor Binding Assays Using CHO-H₁ Cell Membranes

Competition Binding Analysis. Representative H₁ radioligand (the antagonist [³H]mepyramine) displacement curves for trans-PAT and cis-PAB, in comparison with the endogenous agonist histamine and the standard competitive H₁ antagonist triprolidine (Hill et al., 1997), are shown in Fig. 2. Curves are sigmoidal and span three to four log ligand concentration units to achieve complete radioligand displacement, characteristic of competitive displacement of ∼K_D radioligand concentration from a single population of GPCRs. Values for K_0.5± S.E.M. (nanomolar) for triprolidine, trans-PAT, cis-PAB, and histamine are 0.93 ± 0.16, 1.15 ± 0.36, 175.0 ± 16.0, and 18,300 ± 1200, respectively. n_H for the slope of the competitive displacement curve was ∼1.0 (0.98 ± 0.08) for the H₁ antagonist triprolidine. n_H was < 1.0 for trans-PAT, cis-PAB, and histamine (n_H± S.E.M.; 0.87 ± 0.14, 0.77 ± 0.03, and 0.72 ± 0.09, respectively), characteristic of agonist ligand binding at a GPCR, according to the ternary complex model with limiting availability of G protein (De Lean et al., 1980).

Saturation Binding Analysis.Figure 3, A to C, shows representative saturation binding curves for [³H]mepyramine alone and in the presence of triprolidine, cis-PAB, and trans-PAT, at increasing concentration (∼K_0.5, ∼10 times K_0.5 and ∼100 times K_0.5, except for cis-PAB, where maximum solubility was 30 μM). As shown in Fig. 3A, the B_max value for [³H]mepyramine binding in absence of triprolidine was 320 ± 11 fmol/mg protein, and this value is not significantly different in presence of 1.0, 10, and 100 nM triprolidine (ANOVA; p = 0.1). The K_D value for [³H]mepyramine binding increases from 0.51 ± 0.01 nM in the absence of triprolidine to 1.24 ± 0.05, 3.64 ± 0.01, and 16.3 ± 2.50 nM, in the presence of 1.0, 10, and 100 nM triprolidine, respectively (ANOVA; p < 0.003). These results (no change in B_max but decreased affinity of radioligand in presence of increasing concentration of competitor), suggest that triprolidine binds to H₁ receptors in accordance with typical Michaelis-Menten type competitive kinetics. Figure 3B shows that analogous results are obtained using cis-PAB as displacing ligand, i.e., there is no significant change in [³H]mepyramine B_max, but K_D value increases from 0.43 ± 0.02 nM in absence of cis-PAB to 0.52 ± 0.01, 2.50 ± 0.03, and 7.31 ± 0.31 nM in the presence of 0.1, 0.5, and 1.0 μM cis-PAB, respectively (ANOVA; p < 0.0001). Thus, it seems cis-PAB binding to H₁ receptors also is according to typical competitive kinetics. In contrast, Fig. 3C shows that when the displacing ligand is trans-PAT, the [³H]mepyramine B_max value decreases from 260 ± 4.9 fmol/mg protein in absence of trans-PAT to 230 ± 1.1, 160 ± 4.4, and 52 ± 0.6 fmol/mg protein in presence of 1.0, 10, and 100 nM trans-PAT, respectively (ANOVA; p < 0.0001). Also, Fig. 3C shows that K_D for [³H]mepyramine increases from 0.34 ± 0.05 nM in absence of trans-PAT to 1.21 ± 0.32, 1.64 ± 0.31, and 2.61 ± 0.14 nM in presence of 1.0, 10, and 100 nM trans-PAT, respectively (ANOVA; p < 0.002). These results (decreased B_max value and decreased affinity of radioligand in presence of increasing concentrations of displacing ligand), suggest trans-PAT binding to H₁ receptors is complex and not according to typical competitive kinetics. This unique trans-PAT binding interaction with H₁ receptors is not obvious from the results of Fig. 2, where K_D amount of [³H]mepyramine is used to label H₁ receptors, i.e., only one-half the total H₁ receptor population is labeled. Likewise, it seems that when concentration of trans-PAT begins to exceed K_0.5, it binds to H₁ receptors in a manner that is not competitive, decreasing the total number of H₁ receptors that can be labeled by [³H]mepyramine (B_max) and also decreasing apparent H₁ affinity of [³H]mepyramine (K_D).

