Two Distinct Pathways for Histamine H2 Receptor Down-regulation

Pretreatment of Chinese hamster ovary cells expressing the histamine H receptor (CHOrH cells) with histamine resulted in a time-dependent (t ≈ 7 h) and dose-dependent (EC = 18 nM) H receptor down-regulation measured as [I]iodoaminopotentidine binding (44 ± 10% down-regulation). Pretreatment of CHOrH cells with cholera toxin or forskolin also led to H receptor down-regulation. Forskolin time-dependently (t ≈ 7 h) and dose-dependently (EC = 0.3 μM) induced H receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O., Lameh, J., Hogger, P., and Sadée, W.(1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H receptor from the G-protein, we introduced the Leu Ala mutation in the second intracellular loop of the H receptor. The H Leu Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH cells, H receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.

The introduction of molecular biology in the field of histamine receptor research has greatly improved the possibilities to study molecular aspects of histamine receptor proteins. In 1991, Gantz et al. (1) cloned the cDNA encoding the canine histamine H 2 receptor, which was followed by the cloning of both the rat and human homologues (2,3). The deduced amino acid sequence of the H 2 receptor proteins reveals the existence of seven putative transmembrane domains, indicating that this receptor is a member of the large family of G-protein-coupled receptors (GPCR). 1 This family of receptors is known to be readily subjected to regulatory processes in order to control receptor signaling and thus cellular communication (4). Shortterm exposure of receptors to high concentrations of agonists is often followed by a decrease in cellular responsiveness, called desensitization (5). Long-term exposure, on the other hand, results in the reduction of receptor number (6) and is referred to as receptor down-regulation. Since the histamine H 2 receptor is a member of this family of GPCRs, it is not surprising that this receptor is also susceptible to such regulatory mechanisms.
Recently, we have shown that in human U937 cells the endogenously expressed histamine H 2 receptors are indeed rapidly desensitized when exposed to histamine (7). Similar observations have been reported in other cellular systems (8,9). Yet, so far, no detailed information is available on long-term desensitization of the histamine H 2 receptor such as receptor down-regulation. Such processes may become apparent under several pathophysiological conditions (e.g. asthmatic attack or allergic reactions in general), during which histamine is released in large quantities, but might also occur under normal physiological conditions. Recently, Diaz et al. (10) suggested for example that in vivo receptor down-regulation might explain the inverse relationship between H 2 receptor expression and the localization of histamine-synthesizing cells in the rodent gastric wall. The regulation of H 2 receptor expression has gained further interest due to the potential therapeutic application of H 2 receptor agonists in patients suffering from congestive heart failure (11).
Investigation of the regulation of H 2 receptor expression has so far been hampered by the availability of suitable model systems. Cellular systems (7)(8)(9)12) have been used to investigate second messenger responses coupled to the histamine H 2 receptor stimulation, but the used systems such as U937 cells for example do not express a sufficiently high density of H 2 receptors to permit radioligand binding studies, which are essential for the investigation of long-term regulatory mechanisms (7). Following the recent cloning of cDNAs or genes encoding histamine H 2 receptors, cell lines expressing considerable amounts of histamine H 2 receptors can be obtained (13,14). Additionally, the availability of the H 2 receptor gene allows the construction of receptor mutants, which can provide mechanistic insights in phenomena like receptor down-regulation.
In the present study we have examined the effects of longterm exposure of the rat histamine H 2 receptor stably expressed in Chinese hamster ovary (CHO) cells (referred to as CHOrH 2 cells) (13) to H 2 agonists and cAMP mobilizing agents with regard to H 2 receptor protein expression and H 2 receptor mRNA levels. In order to get more insight into the mechanisms underlying H 2 receptor regulation, we constructed a H 2 receptor mutant, in which leucine 124 in the second intracellular loop was substituted by an alanine. This H 2 Leu 124 3 Ala receptor mutant was partially uncoupled from its G-protein and proved to be a suitable tool for investigating the existence of possible cAMP-dependent and independent pathways in the process of agonist-induced H 2 receptor down-regulation.
