©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Distinct Pathways for Histamine H Receptor Down-regulation
H(2) LEU ALA RECEPTOR MUTANT PROVIDES EVIDENCE FOR A cAMP-INDEPENDENT ACTION OF H(2) AGONISTS (*)

(Received for publication, September 11, 1995; and in revised form, January 17, 1996)

Martine J. Smit Edwin Roovers Henk Timmerman Yvonne van de Vrede Astrid E. Alewijnse Rob Leurs (§)

From the Leiden/Amsterdam Center for Drug Research, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Pretreatment of Chinese hamster ovary cells expressing the histamine H(2) receptor (CHOrH(2) cells) with histamine resulted in a time-dependent (t approx 7 h) and dose-dependent (EC = 18 nM) H(2) receptor down-regulation measured as [I]iodoaminopotentidine binding (44 ± 10% down-regulation). Pretreatment of CHOrH(2) cells with cholera toxin or forskolin also led to H(2) receptor down-regulation. Forskolin time-dependently (t approx 7 h) and dose-dependently (EC = 0.3 µM) induced H(2) receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H(2) 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(2) receptor from the G(s)-protein, we introduced the Leu Ala mutation in the second intracellular loop of the H(2) receptor. The H(2) 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(2) cells, H(2) receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.


INTRODUCTION

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) . Short-term 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 long-term 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 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.


MATERIALS AND METHODS

Cell Culture

CHO cells expressing the rat histamine H(2) receptor (CHOrH(2)) (13) and the mutated H(2) Leu Ala receptor (CHOrH(2)LeuAla) were grown at 37 °C in a humidified atmosphere with 5% CO(2) in Dulbecco's modified Eagle's medium (DMEM), containing 10% (v/v) dialyzed fetal calf serum supplemented with 2 mML-glutamine, MEM amino acids, 50 IU/ml penicillin, and 50 µg/ml streptomycin.

Site-directed Mutagenesis

The H(2) receptor mutant, in which leucine 124 was substituted by alanine (Leu Ala) (Fig. 7), was constructed by means of the polymerase chain reaction. The oligonucleotides S1 (5`-GGGAAGCTTGGCCCCAGAATGGAGCCCAATGGCACAGT), corresponding to nucleotides -9 to 21 (2) and a HindIII site (underlined), and AS1 (5`-GGGGGTACCGCGCTGGGTCCGTGACAGCGCAGTAGTTGTTCAAGCTGATCAT), 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 µM S1, 0.4 µM AS2, 40 µM 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 Ala receptor mutant was amplified in 100 µl 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 Ala receptor was subcloned into the eukaryotic pSV expression vector. CHO cells, deficient in dihydrofolate reductase, were stably transfected with 15 µg of pSVrH(2)LeuAla using Transfectam (Promega).


Figure 7: Schematic representation of the rat histamine H(2) receptor. The leucine (L) at position 124, located in the second intracellular loop, was mutated to an alanine (A) by site-directed mutagenesis as described under ``Materials and Methods.''



Membrane Preparation

CHOrH(2) and CHOrH(2)LeuAla 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 times 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 [I]iodoaminopotentidine ([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 [I]APT were incubated with 5-10 µg of membrane proteins in the absence or presence of 1 µM tiotidine in a total volume of 400 µl. 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)LeuAla 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 µM 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 µl 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 -20 °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 µM 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)LeuAla 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 times g at 4 °C. The supernatant, containing protein kinase A, was carefully recovered and frozen in 1 ml aliquots at -80 °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 µl of the binding protein was mixed with 50-100 µl of the CHO homogenate or cAMP standards and 30,000 dpm of [^3H]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.