Stimulation of PLC/IP Formation in CHO-H₁ Cells

Figure 4 shows that histamine stimulates PLC/[³H]IP formation in a concentration-dependent manner in CHO-H₁ cells, with E_max of ∼100 μM (∼900% basal control activity) and EC₅₀ of ∼3 μM; the effect at EC₅₀ is fully blocked by the competitive H₁ antagonist triprolidine (Fig. 4, inset). Also shown in Fig. 4, cis-PAB stimulates formation of [³H]IP in CHO-H₁ cells in a concentration-dependent manner. Although solubility problems limited maximal cis-PAB concentrations to ∼30 μM, it could be determined that E_max is ∼10 μM (∼200% basal control activity; ∼20% histamine E_max) and EC₅₀ is ∼140 nM; the effect at EC₅₀ is fully blocked by triprolidine (Fig. 4, inset). Even at concentrations up to 30 μM, trans-PAT had no effect to stimulate [³H]IP formation in CHO-H₁ cells (Fig. 4).

Although cis-PAB is ∼20-fold more potent than histamine at stimulating [³H]IP formation (EC₅₀ ∼140 nM versus 3.0 μM), the maximal stimulation produced by cis-PAB is ∼20% of the histamine maximal response. These results suggest that in comparison with the endogenous agonist histamine, cis-PAB is a partial agonist regarding H₁-mediated stimulation of PLC/IP formation; accordingly, cis-PAB should seem to be an antagonist in the presence of histamine in this H₁ functional assay. Figure 5 shows that, in fact, histamine H₁-mediated stimulation of PLC/[³H]IP formation is competitively antagonized by cis-PAB, as indicated by a histamine EC₅₀ value that increases as concentration of cis-PAB increases (shift to the right in concentration-response curve), i.e., histamine EC₅₀ is ∼3 μM in absence of cis-PAB, but histamine EC₅₀ increases to ∼8 and ∼25 μM in presence of 0.1 and 1.0 μM cis-PAB, respectively (ANOVA; p < 0.0001). Histamine E_max for stimulation of PLC/IP formation is achieved, regardless of the concentration of cis-PAB present. As described by Kenakin (1993), competitive antagonism by a weak partial agonist (i.e., cis-PAB) can be treated as competitive agonism by the antagonist, because the error in the apparent K_B value introduced by the weak partial agonist is negligible. Assuming competitive antagonism by cis-PAB, the data in Fig. 5 yield an apparent K_B value of 180 nM.

The Fig. 5 inset shows that, as expected, the H₁ antagonist triprolidine also competitively antagonizes histamine stimulation of [³H]IP formation. This result is indicated by histamine EC₅₀ values that increase concomitant with increasing concentration of triprolidine, i.e., the histamine EC₅₀ is ∼4 μM in absence of triprolidine, but increases to ∼40 and ∼300 μM in presence of 0.01 and 0.01 μM triprolidine, respectively (ANOVA; p < 0.0001). For triprolidine, the data in Fig. 5 yield an apparent K_B value of 2.0 nM (nearly 100 times more potent than cis-PAB, consistent with its higher H₁ affinity). Overall, the results in Figs. 4 and 5 confirm the H₁ competitive antagonist activity of triprolidine and identify cis-PAB as a novel potent partial agonist (in comparison with histamine) at H₁ receptors that activate PLC/IP formation.

Results summarized in Fig. 6 show that in contrast to the competitive antagonism observed for cis-PAB and triprolidine (Fig. 5), trans-PAT antagonism of histamine H₁-mediated stimulation of PLC/[³H]IP formation apparently is not competitive. For instance, the histamine EC₅₀ is ∼3 μM in the absence of trans-PAT, but the EC₅₀ value increases to ∼10, 17, and 27 μM in presence of 0.01, 0.1, and 1.0 μM trans-PAT, respectively (ANOVA; p < 0.0001). Also, the histamine E_max decreases from 100% response in the absence of trans-PAT to ∼ 95, 60, and 50% response in presence of increasing concentrations of trans-PAT (ANOVA; p < 0.0001). As shown in the Fig. 6 inset, trans-PAT antagonism of cis-PAB H₁-mediated stimulation of PLC/[³H]IP formation seems to be competitive. For instance, whereas the cis-PAB EC₅₀ is ∼85 nM in the absence of trans-PAT, the EC₅₀ value increases to ∼850 and 2600 nM in the presence of 0.01 and 0.1 μM trans-PAT, respectively (ANOVA; p < 0.0001). Also, consistent with competitive antagonism, the cis-PAB E_max is unchanged in the absence or presence of trans-PAT (ANOVA; p = 0.2).