Site-directed Mutagenesis-The H 2 receptor mutant, in which leucine 124 was substituted by alanine (Leu 124 3 Ala) (Fig. 7), was constructed by means of the polymerase chain reaction. The oligonucleotides S1 (5Ј-GGGAAGCTTGGCCCCAGAATGGAGCCCAATGGCACAGT), corresponding to nucleotides Ϫ9 to 21 (2) and a HindIII site (underlined), and AS1 (5Ј-GGGGGTACCGCGCTGGGTCCGTGACAGCGCAGTAG-TTGTTCAAGCTGATCAT), corresponding to nucleotides 358 to 383 of the complementary strand (2) containing a KpnI site (underlined) with two nucleotide changes, were synthesized on an Applied Biosystems DNA synthesizer (model 381A). Using 100 ng of pSVrH 2 (13) as a template, 0.4 M S1, 0.4 M AS2, 40 M dNTPs, and 2.5 units of Amplitaq according to the manufacturer's protocol (Perkin Elmer), a 392-base pair DNA fragment of the H 2 Leu 124 3 Ala receptor mutant was amplified in 100 l using 30 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 1.5 min and a final extension at 72°C for 10 min. The obtained PCR product was gel-purified and restricted with HindIII/ KpnI (Boehringer). This fragment was cloned into the plasmid pSP73 (Promega) containing the wild-type rH 2 receptor, which was restricted with KpnI and HindIII. Thereafter, the PCR-amplified sequence was verified using the dideoxy chain termination method with the Sequenase kit (U. S. Biochemical Corp.). Subsequently, the coding sequence of the mutated H 2 Leu 124 3 Ala receptor was subcloned into the eukaryotic pSV expression vector. CHO cells, deficient in dihydrofolate reductase, were stably transfected with 15 g of pSVrH 2 Leu 124 Ala using Transfectam (Promega).
Membrane Preparation-CHOrH 2 and CHOrH 2 Leu 124 Ala cells were washed three times with cold phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ) and harvested with a cell scraper and recovered by a 10-min centrifugation at 500 ϫ g. Cells were homogenized in ice-cold 50 mM Na 2 /potassium phosphate buffer (pH 7.4) with a Polytron homogenizer (5 s, maximal speed) and used for radioligand binding studies. Protein concentrations were determined according to Bradford using bovine serum albumin as a standard (15).
Histamine H 2 Receptor Binding-The radiolabeled H 2 receptor antagonist [ 125 I]iodoaminopotentidine ([ 125 I]APT) was synthesized as described previously (14). Triplicate assays were performed in polyethylene tubes in 50 mM Na 2 /potassium phosphate buffer containing gelatin (0.1%) to prevent adsorption of the radioligand. In saturation studies, increasing concentrations of [ 125 I]APT were incubated with 5-10 g of membrane proteins in the absence or presence of 1 M tiotidine in a total volume of 400 l. After 90 min at 30°C, the incubations were stopped by rapid dilution with 3 ml of ice-cold 20 mM Na 2 /potassium phosphate buffer (pH 7.4) supplemented with 0.1% bovine serum albumin. The bound radioactivity was subsequently separated by filtration with a Brandel cell harvester (Semat) through Whatman GF/B glass fiber 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 counted with a LKB-␥-counter at an efficiency of 63%. The binding data were evaluated by use of LIGAND, a nonlinear, weighted, least squares curve-fitting procedure (16). Changes in H 2 receptor density were denoted as a percentage down-regulation compared to nontreated control cells. During the 24-h incubation of cells with various histamine ligands or other compounds, cells were maintained in medium without fetal calf serum.
Cyclic AMP Production-CHOrH 2 and CHOrH 2 Leu 124 Ala cells were seeded in 24-well plates and cultured overnight in culture medium. Cells were washed twice with DMEM, supplemented with 50 mM HEPES (pH 7.4 at 37°C), and preincubated for 30 min at 37°C. Thereafter, the medium was aspirated, appropriate drugs in DMEM/HEPES supplemented with 300 M phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were added, and the cells were incubated for 10 min at 37°C. The reaction was stopped by the rapid aspiration of the culture medium and the addition of 200 l of 0.1 N cold HCl. The cells were kept on ice and disrupted by sonification (5 s, 50 watts, Labsonic 1510, Braun-Melsungen). The resulting homogenate was frozen at Ϫ20°C or directly neutralized with 1 N NaOH and assayed for the presence of cAMP. In order to determine the long-term effects of histamine treatment on H 2 receptor signaling, CHOrH 2 cells were preincubated with 100 M histamine for 24 h in DMEM without fetal calf serum. Thereafter, these cells were thoroughly washed and preincubated for 1 h in DMEM/HEPES at 37°C before actual incubation with the indicated drugs.