RNA Slot Blot Analysis

RNA was analyzed by means of mRNA slot blot assay as described by Zhang et al.(18) , with minor modifications. Briefly, RNA was isolated according to the method of Chomczynski and Sacchi(19) , using Trizol reagent (Life Technologies, Inc.), and RNA was slot-blotted on a nitrocellulose filter (GeneScreen Plus, DuPont NEN) and prehybridized for 2 h at 65 °C in 7% SDS, 0.5 M Na(2)HPO(4), 1 mM EDTA (pH 7.2). Thereafter, filters were hybridized overnight with a radioactive labeled 48-mer antisense rat H(2) receptor oligonucleotide (5`-GATGGTGGCTGCCTTCCAGGAGCTGATGTGGTTGATCCGTTTGGCCTG-3`, corresponding to nucleotides 631 to 678) or antisense rat beta-actin oligonucleotide (5`-CTCCTGCTTGCTGATCCACATCTGCTGGAAGGTGGACAGTGAGGCCAG-3`, corresponding to nucleotides 3040 to 3087) at 65 °C in 7% SDS, 0.5 M Na(2)PO(4), and 1 mM EDTA (pH 7.2). The rat H(2) receptor oligonucleotide (1.5 pmol) was P-labeled by 3`-end tailing using 16 pmol of [alpha-P]dATP (3000 Ci/mmol; Amersham) and 1 unit of terminal deoxynucleotidyltransferase (Boehringer) for 20 min at 37 °C. The beta-actin oligonucleotide (5 pmol) was labeled using 10 pmol of [-P]dATP (3000 Ci/mmol; Amersham) and 4 units of polynucleotide kinase (Boehringer) for 30 min at 37 °C. The blots were washed twice for 5 min at room temperature in 2 times SSC (0.3 M NaCl, 0.03 M Na(3)C(6)H(5)O(7)bullet2H(2)O) supplemented with 0.1% SDS, which was followed by two 45-min washes at 65 °C with 2 times SSC supplemented with 0.1% SDS. The blots were exposed to a PhosphorScreen (Molecular Dynamics) and signals were quantified with a PhosphorImager 425 (Molecular Dynamics) using the computer program ImageQuant (Molecular Dynamics). H(2) receptor mRNA levels were expressed as the ratio of the values of the H(2) receptor mRNA signals and the corresponding beta-actin signals.

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 µM histamine or with 10 µM forskolin for 1 h in DMEM. Thereafter, actinomycin D (10 µg/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.

Chemicals

Histamine dihydrochloride, isobutylmethylxanthine (IBMX), cyclic AMP (cAMP), forskolin, 1,9-dideoxyforskolin, cholera toxin, and GTPS were obtained from Sigma. Actinomycin D was purchased from Boehringer Mannheim. [2,8-^3H]cAMP (40 Ci/mmol) was obtained from Amersham. H-89 dihydrochloride and KT5720 were purchased from Calbiochem. Dimaprit dihydrobromide, homo- and nordimaprit dihydrochloride, amthamine dihydrobromide, amselamine dihydrobromide, and aminopotentidine were taken from laboratory stock. Gifts of cimetidine (SmithKline Beecham), ranitidine dihydrochloride (Glaxo), tiotidine (Imperial Chemical Industries), CHO cells expressing the rat H(2) receptor, and pSVrH(2) vector (Dr. J.-C. Schwartz) are gratefully acknowledged.

Statistical Analysis

All data shown are expressed as mean ± S.E. of at least three independent experiments. Statistical analysis was carried out by Student's t-test. p values < 0.05 were considered to indicate a significant difference.


RESULTS

Histamine-induced H(2) Receptor Down-regulation

Exposure of CHOrH(2) cells (13) to 100 µM histamine for prolonged periods of time resulted in a time-dependent decrease of [I]APT binding (Fig. 1A). Maximum reduction of [I]APT binding was observed after a 16-h incubation of cells with 100 µM histamine. Under this condition, histamine induced 44 ± 10% (p < 0.05) reduction of [I]APT binding. Half-maximum reduction of the [I]APT binding was recorded at an incubation period of approximately 7 h. A 24-h incubation of CHOrH(2) cells with increasing concentrations of histamine led to a dose-dependent reduction of [I]APT binding (EC value = 18 ± 6 nM, mean ± S.E., n = 7) (Fig. 1B). The observed reduction of [I]APT binding was not reflected by a change in affinity of [I]APT for the H(2) receptor, as its dissociation constant (K(d)), determined by means of saturation studies, remained unaffected in CHOrH(2) cells incubated with 100 µM histamine for 24 h (Table 1). Exposure of CHOrH(2) cells to histamine resulted only in a marked decrease of the total number of [I]APT binding sites (B(max)) (Table 1).