Stimulation of AC/cAMP Formation in CHO-H₁ Cells

Results in Fig. 7 show that histamine stimulates AC/cAMP formation over basal control levels in a concentration-dependent manner in CHO-H₁ cells, with E_max ∼100 μM (∼170% basal control activity) and EC₅₀ ∼2.1 μM. Moreover, the novel H₁ ligand trans-PAT also stimulates AC/cAMP in this assay system with efficacy and potency similar to histamine. Although solubility problems limited maximal trans-PAT concentrations to ∼1.0 mM, it could be determined that E_max is ∼160% basal level at 100 μM and EC₅₀ is ∼2.0 μM. Maximal response for both histamine and trans-PAT is comparable with the AC activator forskolin (∼173% basal at 1.0 μM) here, and the histamine response also is comparable with that observed in bovine adrenal cells (∼200%) (Marley et al., 1991). At EC₅₀, both histamine and trans-PAT H₁-mediated increases in cAMP are significantly different than basal (p < 0.01), and effects of both ligands are fully blocked by the H₁ antagonist triprolidine (data not shown). At concentrations 0.01 to 100 μM, cis-PAB alone had no effect on cAMP formation, but it could competitively antagonize H₁-mediated stimulation of AC/cAMP formation produced by trans-PAT (Fig. 7, inset) and histamine (data not shown).

Discussion

Results of these studies indicate that the novel H₁ ligands trans-PAT and cis-PAB can display mutually opposing activity and selectively activate and block different H₁-linked intracellular signaling pathways, i.e., the AC/cAMP and PLC/IP signaling cascades. The endogenous agonist histamine, on the other hand, nonselectively activates both H₁ pathways. The literature now documents many observations of the same GPCR activating different intracellular signaling pathways. For example, adrenergic α_1B receptors, which predominantly couple to Gα_q/PLC to stimulate IP formation, also can couple to Gα_S/AC to stimulate cAMP formation in CHO cells expressing α_1B cDNA (Horie et al., 1995). The reverse also is observed, e.g., histamine H₂ receptors, which predominantly couple to Gα_S/AC/cAMP, also can couple to Gα_q/PLC/IP in COS cells expressing H₂ cDNA (Kuhn et al., 1996). The 5-HT_2C receptor, that is phylogenetically closely related to H₁ receptors (Smit et al., 1999), predominantly couples to Gα_q to activate PLC but also can modulate AC/cAMP formation through Gα_i (Cussac et al., 2002). Recent evidence also suggests that H₁ receptors may mediate antinociception via G_i/o signaling in vivo (Galeotti et al., 2002). GPCRs are not limited to just two different G protein partners; many of the 30 or so different 5-HT receptors couple with several different G proteins and can modulate a half-dozen different intracellular signaling pathways (Raymond et al., 2001).

Examples of GPCR signaling promiscuity have been demonstrated using native intact cells, primary cultures, and in vivo animal models (Allgeier et al., 1994; Arey et al., 1997; Galeotti et al., 2002), suggesting that multifunctional signaling among GPCRs is not necessarily an artifact of receptor overexpression in clonal cell lines. On the other hand, receptor and/or G protein overexpression might reveal both qualitative and quantitative ligand-GPCR responses that might not be detected in native cell and tissue preparations. Nevertheless, whereas the H₁-linked AC/cAMP signaling revealed here for trans-PAT may be due, in part, to receptor and/or G protein overexpression, the lack of effect for cis-PAB to activate H₁/AC/cAMP signaling in comparison with trans-PAT and histamine in the same assay system indicates that the effect is ligand-specific. Clearly, too, the activity of trans-PAT as a functionally selective full agonist regarding H₁/AC/cAMP signaling is a concentration-dependent phenomenon. Thus, whereas trans-PAT binds to the H₁ receptor (Fig. 2) and blocks H₁/PLC/IP signaling (Fig. 3C) at concentrations as low as ∼1.0 nM, significant activation of H₁/AC/cAMP signaling does not occur until concentration of trans-PAT approaches ∼1.0 μM (Fig. 7). Whereas the physiological relevance of concentration-dependent functional selectivity is presently unclear, preliminary results presented in this article suggest that specific ligands can be designed to selectively activate or block specific intracellular signaling pathways for multifunctional signaling GPCRs.