Cyclic AMP Assay-The amount of cAMP in the CHOrH 2 and CHOrH 2 Leu 124 Ala cells was determined according to Nordstedt and Fredholm (17), with some minor modifications. Briefly, a protein kinase A-containing fraction was isolated from bovine adrenal glands. Adrenal cortex was homogenized in 10 volumes of 100 mM Tris-HCl, 250 mM NaCl, 10 mM EDTA, 0.25 M sucrose, and 0.1% 2-mercaptoethanol (pH 7.4 at 4°C, buffer A) using an Omni-Sorval mixer (30 s, maximal speed) and a Polytron homogenizer (10 s, maximal speed). The homogenate was centrifuged for 60 min at 30,000 ϫ g at 4°C. The supernatant, containing protein kinase A, was carefully recovered and frozen in 1 ml aliquots at Ϫ80°C. Before use, the binding protein was diluted 5-fold in ice-cold buffer A without sucrose and 2-mercaptoethanol and kept on ice. Subsequently, 200 l of the binding protein was mixed with 50 -100 l of the CHO homogenate or cAMP standards and 30,000 dpm of [ 3 H]cAMP. After incubation for 150 min at 4°C, the mixture was rapidly diluted with 3 ml of ice-cold 50 mM Tris-HCl (pH 7.4 at 4°C) and filtered through Whatman GF/B filters using a Brandel cell-harvester (Semat). The radioactivity retained on the filters was measured by liquid scintillation counting.
Analysis of H 2 Receptor mRNA Stability-H 2 receptor mRNA levels were determined after incubation of the CHOrH 2 cells with actinomycin D to block transcription as described previously (20). Cells were preincubated with or without 100 M histamine or with 10 M forskolin for 1 h in DMEM. Thereafter, actinomycin D (10 g/ml) was added. Cells were harvested from 0 to 90 min after addition of actinomycin D. Total RNA was extracted at each time point, and H 2 receptor mRNA was quantified by means of the mRNA slot blot assay as described above.
Statistical Analysis-All data shown are expressed as mean Ϯ S.E. of at least three independent experiments. Statistical analysis was carried out by Student's t-test. p values Ͻ 0.05 were considered to indicate a significant difference.  (Table I). Exposure of CHOrH 2 cells to histamine resulted only in a marked decrease of the total number of [ 125 I]APT binding sites (B max ) ( Table I).

Histamine-induced H 2 Receptor Down-regulation-Exposure
The recently described selective H 2 receptor agonists amsel-amine and amthamine (21,22) induced cAMP production in CHOrH 2 cells, with EC 50 values lower and maximum responses comparable to histamine (Table II) (Table II). The reduced ability of these dimaprit analogues to induce a cAMP response was paralleled by a lack of H 2 receptor downregulation after 24 h of incubation of CHOrH 2 cells with 100 M concentrations of the analogues (Table II).
Effect of Long-term Histamine Treatment on Histamine-and Forskolin-induced Signaling in CHOrH 2 Cells-Long-term exposure (24 h) of CHOrH 2 cells with 100 M histamine resulted in a rightward shift of the dose-response curve of the histamine-induced cAMP production (EC 50 of histamine-induced cAMP response in nontreated cells: 36 Ϯ 3 nM, mean Ϯ S.E., n ϭ 7, and histamine-treated cells: 1.2 Ϯ 0.05 M, mean Ϯ S.E., n ϭ 4) (Fig. 2A). The forskolin-induced rise in cAMP was not found to be affected as no change in dose dependence or im- Significant difference (p Ͻ 0.05) from control, represented by nontreated cells.
b The E max value of nordimaprit was determined using a concentration of 1 mM.
c Determined at 100 M.

Down-regulation of the Histamine H 2 Receptor
pairment of the maximal forskolin-induced cAMP response was observed after a 24-h pretreatment of cells with 100 M histamine (Fig. 2B).