Figure 1: Time- and dose-dependent decrease of [I]APT binding in CHOrH(2) cells by histamine. A, CHOrH(2) cells were incubated with 100 µM histamine for the indicated times, and [I]APT binding in membranes was measured. The [I]APT binding is expressed as a percentage of [I]APT binding measured in nontreated cells. The data shown represent the mean ± S.E. of 4 independent experiments. B, dose-dependent decrease of [I]APT binding induced by histamine. CHOrH(2) cells were exposed to various concentrations of histamine for 24 h. The data represent the mean ± S.E. of 7 independent experiments.





The recently described selective H(2) receptor agonists amselamine and amthamine (21, 22) induced cAMP production in CHOrH(2) cells, with EC values lower and maximum responses comparable to histamine (Table 2). Long-term exposure (24 h) of CHOrH(2) cells to amselamine or amthamine resulted in a dose-dependent decrease of [I]APT binding with EC values, which were correlated with their respective EC values for the cAMP response. Amselamine and amthamine induced a maximal decrease of [I]APT binding sites of 50 ± 3% and 43 ± 4% respectively. As shown in Table 2, 24-h incubation of CHOrH(2) cells with 100 µM dimaprit, which exhibits a lower potency as compared to histamine, induced a 40 ± 3% decrease of [I]APT binding sites. Dimaprit's structural analogues, homodimaprit and nordimaprit, showed strongly reduced capacities to generate cAMP with EC values of 1.4 ± 0.9 µM and higher than 10 µM, respectively (Table 2). The reduced ability of these dimaprit analogues to induce a cAMP response was paralleled by a lack of H(2) receptor down-regulation after 24 h of incubation of CHOrH(2) cells with 100 µM concentrations of the analogues (Table 2).



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 µM histamine resulted in a rightward shift of the dose-response curve of the histamine-induced cAMP production (EC of histamine-induced cAMP response in nontreated cells: 36 ± 3 nM, mean ± S.E., n = 7, and histamine-treated cells: 1.2 ± 0.05 µM, mean ± S.E., n = 4) (Fig. 2A). The forskolin-induced rise in cAMP was not found to be affected as no change in dose dependence or impairment of the maximal forskolin-induced cAMP response was observed after a 24-h pretreatment of cells with 100 µM histamine (Fig. 2B).


Figure 2: Effect of long-term histamine treatment on histamine- and forskolin-induced signaling in CHOrH(2) cells. CHOrH(2) cells were treated with (open circles) or without (filled circles) 100 µM histamine for 24 h in DMEM without fetal calf serum. Thereafter, cells were washed several times and incubated for 1 h with DMEM supplemented with 25 mM HEPES, pH 7.4. CHOrH(2) cells were subsequently incubated with increasing concentrations of histamine (A) or forskolin (B) for 10 min at 37 °C in DMEM in the presence of 300 µM IBMX and 25 mM HEPES, pH 7.4. The data represent the mean ± S.E. of 4 independent experiments.



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 µM forskolin led to a marked reduction of 58 ± 2% [I]APT binding (Fig. 3B). Again, no major change in affinity of [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 µM forskolin (Table 1). Maximum and half-maximum down-regulation was recorded after 16 h and approximately 7 h of incubation of CHOrH(2) cells with 10 µM 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 value of 0.3 ± 0.06 µM (mean ± S.E., n = 4) (Fig. 3C). Concentrations up to 10 µM 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).


Figure 3: Forskolin- and 1,9-dideoxyforskolin-induced cAMP production and decrease of [I]APT binding in CHOrH(2) cells. A, dose-dependent increase of the cAMP production by forskolin (filled circles) and 1,9-dideoxyforskolin (open circles). CHOrH(2) cells were incubated with the indicated drugs with increasing concentrations for 10 min at 37 °C in DMEM in the presence of 300 µM IBMX and 25 mM HEPES, pH 7.4. Data represent the mean ± S.E. of 6 independent experiments. B, forskolin-induced decrease of [I]APT binding in CHOrH(2) cells. CHOrH(2) cells were incubated with 10 µM forskolin for the indicated times, and [I]APT binding was measured. The [I]APT binding is expressed as a percentage of [I]APT binding measured in nontreated cells. C, dose-dependent decrease of [I]APT binding in CHOrH(2) cell membranes induced by forskolin. CHOrH(2) cells were exposed to various concentrations of forskolin for 24 h. The H(2) receptor density was determined as described. D, effect of 1,9 dideoxyforskolin (ddF) and forskolin (FORS) on [I]APT binding. CHOrH(2) cells were exposed to 1 and 10 µM 1,9-dideoxyforskolin (filled bars) and forskolin (open bars), respectively, for 24 h and examined for [I]APT binding. The asterisks indicate a significant difference (p < 0.05) from control, represented by the [I]APT binding measured in untreated cells. Data from B, C, and D were calculated as mean ± S.E. from 4 independent experiments.



