(Received for publication, September 11, 1995; and in revised form, January 17, 1996)
From the
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 receptor, which was followed by the cloning of both the
rat and human homologues(2, 3) . The deduced amino
acid sequence of the H
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). (
)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
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 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
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
receptor expression and the localization of
histamine-synthesizing cells in the rodent gastric wall. The regulation
of H
receptor expression has gained further interest due to
the potential therapeutic application of H
receptor
agonists in patients suffering from congestive heart
failure(11) .
Investigation of the regulation of H 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
receptor stimulation, but the used systems such as U937 cells for
example do not express a sufficiently high density of H
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
receptors, cell lines expressing
considerable amounts of histamine H
receptors can be
obtained(13, 14) . Additionally, the availability of
the H
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 receptor stably expressed in Chinese hamster ovary (CHO) cells
(referred to as CHOrH
cells) (13) to H
agonists and cAMP mobilizing agents with regard to H
receptor protein expression and H
receptor mRNA
levels. In order to get more insight into the mechanisms underlying
H
receptor regulation, we constructed a H
receptor mutant, in which leucine 124 in the second intracellular
loop was substituted by an alanine. This H
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
receptor down-regulation.
Figure 7:
Schematic representation of the rat
histamine H 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.''
Figure 1:
Time- and dose-dependent decrease of
[I]APT binding in CHOrH
cells by
histamine. A, CHOrH
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
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 receptor agonists amselamine and amthamine (21, 22) induced cAMP production in CHOrH
cells, with EC
values lower and maximum responses
comparable to histamine (Table 2). Long-term exposure (24 h) of
CHOrH
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
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
receptor down-regulation after 24 h of incubation of CHOrH
cells with 100 µM concentrations of the analogues (Table 2).
Figure 2:
Effect of long-term histamine treatment on
histamine- and forskolin-induced signaling in CHOrH cells.
CHOrH
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
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.
Figure 3:
Forskolin- and
1,9-dideoxyforskolin-induced cAMP production and decrease of
[I]APT binding in CHOrH
cells. A, dose-dependent increase of the cAMP production by forskolin (filled circles) and 1,9-dideoxyforskolin (open
circles). CHOrH
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
cells.
CHOrH
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
cell membranes induced by forskolin.
CHOrH
cells were exposed to various concentrations of
forskolin for 24 h. The H
receptor density was determined
as described. D, effect of 1,9 dideoxyforskolin (ddF)
and forskolin (FORS) on [
I]APT
binding. CHOrH
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-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
cells resulted in
a maximum down-regulation of H
receptors of 46 ± 3%.
Finally, exposure of CHOrH
cells for 24 h to 300 µM IBMX, a cAMP phosphodiesterase inhibitor, also resulted in an
attenuation of [
I]APT binding (26 ± 7%
H
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
cells. The
CHOrH
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.
Figure 5:
Histamine- and forskolin-induced
modulation of H receptor mRNA levels. CHOrH
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
receptor mRNA stability.
CHOrH
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 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
receptor mRNA content. The H
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
receptor mRNA levels (inset, Fig. 5). Similar results were obtained after forskolin treatment (inset, Fig. 5).
Figure 6:
Differences between histamine- and
forskolin-induced H receptor down-regulation. A,
effect of short-term exposure of CHOrH
cells to histamine
and forskolin on [
I]APT binding to CHOrH
membranes. CHOrH
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
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
receptor down-regulation.
CHOrH
cells were incubated for 24 h with histamine (HA) or forskolin (FORS) in the presence of H-89. The
effect of incubation of CHOrH
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 receptor down-regulation, we incubated the
CHOrH
cells with the protein kinase A inhibitor H-89.
However, long-term exposure (24 h) of CHOrH
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. (
)Taking into account that H-89 itself induces a reduction
in [
I]APT binding sites, CHOrH
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
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.
Figure 8:
Binding of histamine to the wild-type and
H 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 GTP
S in
CHOrH
cells (circles) and
CHrH
Leu
Ala 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
Leu
Ala cells (Fig. 9A).
The EC
value of the histamine-induced cAMP response in
CHOrH
Leu
Ala 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
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
Leu
Ala
cells (E
in CHOrH
cells: 40 ±
4 pmol/well, E
in
CHOrH
Leu
Ala 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
cells (filled circles) and
CHOrH
Leu
Ala 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
receptor down-regulation (open
circles) and cAMP production (filled circles, see also
above). CHOrH
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.
In the CHOrHLeu
Ala cells, the
maximal histamine-induced down-regulation was more pronounced (68
± 4%, mean ± S.E., n = 4) than was
observed for the CHOrH
cells (43 ± 4%, mean ±
S.E., n = 7). The forskolin (10
µM)-induced H
receptor down-regulation was
also found to be more pronounced in the
CHOrH
Leu
Ala cells (67 ± 1%, mean
± S.E., n = 3) than in CHOrH
cells
(58 ± 2%, mean ± S.E., n = 4).