Role of cAMP in the Process of H 2 Receptor
Down-regulation-Forskolin, which directly activates adenylyl cyclase, dose dependently induced the formation of cAMP in CHOrH 2 cells (Fig. 3A). Prolonged exposure (incubation periods ranging from 4 to 32 h) of CHOrH 2 cells with 10 M forskolin led to a marked reduction of 58 Ϯ 2% [ 125 I]APT binding (Fig. 3B). Again, no major change in affinity of [ 125 I]APT for the H 2 receptor was apparent, only a decrease in B max was observed when CHOrH 2 cells were incubated for 24 h with 10 M forskolin (Table I). Maximum and half-maximum down-regulation was recorded after 16 h and approximately 7 h of incubation of CHOrH 2 cells with 10 M forskolin, respectively (Fig. 3B). The H 2 receptor binding sites appeared to be dose dependently down-regulated by increasing concentrations of forskolin, with an EC 50 value of 0.3 Ϯ 0.06 M (mean Ϯ S.E., n ϭ 4) (Fig. 3C). Concentrations up to 10 M of the inactive analogue 1,9-dideoxyforskolin, which does not generate cAMP in CHOrH 2 cells (Fig. 3A), did not attenuate the H 2 receptor density after 24 h of pretreatment (Fig. 3D).
H 2 Receptor mRNA Levels and Stability in Control, Histamine-treated, and Forskolin-treated CHOrH 2 Cells-Exposure of CHOrH 2 cells to 100 M histamine for increasing periods of time resulted in a rapid transient decrease of H 2 receptor mRNA (maximum reduction of 71 Ϯ 4%, mean Ϯ S.E., n ϭ 4) (Fig. 5). This effect was at its peak after 4 h of incubation of cells with histamine (100 M), while the amount of H 2 receptor mRNA returned to approximately 50% of control after 12 h of histamine treatment. Long-term incubation of CHOrH 2 cells with 10 M forskolin also induced a time-dependent transient decrease (maximum reduction: 75 Ϯ 7%, mean Ϯ S.E., n ϭ 4) of H 2 receptor mRNA to levels similar to those observed after histamine treatment (Fig. 5).
To study the role of mRNA stability, CHOrH 2 cells were incubated for 1 h in the absence or presence of histamine (100 M) or forskolin (10 M), whereafter actinomycin D (10 g/ml) was added to block mRNA transcription. Cells were collected at different time intervals ranging from 0 to 90 min after addition of actinomycin D and were analyzed for H 2 receptor mRNA content. The H 2 receptor mRNA in nontreated cells was hardly affected during the 90 min of incubation with actinomycin D (inset, Fig. 5). Incubation of cells with 100 M histamine, however, resulted in a significant breakdown of H 2 receptor mRNA levels (inset, Fig. 5). Similar results were obtained after forskolin treatment (inset, Fig. 5).
Differences between Histamine-and Forskolin-induced H 2 Receptor Down-regulation-As can be seen in Fig. 5, 4 h of incubation of cells with 100 M histamine resulted in a marked down-regulation (71 Ϯ 4%) of the H 2 receptor mRNA, whereas after 4 h of incubation of CHOrH 2 cells and direct measurement of [ 125 I]APT binding, no significant down-regulation of H 2 receptors was observed (Fig. 1A). Yet, when CHOrH 2 cells were incubated with 100 M histamine for 1, 2, or 4 h, extensively washed and further incubated in serum-free medium without histamine for 23, 22, and 20 h, respectively, a significant reduction of H 2 receptor binding sites was observed (Fig. 6A). Interestingly, similar experiments in which CHOrH 2 cells were incubated for 1, 2, or 4 h with 10 M forskolin followed by extensive washing and further incubation of cells in serum-free medium, showed a clearly delayed reduction in the number of H 2 receptor binding sites (Fig. 6A).