Cholera toxin (CTX), which irreversibly activates the G(s)-protein, thereby generating cAMP, also induced a dose-dependent decrease of [I]APT binding sites when incubated for 24 h (EC = 32 ± 1 ng/ml, mean ± S.E., n = 4, Fig. 4). CTX pretreatment of CHOrH(2) cells resulted in a maximum down-regulation of H(2) receptors of 46 ± 3%. Finally, exposure of CHOrH(2) cells for 24 h to 300 µM IBMX, a cAMP phosphodiesterase inhibitor, also resulted in an attenuation of [I]APT binding (26 ± 7% H(2) receptor down-regulation, n = 4, mean ± S.E., p < 0.05).


Figure 4: Effect of long-term treatment with CTX on [I]APT binding in CHOrH(2) cells. The CHOrH(2) cells were treated with increasing concentrations of CTX for 24 h. [I]APT binding is expressed as a percentage of [I]APT binding measured in nontreated cells studied under the same conditions and was measured as described earlier. The data shown represent the mean ± S.E. of 4 independent experiments.



H(2) Receptor mRNA Levels and Stability in Control, Histamine-treated, and Forskolin-treated CHOrH(2) Cells

Exposure of CHOrH(2) cells to 100 µM histamine for increasing periods of time resulted in a rapid transient decrease of H(2) receptor mRNA (maximum reduction of 71 ± 4%, mean ± S.E., n = 4) (Fig. 5). This effect was at its peak after 4 h of incubation of cells with histamine (100 µM), 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 µM forskolin also induced a time-dependent transient decrease (maximum reduction: 75 ± 7%, mean ± S.E., n = 4) of H(2) receptor mRNA to levels similar to those observed after histamine treatment (Fig. 5).


Figure 5: Histamine- and forskolin-induced modulation of H(2) receptor mRNA levels. CHOrH(2) cells were incubated for the indicated times with 100 µM histamine (filled circles) or 10 µM forskolin (open circles). Cells were harvested, and total RNA was extracted and quantified by means of a RNA slot blot assay as described under ``Materials and Methods.'' The results displayed are the mean ± S.E. of two separate experiments, performed in duplicate. Inset, effect of histamine treatment and forskolin treatment on H(2) receptor mRNA stability. CHOrH(2) cells were incubated with (open circles) or without (open squares) 100 µM histamine or with 10 µM forskolin (filled squares) for 1 h, before actinomycin D (10 µg/ml) was added. Cells were harvested at 0, 15, 30, 60, and 90 min after the addition of actinomycin D. The data are the mean ± S.E. of three separate experiments, each performed in duplicate. The asterisks indicate a significant difference (p < 0.05) from control, represented by untreated cells.



To study the role of mRNA stability, CHOrH(2) cells were incubated for 1 h in the absence or presence of histamine (100 µM) or forskolin (10 µM), whereafter actinomycin D (10 µg/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 µM 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 µM histamine resulted in a marked down-regulation (71 ± 4%) of the H(2) receptor mRNA, whereas after 4 h of incubation of CHOrH(2) cells and direct measurement of [I]APT binding, no significant down-regulation of H(2) receptors was observed (Fig. 1A). Yet, when CHOrH(2) cells were incubated with 100 µM 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 µM 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).