Molecular mechanisms to account for GPCR multifunctional signaling involve the concept of “GPCR permissiveness” that assumes a high degree of flexibility in the interactions between a ligand, receptor, and G protein (Raymond, 1995). These interactions occur mainly between the G proteins and the second and third intracellular loops and carboxy-terminal tail of the receptor. Some factors that influence this interaction include receptor-G protein ratios and amounts, alternative GPCR splicing, and conformational changes in the G protein and/or receptor. In the present study, amount and ratio of H₁ receptors and G protein types expressed in the CHO-H₁ cells probably is a factor in signaling. This is suggested by the robust potency and efficacy of histamine to stimulate H₁-mediated PLC/IP formation (Fig. 3) such that EC₅₀ (∼2.6 μM) is well below K_0.5 (∼18 μM), and the maximal effect is nearly a 10-fold increase over basal IP formation. In contrast, a 3-fold increase in basal IP formation by histamine is the maximum we found reported using mammalian brain, adrenal, retina, and ileum tissue preparations (Hill et al., 1997). Receptor splice variants are not likely to play a role in the ligand-directed signaling results observed here because the CHO-H₁ cells were transfected with H₁ cDNA, and, in any event, we could find no evidence in the literature for the existence of H₁ splice variants. Receptor conformational changes, however, likely are a factor for the current results, given that ligand chemical structure is among the most important molecular determinants for GPCR conformation that leads to activation of signaling (Kenakin, 2001). Furthermore, there is the phenomenon of spontaneous “precoupling of receptor-Gα protein complexes” (Leff et al., 1997) that explains observed GPCR constitutive activity, now well documented, including for H₁ receptors (Bakker et al., 2000). Such spontaneous receptor-Gα protein coupling suggests that ligand binding influences stabilization of fluctuating GPCR-Gα conformations and that ligand chemical structural parameters determine which conformation will be stabilized, induced, or selected (Kenakin, 2001).

The molecular determinants for trans-PAT versus cis-PAB differential binding to the H₁ active site that leads to differential activation of the AC/cAMP versus PLC/IP signaling pathways likely are due, in large part, to stereochemical factors. The H₁ receptor is known to be highly sensitive to the stereochemistry of PAT/PAB-type ligands. For example, cis-PAT is an H₁ antagonist that blocks H₁-medited stimulation of tyrosine hydroxylase and dopamine synthesis by trans-PAT in rat forebrain (Choksi et al., 2000). Likewise, rank order of PAT isomer H₁ affinity depends on stereochemistry ([1R,3S]-[-]-trans > [1S,3S]-[-]-cis > [1S,3R]-[+]-trans > [1R,3R]-[+]-cis) and varies ∼50-fold (K_0.5 ∼1–50 nM), with S-chirality at the C3 amine position (Fig. 1) being the most important structural determinate for binding (Buchlotz et al., 1998, 1999). Interestingly, only the (-)-trans-PAT isomer has H₁ agonist activity (AC activation); the other isomers are H₁ antagonists regarding PLC and AC activation (Booth et al., 2002; Moniri and Booth, 2004). Previous molecular modeling studies (Bucholtz et al., 1999) suggest that the protonated amine moiety of PAT/PAB-type compounds forms an ionic bond with the H₁ Asp¹¹⁶ residue (guinea pig numbering) in transmembrane helix (TMH) 3. The equivalent TMH3 Asp¹¹⁶ residue is highly conserved among biogenic amine neurotransmitter GPCRs, and mutagenesis studies suggest this residue interacts with a positively charged amine moiety of endogenous agonists and other ligands (Savarese and Fraser, 1992), including for the H₁ receptor (Ohta et al., 1994) and the serotonin 5-HT₂ receptor family (Wang et al., 1993; Kristiansen et al., 1996) that is phylogenetically closely related to H₁ (Smit et al., 1999). Whereas the main structural difference between trans-PAT and cis-PAB is stereochemical, there also is added conformational flexibility in the hexane (PAT) versus heptane (PAB) ring systems, and molecular modeling studies indicate both structural parameters influence orientation of the dimethylamino and appended phenyl moieties (Bucholtz et al., 1999). Thus, in addition to differential interactions of the trans-PAT and cis-PAB amine moieties with H₁ TMH 3 residue(s), the phenyl moieties of these ligands may form differential π-π electron binding interactions with aromatic amino acids of the H₁ receptor binding pocket. In this regard, mutational analysis and molecular dynamics simulations of the H₁ receptor (Elz et al., 2000) and the related 5-HT_2A serotonin receptor (Kroeze et al., 2002) suggest that π-π stacking interactions occur between bound aromatic-containing ligands and aromatic amino acid residues in TMH 5 and 6. Mutagenesis studies to probe the molecular interactions of trans-PAT versus cis-PAB with H₁ active site amino acid residues currently are underway in our laboratory.