In order to eliminate the cAMP-dependent pathway of H 2 receptor down-regulation, we incubated the CHOrH 2 cells with the protein kinase A inhibitor H-89. However, long-term exposure (24 h) of CHOrH 2 cells to 10 M H-89 resulted already in a 55 Ϯ 2% (mean Ϯ S.E., n ϭ 6) decrease of [ 125 I]APT binding sites. Similar data were obtained with another protein kinase A inhibitor KT5720. 2 Taking into account that H-89 itself induces a reduction in [ 125 I]APT binding sites, CHOrH 2 cells were exposed to 1 M histamine and 1 M forskolin in the presence of 10 M H-89 for 24 h. As can be seen in Fig. 6B (Table III). In contrast, the introduced Leu 124 3 Ala mutation significantly affected the agonist binding characteristics. In CHOrH 2 cells, histamine displacement curves were shallow and could be analyzed best by a two-site model (Fig. 8, Table III). The addition of 10 M GTP␥S resulted in a steepening and a rightward shift of the histamine displacement curve, which could be analyzed best by a single site model with a K i value of 0.18 Ϯ 0.02 mM (Fig. 8). In CHOrH 2 Leu 124 Ala cells, however, the displacement curve of histamine was analyzed best by a single site model, leading to a K i value (0.21 Ϯ 0.02 mM) that corresponded to the low affinity site of the wild-type receptor (Fig. 8, Table III). The addition of 10 M GTP␥S did not result in a rightward shift of the displacement curve of histamine (Fig. 8, Table III).
Moreover, the Leu 124 3 Ala mutation also affected the ability of histamine to induce the formation of cAMP in CHOrH 2 Leu 124 Ala cells (Fig. 9A). The EC 50 value of the histamine-induced cAMP response in CHOrH 2 Leu 124 Ala cells was approximately 162-fold higher (11 Ϯ 3 M, mean Ϯ S.E., n ϭ 7) than the observed EC 50 value of the histamine-induced cAMP response in CHOrH 2 cells (66 Ϯ 29 nM, mean Ϯ S.E., n ϭ 6) measured under the same conditions. The maximum hista- mine-induced response was also found to be affected in CHOrH 2 Leu 124 Ala cells (E max in CHOrH 2 cells: 40 Ϯ 4 pmol/ well, E max in CHOrH 2 Leu 124 Ala cells: 18 Ϯ 1 pmol/well).
Histamine-induced Down-regulation of Rat H 2 Leu 124 3 Ala Receptors-Long-term exposure (24 h) of CHOrH 2 Leu 124 Ala cells to increasing concentrations of histamine resulted in a dose-dependent reduction of [ 125 I]APT binding sites (Fig. 9B). Whereas in CHOrH 2 cells an EC 50 of 18 Ϯ 6 nM (mean Ϯ S.E., n ϭ 7) was observed for histamine, in CHOrH 2 Leu 124 Ala cells histamine induced down-regulation with an EC 50 value of 288 Ϯ 89 nM (mean Ϯ S.E., n ϭ 4). Comparing the histamineinduced cAMP production and H 2 Leu 124 3 Ala receptor downregulation (Fig. 9B), a discrepancy in dose relationships is observed. Almost 40-fold higher concentrations of histamine are required to induce cAMP production, compared to receptor down-regulation. Pretreatment of CHOrH 2 Leu 124 Ala cells for 24 h with 1 M histamine resulted in a significant degree of H 2 receptor down-regulation (51 Ϯ 2%, mean Ϯ S.E., n ϭ 4), whereas no significant cAMP production was observed after 10 min of incubation (Fig. 9B). Even after 24 h of incubation of CHOrH 2 Leu 124 Ala cells with 1 M histamine, no significant increase in cAMP was observed (data not shown). Moreover, even at 0.1 M histamine, significant H 2 receptor down-regulation was observed.

DISCUSSION
In the present study we have demonstrated that the rat histamine H 2 receptor density in CHO cells is reduced about 50% by long-term exposure to histamine or selective H 2 agonists. Long-term treatment of CHOrH 2 cells with histamine resulted in a time-dependent (t1 ⁄2 Ϸ 7 h at a concentration of 100 M) and dose-dependent (EC 50 ϭ 18 nM at 24 h of incubation) decrease in the number of H 2 receptor binding sites. Yet, incubation of CHOrH 2 cells with homo-and nordimaprit, two side chain homologues of the H 2 agonist dimaprit with weak H 2 agonistic activity ((23), present study), did not significantly reduce the number of H 2 receptors. These findings show that the observed H 2 agonist-induced down-regulation is a H 2 receptor-mediated process. Long-term exposure of CHOrH 2 cells to histamine resulting in a reduction of H 2 receptor binding sites is paralleled by a decrease of H 2 receptor responsiveness, characterized by a 34-fold shift of the histamine dose-response curve. The observed shift cannot be ascribed to decreased adenylyl cyclase activity as forskolin dose-response curves remained unaffected after long-term histamine exposure.