Figure 6: Differences between histamine- and forskolin-induced H(2) receptor down-regulation. A, effect of short-term exposure of CHOrH(2) cells to histamine and forskolin on [I]APT binding to CHOrH(2) membranes. CHOrH(2) cells were incubated for the indicated times, extensively washed, and incubated in serum-free medium without the stimulating agent up to 24 h. For comparison, the effect of 24-h exposure to histamine or forskolin is shown. The effect of incubation of CHOrH(2) cells with 100 µM histamine (filled bars) or 10 µM forskolin (open bars) on [I]APT binding is expressed as the percentage of [I]APT binding sites of nontreated cells. Data shown are the mean ± S.E. from 4 experiments. The asterisks indicate a significant difference (p < 0.05) from control, represented by nontreated cells. B, effect of the protein kinase A inhibitor H-89 on histamine- and forskolin-induced H(2) receptor down-regulation. CHOrH(2) cells were incubated for 24 h with histamine (HA) or forskolin (FORS) in the presence of H-89. The effect of incubation of CHOrH(2) cells with 10 µM H-89 and 100 µM histamine or 10 µM forskolin on [I]APT binding is expressed as the percentage of [I]APT binding sites of cells treated with 10 µM H-89 alone. Data shown are mean ± S.E. from 4 experiments. The asterisks indicate a significant difference (p < 0.05) from control, represented by cells treated with H-89.



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 µM H-89 resulted already in a 55 ± 2% (mean ± S.E., n = 6) decrease of [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 [I]APT binding sites, CHOrH(2) cells were exposed to 1 µM histamine and 1 µM forskolin in the presence of 10 µM H-89 for 24 h. As can be seen in Fig. 6B, the forskolin-induced effect is inhibited by co-incubation with 10 µM H-89 as no H(2) receptor down-regulation is observed. Yet, long-term incubation of cells with histamine and H-89 still induces down-regulation, suggesting that a cAMP-independent pathway is responsible for the histamine-induced down-regulation.

Functional Analysis of the Leu Ala Mutation of the Rat Histamine H(2) Receptor

Using the polymerase chain reaction, leucine 124 in the second intracellular loop of the rat histamine H(2) receptor (Fig. 7) was mutated into an alanine residue. Transfection of the H(2) Leu Ala receptor cDNA into CHO cells, resulted in the formation of several clonal cell lines expressing [I]APT binding sites. A clonal cell line expressing amounts of [I]APT binding comparable to those of the CHOrH(2) cells was chosen for further analysis and referred to as CHOrH(2)LeuAla (CHOrH(2) cells: 975 ± 12 fmol/mg of protein, CHOrH(2)LeuAla cells: 980 ± 7 fmol/mg of protein, mean ± S.E., n = 3). There were no major differences in the binding of the H(2) antagonists to the wild-type receptor or the mutated receptor. The affinity of [I]APT for the mutated receptor was hardly affected (K(d) of [I]APT in CHOrH(2) cells: 0.43 ± 0.06 nM, in CHOrH(2)LeuAla cells: 0.61 ± 0.03 nM, mean ± S.E., n = 3). Moreover, cimetidine and ranitidine had similar K(i) values for both receptors (Table 3). In contrast, the introduced Leu 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 3). The addition of 10 µM GTPS 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 ± 0.02 mM (Fig. 8). In CHOrH(2)LeuAla cells, however, the displacement curve of histamine was analyzed best by a single site model, leading to a K(i) value (0.21 ± 0.02 mM) that corresponded to the low affinity site of the wild-type receptor (Fig. 8, Table 3). The addition of 10 µM GTPS did not result in a rightward shift of the displacement curve of histamine (Fig. 8, Table 3).




Figure 8: Binding of histamine to the wild-type and H(2) Leu Ala receptor. Displacement of binding of 0.3 nM [I]APT by increasing concentrations of histamine in the presence (open symbols) and absence (filled symbols) of 10 µM GTPS in CHOrH(2) cells (circles) and CHrH(2)LeuAla cells (squares). Mean values of triplicate determinations of a typical experiment out of at least three are shown.



Moreover, the Leu Ala mutation also affected the ability of histamine to induce the formation of cAMP in CHOrH(2)LeuAla cells (Fig. 9A). The EC value of the histamine-induced cAMP response in CHOrH(2)LeuAla cells was approximately 162-fold higher (11 ± 3 µM, mean ± S.E., n = 7) than the observed EC value of the histamine-induced cAMP response in CHOrH(2) cells (66 ± 29 nM, mean ± S.E., n = 6) measured under the same conditions. The maximum histamine-induced response was also found to be affected in CHOrH(2)LeuAla cells (E(max) in CHOrH(2) cells: 40 ± 4 pmol/well, E(max) in CHOrH(2)LeuAla cells: 18 ± 1 pmol/well).