Presently, we have not succeeded in separating the (5R,7S) and (5S,7R) enantiomers of (±)-cis-PAB. We note that the nearly 5-fold lower efficacy of cis-PAB in comparison to histamine with regard to H₁-mediated activation of PLC/IP formation (Fig. 5) might involve antagonism of one cis-PAB enantiomer by the other, analogous to (+)-trans-PAT antagonism of (-)-trans-PAT regarding H₁-mediated activation of tyrosine hydroxylase and dopamine synthesis (Booth et al., 1999; Choksi et al., 2000). Based on studies with PAT, we predict that (5S,7R)-cis-PAB will be the more active enantiomer at H₁ receptors because this isomer has the critical dimethylamino moiety in the same configuration as (1R,3S)-(-)-trans-PAT (stereochemical nomenclature rules result in differential R,S designation for the amine moieties of the two molecules even though they share the same three-dimensional configuration; Fig. 1).

The binding interaction between trans-PAT and the H₁ receptor seems to be complex given that it is not competitive with respect to antagonism of H₁ saturation binding by [³H]mepyramine (Fig. 3C) and H₁/PLC/IP functional activation by histamine (Fig. 6). The H₁/PLC/IP functional interaction between trans-PAT and cis-PAB is typically competitive (Fig. 6, inset), suggesting that there is overlap in the H₁ binding pharmacophores of trans-PAT and cis-PAB. Here again, mutagenesis studies will be helpful to sort H₁ receptor molecular recognition determinants for trans-PAT versus cis-PAB functionally selective binding that presumably leads to differential activation of AC/cAMP versus PLC/IP signaling.

To the best of our knowledge, the present results are the first to show that different but structurally related ligands can be developed to selectively activate or block two different intracellular signaling pathways for the same GPCR, i.e., H₁ receptors linked to AC/cAMP and PLC/IP signaling. Previously, it has been shown that a novel ligand ([Gly¹,Arg¹⁹]hPTH-[1-28]) could be designed to selectively activate one of the two signaling pathways (AC/cAMP versus PLC/IP) associated with the type 1 parathyroid GPCR (Takasu et al., 1999). The PAT/PAB-type probes, however, will be uniquely useful to study molecular determinants of switching mechanisms involved in GPCR multifunctional signaling. Such functionally selective ligands targeted to GPCRs also hold promising pharmacotherapeutic utility. For example, activation of H₁-linked PLC/IP signaling in peripheral smooth muscle and endothelial tissues can present clinically as respiratory distress (bronchial constriction), diarrhea (gastrointestinal contractions), and edema and hypotension (increased vascular permeability), especially associated with an allergic response. trans-PAT, however, is a lipophilic molecule that can penetrate brain tissue to selectively activate H₁-linked AC/cAMP signaling, presumably leading to modulation of tyrosine hydroxylase activity and catecholamine neurotransmitter synthesis (Choksi et al., 2000; Moniri and Booth, 2004). Because most untoward cardiovascular-, respiratory-, and gastrointestinal H₁-mediated effects proceed via the PLC/IP pathway (Hill et al., 1997), PAT-type drugs that selectively enhance H₁-mediated AC/cAMP signaling in brain provide a mechanistic basis for exploiting H₁ receptors as pharmacotherapeutic targets in neuropsychiatric and neurodegenerative disorders involving alterations in catecholamine neurotransmission.