As was found for the ␤ 2 -adrenergic receptor (24), a cAMPdependent pathway can also regulate the H 2 receptor density. Forskolin, generating cAMP upon addition, time dependently (t1 ⁄2 Ϸ 7 h at a concentration of 10 M) and dose dependently (EC 50 ϭ 0.3 M at 24 h of incubation) induced H 2 receptor down-regulation. CTX and IBMX, agents that also elevate intracellular levels of cAMP in CHOrH 2 cells, induced downregulation of the H 2 receptor as well. Thus, the H 2 receptor does not need to be stimulated by an agonist in order to be down-regulated. This mechanism might be involved in heterologous H 2 receptor down-regulation as previously shown for other GPCRs (see Refs. 4 and 25). The time course of the forskolin-induced decrease of H 2 receptor number in CHOrH 2 cells parallels the time-dependent decrease of H 2 receptors induced by histamine. For both histamine and forskolin, halfmaximal H 2 receptor down-regulation is reached after approximately 7 h of incubation. Moreover, the maximum decrease of H 2 receptor numbers induced by forskolin is comparable to the maximum agonist-mediated H 2 receptor down-regulation.
Agonist-induced receptor down-regulation is a commonly occurring regulatory process of the large family of GPCRs (see for reference reviews, Refs. 4 and 25). Enhanced degradation and/or decreased synthesis of the receptor protein are thought to contribute to receptor down-regulation (4,25). Agonist-induced down-regulation of GPCRs is often accompanied by a decline of receptor mRNA levels, presumably contributing to the overall reduction in receptor number and responsiveness (26). Indeed, incubation of CHOrH 2 cells with histamine or forskolin resulted in a transient decrease of H 2 receptor mRNA levels (70% reduction) within 4 h, which was followed by a gradual increase of H 2 receptor mRNA to 50% of control mRNA levels in the following hours. The reduced H 2 receptor mRNA levels, 50% of the control levels, at later time points are considered to represent a new steady-state level of receptor mRNA to maintain the down-regulated state of H 2 receptors. The reduction of H 2 receptor mRNA is most likely explained by post-transcriptional events, such as receptor mRNA destabilization. For example, the ␤ 2 -adrenergic receptor and thrombin receptor in DDT 1 MF-2 smooth muscle cells, the endothelin ET B receptor in ROS17/2 rat osteosarcoma cells, and also for the ␤ 2 -adrenergic receptor and muscarine m1 receptor expressed into CHW and CHO cells, respectively, the decline in receptor mRNA has been ascribed to destabilization of the mRNA (24,(27)(28)(29)(30). In the presence of actinomycin D, breakdown of the H 2 receptor mRNA in CHOrH 2 cells was stimulated significantly upon histamine-treated and forskolin-treated compared to nontreated cells. Recently, it was shown that a so-called M r ϭ 35,000 ␤-adrenergic receptor mRNA-binding protein, involved in the destabilization of ␤ 2 -adrenergic receptor mRNA, also recognizes other GPCR transcripts (29). As such, our observations of H 2 receptor mRNA destabilization fit well in an appar-  ently general mechanism of ␤-adrenergic receptor mRNA binding protein-mediated regulation of GPCR mRNA (29,31). For the ␤ 2 -adrenergic receptor, the most extensively studied GPCR, receptor down-regulation is ascribed to two pathways: an agonist-dependent, protein kinase A-independent, and a protein kinase A-dependent process (4,25). Evidence for a protein kinase A-independent pathway was obtained by studies which showed unaffected profiles of ␤ 2 -adrenergic receptor down-regulation in mutant S49 mouse lymphoma cells defective in signal transduction components (32)(33)(34)(35). Receptor-G s coupling seems to be important for the process of ␤ 2 -adrenergic receptor down-regulation, since defects in this coupling introduced by mutations of the receptor or G s -protein have lead to impaired ␤ 2 -adrenergic receptor down-regulation (33)(34)(35)(36)(37). Agents responsible for the elevation of intracellular levels of cAMP, such as forskolin and IBMX, or cAMP analogues, e.g. dibutyryl cAMP, were shown