Figure 9: Effects of Leu Ala mutation on histamine-induced cAMP production and down-regulation. A, dose-dependent increase of the cAMP production by histamine in CHOrH(2) cells (filled circles) and CHOrH(2)LeuAla cells (open circles). Cells were incubated with increasing concentrations of histamine for 10 min at 37 °C in DMEM in the presence of 300 µM IBMX and 25 mM HEPES, pH 7.4. The data shown represent the mean ± S.E. for, respectively, 6 and 7 independent experiments. B, effects of Leu Ala mutation on histamine-induced H(2) receptor down-regulation (open circles) and cAMP production (filled circles, see also above). CHOrH(2)Leu cells were exposed to increasing concentrations of histamine for 24 h, and [I]APT binding in membranes was measured. The [I]APT binding is expressed as a percentage of [I]APT binding measured in nontreated cells. The data shown represent the mean ± S.E. of 4 experiments. The asterisk and number sign indicate a significant difference (p < 0.05) from control, represented by untreated cells and basal cAMP levels respectively.



Histamine-induced Down-regulation of Rat H(2) Leu Ala Receptors

Long-term exposure (24 h) of CHOrH(2)LeuAla cells to increasing concentrations of histamine resulted in a dose-dependent reduction of [I]APT binding sites (Fig. 9B). Whereas in CHOrH(2) cells an EC of 18 ± 6 nM (mean ± S.E., n = 7) was observed for histamine, in CHOrH(2)LeuAla cells histamine induced down-regulation with an EC value of 288 ± 89 nM (mean ± S.E., n = 4). Comparing the histamine-induced cAMP production and H(2) Leu Ala receptor down-regulation (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)LeuAla cells for 24 h with 1 µM histamine resulted in a significant degree of H(2) receptor down-regulation (51 ± 2%, mean ± S.E., n = 4), whereas no significant cAMP production was observed after 10 min of incubation (Fig. 9B). Even after 24 h of incubation of CHOrH(2)LeuAla cells with 1 µM histamine, no significant increase in cAMP was observed (data not shown). Moreover, even at 0.1 µM histamine, significant H(2) receptor down-regulation was observed.

In the CHOrH(2)LeuAla cells, the maximal histamine-induced down-regulation was more pronounced (68 ± 4%, mean ± S.E., n = 4) than was observed for the CHOrH(2) cells (43 ± 4%, mean ± S.E., n = 7). The forskolin (10 µM)-induced H(2) receptor down-regulation was also found to be more pronounced in the CHOrH(2)LeuAla cells (67 ± 1%, mean ± S.E., n = 3) than in CHOrH(2) cells (58 ± 2%, mean ± S.E., n = 4).


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 (t approx 7 h at a concentration of 100 µM) and dose-dependent (EC = 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 beta(2)-adrenergic receptor(24) , a cAMP-dependent pathway can also regulate the H(2) receptor density. Forskolin, generating cAMP upon addition, time dependently (t approx 7 h at a concentration of 10 µM) and dose dependently (EC = 0.3 µM 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 down-regulation 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 (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, half-maximal 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, (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 beta(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 beta(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) = 35,000 beta-adrenergic receptor mRNA-binding protein, involved in the destabilization of beta(2)-adrenergic receptor mRNA, also recognizes other GPCR transcripts(29) . As such, our observations of H(2) receptor mRNA destabilization fit well in an apparently general mechanism of beta-adrenergic receptor mRNA binding protein-mediated regulation of GPCR mRNA(29, 31) .

For the beta(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 beta(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 beta(2)-adrenergic receptor down-regulation, since defects in this coupling introduced by mutations of the receptor or G(s)-protein have lead to impaired beta(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 beta(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 beta(2)-adrenergic receptor was much slower than the beta-agonists-induced down-regulation, suggesting that distinct pathways can lead to down-regulation of the beta(2)-adrenergic receptor (24) . Yet, protein kinase A-dependent phosphorylation of the beta(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, beta(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 down-regulated 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 (<4 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 cAMP-independent processes as the histamine-induced down-regulation was found to be more pronounced than the forskolin-induced H(2) receptor down-regulation (39) .