In the present study we have demonstrated that the rat
histamine H receptor density in CHO cells is reduced about
50% by long-term exposure to histamine or selective H
agonists. Long-term treatment of CHOrH
cells with
histamine resulted in a time-dependent (t
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
receptor binding sites. Yet,
incubation of CHOrH
cells with homo- and nordimaprit, two
side chain homologues of the H
agonist dimaprit with weak
H
agonistic activity ((23), present study), did not
significantly reduce the number of H
receptors. These
findings show that the observed H
agonist-induced
down-regulation is a H
receptor-mediated process. Long-term
exposure of CHOrH
cells to histamine resulting in a
reduction of H
receptor binding sites is paralleled by a
decrease of H
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 -adrenergic
receptor(24) , a cAMP-dependent pathway can also regulate the
H
receptor density. Forskolin, generating cAMP upon
addition, time dependently (t
7 h at a
concentration of 10 µM) and dose dependently (EC
= 0.3 µM at 24 h of incubation) induced
H
receptor down-regulation. CTX and IBMX, agents that also
elevate intracellular levels of cAMP in CHOrH
cells,
induced down-regulation of the H
receptor as well. Thus,
the H
receptor does not need to be stimulated by an agonist
in order to be down-regulated. This mechanism might be involved in
heterologous H
receptor down-regulation as previously shown
for other GPCRs (see (4) and (25) ). The time course
of the forskolin-induced decrease of H
receptor number in
CHOrH
cells parallels the time-dependent decrease of
H
receptors induced by histamine. For both histamine and
forskolin, half-maximal H
receptor down-regulation is
reached after approximately 7 h of incubation. Moreover, the maximum
decrease of H
receptor numbers induced by forskolin is
comparable to the maximum agonist-mediated H
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 cells with histamine or forskolin resulted in a
transient decrease of H
receptor mRNA levels (70%
reduction) within 4 h, which was followed by a gradual increase of
H
receptor mRNA to 50% of control mRNA levels in the
following hours. The reduced H
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
receptors. The reduction of H
receptor mRNA is most likely explained by post-transcriptional
events, such as receptor mRNA destabilization. For example, the
-adrenergic receptor and thrombin receptor in
DDT
MF-2 smooth muscle cells, the endothelin ET
receptor in ROS17/2 rat osteosarcoma cells, and also for the
-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
receptor mRNA in CHOrH
cells was stimulated
significantly upon histamine-treated and forskolin-treated compared to
nontreated cells. Recently, it was shown that a so-called M
= 35,000
-adrenergic receptor
mRNA-binding protein, involved in the destabilization of
-adrenergic receptor mRNA, also recognizes other GPCR
transcripts(29) . As such, our observations of H
receptor mRNA destabilization fit well in an apparently general
mechanism of
-adrenergic receptor mRNA binding protein-mediated
regulation of GPCR mRNA(29, 31) .
For the
-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
-adrenergic receptor
down-regulation in mutant S49 mouse lymphoma cells defective in signal
transduction
components(32, 33, 34, 35) .
Receptor-G
coupling seems to be important for the process
of
-adrenergic receptor down-regulation, since defects
in this coupling introduced by mutations of the receptor or
G
-protein have lead to impaired
-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
-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
-adrenergic receptor was much slower than the
-agonists-induced down-regulation, suggesting that distinct
pathways can lead to down-regulation of the
-adrenergic receptor (24) . Yet, protein
kinase A-dependent phosphorylation of the
-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,
-adrenergic receptor receptor down-regulation seems to
require receptor-G
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 cells,
comparable time courses and a maximum extent of histamine-induced and
forskolin-induced H
receptor down-regulation as well as
H
mRNA down-regulation suggest the involvement of cAMP in
the process of agonist-induced H
receptor down-regulation.
The initial reduction of H
receptor mRNA upon histamine or
forskolin exposure can, however, not explain the 50% reduction of the
H
receptor numbers, since relatively short (<4 h)
treatments of CHOrH
cells with histamine or forskolin
followed by a wash-out up to 24 h led to a more pronounced H
receptor down-regulation upon histamine than forskolin exposure.
Thus, apparently there is no direct link between H
receptor
mRNA and H
receptor expression. Moreover, these data are a
first indication that histamine and forskolin induce H
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
receptor down-regulation. Moreover,
recently we have shown that down-regulation of H
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
receptor down-regulation (39) .
In order to assess the role of cAMP in the process of
agonist-induced H receptor down-regulation in CHOrH
cells directly, we constructed a mutant H
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
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
receptor expression nor on H
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
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
-adrenergic
receptor.
Interestingly, long-term exposure of
CHOrHLeu
Ala 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
receptor down-regulation in
CHOrH
Leu
Ala 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
-adrenergic receptor
down-regulation(32, 33, 34, 35) .
However, it should be noted that the EC
value of
histamine-induced H
receptor down-regulation was shifted
16-fold to the right for the H
Leu
Ala
receptor compared to the wild-type receptor. These data suggest that
agonist-induced H
receptor down-regulation depends on
intact receptor-G-protein coupling. As already stated earlier, previous
findings for the
-adrenergic receptor have shown that
defective receptor-G
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
Leu
Ala receptor
were found to be more pronounced than for the wild-type H
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
receptor protein, causing an
uncoupling from the G
-protein but also an increase of the
accessibility of molecular entities involved in receptor degradation.
Recent studies with the parathyroid hormone receptor (41) and
-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 receptor is
down-regulated by prolonged treatment with H
agonists.
Elevation of cAMP by long-term incubation of CHOrH
cells
with forskolin, CTX, and IBMX, is also shown to induce H
receptor down-regulation. These data suggest the involvement of
protein kinase A in the process of H
receptor
down-regulation and provides a mechanism for heterologous H
receptor regulation. Also, H
receptor mRNA levels
were rapidly down-regulated upon both histamine treatment and forskolin
treatment. However, the agonist-induced and forskolin-induced H
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
receptor, by an alanine generated a mutant receptor with
impaired ability to couple to its G-protein. Interestingly, the H
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
receptor
down-regulation. Thus, H
receptor down-regulation appears
to be induced by two distinct pathways, a cAMP-dependent and
cAMP-independent pathway.
This paper is dedicated to Prof. Dr. E. Mutschler on the occasion of his 65th birthday.