to induce ␤ 2 -adrenergic receptor down-regulation as well, providing evidence for the existence of cAMP-dependent receptor down-regulation (24,25,36). In CHW cells, the time course of the cAMP-promoted down-regulation of the ␤ 2 -adrenergic receptor was much slower than the ␤-agonists-induced down-regulation, suggesting that distinct pathways can lead to down-regulation of the ␤ 2 -adrenergic receptor (24). Yet, protein kinase A-dependent phosphorylation of the ␤ 2 -adrenergic receptor appears to enhance down-regulation, since receptor mutants lacking protein kinase A phosphorylation sites showed impaired agonist-induced down-regulation (24). Taken together, ␤ 2 -adrenergic receptor receptor down-regulation seems to require receptor-G s coupling for the initial loss of receptor binding sites, while the cAMP-dependent decrease of receptor mRNA levels serves to maintain the downregulated state by establishing a new steady-state of receptor expression (25). The underlying biochemical mechanisms responsible for each of these events is, however, unclear so far.
In our study on CHOrH 2 cells, comparable time courses and a maximum extent of histamine-induced and forskolin-induced H 2 receptor down-regulation as well as H 2 mRNA down-regulation suggest the involvement of cAMP in the process of agonist-induced H 2 receptor down-regulation. The initial reduction of H 2 receptor mRNA upon histamine or forskolin exposure can, however, not explain the 50% reduction of the H 2 receptor numbers, since relatively short (Ͻ4 h) treatments of CHOrH 2 cells with histamine or forskolin followed by a wash-out up to 24 h led to a more pronounced H 2 receptor down-regulation upon histamine than forskolin exposure. Thus, apparently there is no direct link between H 2 receptor mRNA and H 2 receptor expression. Moreover, these data are a first indication that histamine and forskolin induce H 2 receptor down-regulation by different mechanisms. The existence of a cAMP-dependent and cAMP-independent pathway was further corroborated by the fact that the protein kinase A inhibitor H-89 (38) inhibited the forskolin-induced, but not the histamine-induced, H 2 receptor down-regulation. Moreover, recently we have shown that down-regulation of H 2 receptors stably expressed into human embryonal kidney cells (HEK-293 cells) is mediated via cAMP-dependent and cAMPindependent processes as the histamine-induced downregulation was found to be more pronounced than the forskolininduced H 2 receptor down-regulation (39).
In order to assess the role of cAMP in the process of agonistinduced H 2 receptor down-regulation in CHOrH 2 cells directly, we constructed a mutant H 2 receptor which showed impaired G-protein coupling. Recently, Moro et al. (40) have shown that hydrophobic amino acids within a highly conserved GPCR motif DRYXXV(I)XXPL (X is any amino acid and L is leucine or other lipophilic amino acid) in the second intracellular loop are involved in receptor-G-protein coupling (40). In the H 2 receptor protein, a DRYCAVTDPL sequence is found at an equivalent position of the highly conserved motif (2). Substitution of the Leu 124 residue by an alanine residue had no effect on H 2 receptor expression nor on H 2 antagonist binding properties. However, the mutation induced a marked impairment of the ability of the receptor to physically couple to its G-protein as assessed by alterations in its agonist-binding parameters (disappearance high affinity binding site, no detectable GTP␥S shift). The physical uncoupling of the H 2 Leu 124 3 Ala mutant was paralleled by a functional uncoupling, characterized by an impairment of the histamine-induced cAMP production (160fold reduction of the EC 50 value and 55% decrease of the maximal cAMP response). These findings are in agreement with the functional uncoupling reported by Moro et al. (40) after mutation of a hydrophobic amino acid at similar position in the muscarine m1, m3, and ␤ 2 -adrenergic receptor.
Interestingly, long-term exposure of CHOrH 2 Leu 124 Ala cells to 0.1 M and 1 M histamine, concentrations that do not elicit