In order to assess the role of cAMP in the process of agonist-induced 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 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 GTPS shift). The physical uncoupling of the H(2) Leu Ala mutant was paralleled by a functional uncoupling, characterized by an impairment of the histamine-induced cAMP production (160-fold reduction of the EC 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 beta(2)-adrenergic receptor.

Interestingly, long-term exposure of CHOrH(2)LeuAla cells to 0.1 µM and 1 µM histamine, concentrations that do not elicit cAMP production, resulted in a significant reduction of [I]APT binding sites, indicating that a cAMP-independent pathway is involved in the observed agonist-induced H(2) receptor down-regulation in CHOrH(2)LeuAla cells. Previous findings in mutant S49 mouse lymphoma cells defective in signal transduction components also showed the existence of cAMP-independent pathways in the agonist-induced beta(2)-adrenergic receptor down-regulation(32, 33, 34, 35) . However, it should be noted that the EC value of histamine-induced H(2) receptor down-regulation was shifted 16-fold to the right for the H(2) Leu Ala receptor compared to the wild-type receptor. These data suggest that agonist-induced H(2) receptor down-regulation depends on intact receptor-G-protein coupling. As already stated earlier, previous findings for the beta(2)-adrenergic receptor have shown that defective receptor-G(s) coupling leads to impaired receptor down-regulation(33, 34, 35, 36, 37) . Remarkably, both the maximum histamine-induced and forskolin-induced down-regulation of H(2) Leu Ala receptor were found to be more pronounced than for the wild-type H(2) receptor, suggesting that the mutated receptor has become more susceptible to receptor down-regulation. Although we do not have an explanation for this finding, we hypothesize that the Leu Ala mutation induces a conformational change in the second intracellular loop of the H(2) receptor protein, causing an uncoupling from the G(s)-protein but also an increase of the accessibility of molecular entities involved in receptor degradation. Recent studies with the parathyroid hormone receptor (41) and beta(1)-adrenergic receptor (42) support this hypothesis. Small changes in the conformation of intracellular receptor domains have been shown to augment receptor internalization(41, 42) . Unfortunately, no data on receptor down-regulation are available for these mutant receptors(41, 42) .

In conclusion, for the first time we have demonstrated that the histamine H(2) receptor is down-regulated by prolonged treatment with H(2) agonists. Elevation of cAMP by long-term incubation of CHOrH(2) cells with forskolin, CTX, and IBMX, is also shown to induce H(2) receptor down-regulation. These data suggest the involvement of protein kinase A in the process of H(2) receptor down-regulation and provides a mechanism for heterologous H(2) receptor regulation. Also, H(2) receptor mRNA levels were rapidly down-regulated upon both histamine treatment and forskolin treatment. However, the agonist-induced and forskolin-induced H(2) receptor down-regulation do appear to be differentially regulated, by a cAMP-dependent and cAMP-independent pathway. Substitution of the hydrophobic amino acid leucine 124, located within the highly conserved G-protein coupling motif DRYXXV(I)XXPL in the second intracellular loop of the H(2) receptor, by an alanine generated a mutant receptor with impaired ability to couple to its G-protein. Interestingly, the H(2) Leu Ala mutant receptor was still down-regulated by histamine, at concentrations which showed no increase of cAMP, thereby providing additional evidence for a cAMP-independent pathway in the process of agonist-induced H(2) receptor down-regulation. Thus, H(2) receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Prof. Dr. E. Mutschler on the occasion of his 65th birthday.

§
Supported by a fellowship of the Royal Netherlands Academy of Arts and Sciences. To whom correspondence should be addressed. Tel.: 31-204447579; Fax: 31-204447610; leurs{at}chem.vu.nl.

(^1)
The abbreviations used are: GPCR, G-protein-coupled receptor; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; [I]APT, [I]iodoaminopotentidine; IBMX, isobutylmethylxanthine; CTX, cholera toxin; PCR, polymerase chain reaction; GTPS, guanosine 5`-O-(thiotriphosphate).

(^2)
M. J. Smit, unpublished observations.


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