(Received for publication, January 24, 1997, and in revised form, April 21, 1997)
From the To evaluate the role of the histamine
H2 receptor C terminus in signaling, desensitization, and
agonist-induced internalization, canine H2 receptors with truncated C
termini were generated. Wild-type (WT) and truncated receptors were
tagged at their N termini with a hemagglutinin (HA) epitope and
expressed in COS7 cells. Most of the C-terminal intracellular tail
could be truncated (51 of 70 residues, termed T308
mutant) without loss of functions: cAMP production, tiotidine binding,
and plasma membrane targeting. In fact, the T308 mutant
produced more cAMP than the WT when cell-surface expression per cell
was equivalent. Pretreatment of cells with 10 As observed in a number of guanine nucleotide-binding
protein-coupled receptors (GPCRs),1 cAMP
responses occurring via the histamine H2 receptor are rapidly desensitized after agonist stimulation (1-5). The H2 receptor also
exhibits an agonist-induced internalization from the cell surface (2,
6). However, the mechanisms underlying these phenomena have yet to be
identified. Recently, a number of studies focusing on GPCRs have
examined the role of the C terminus in agonist-induced desensitization
and internalization, with varying results. For example, C-terminal
truncation of the angiotensin II receptor inhibited agonist-induced
internalization of this receptor, but not desensitization of the
calcium response mediated via the receptor (7). On the other hand,
C-terminal truncation of [3H]Tiotidine was purchased from
NEN Life Science Products. Goat anti-mouse 125I-IgG was
purchased from ICN (Costa Mesa, CA). Cimetidine was obtained from
Sigma. Anti-hemagglutinin (anti-HA) monoclonal antibody 12CA5 was
purchased from Boehringer Mannheim (Germany). DEAE-dextran was obtained
from Pharmacia Biotech Inc. (Uppsala, Sweden).
COS7 cells were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum, 100 units/ml
penicillin G, and 0.1 mg/ml streptomycin sulfate.
cDNAs for mutant canine H2 receptors with
mutations involving C-terminal truncation of 70, 51, 46, 39, 30, and 15 amino acids, respectively, were constructed by polymerase chain
reactions. They were termed T289, T308,
T313, T320, T329, and
T344, respectively. cDNAs for mutant receptors, in
which Thr315, Ser316, or both, were replaced
with Ala (A315, A316, and
A315,316), were also constructed. The HA epitope
(YPYDVPDYA) was inserted into the extracellular N termini of the
wild-type (WT) and mutant receptors. After confirming the sequences,
the cDNAs encoding the WT histamine H2 receptor and its truncated
mutants with or without the HA-tag were subcloned into pCAGGS. COS7
cells were transfected with varying amounts of plasmid DNAs by the
DEAE-dextran method.
For immunofluorescence microscopy, COS7 cells were fixed in
3% formaldehyde/phosphate-buffered saline (PBS), scraped off the dish
with a rubber blade, and embedded in 10% gelatin/PBS. Semithin frozen
sections (1 µm thick) were cut and incubated with anti-HA antibody or
antibody against the C terminus of the canine H2 receptor (anti-H2RCT antibody) (2). These sections were then
incubated with rhodamine-labeled affinity-purified goat anti-mouse or
anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) (13).
At 24 h
post-transfection, COS7 cells expressing the WT and truncated H2
receptors with the HA epitope or parental COS7 cells were plated onto
24-well plates at a density of 105 cells/well. At 48 h
post-transfection, cells were incubated in Hepes-tyrode's buffer (140 mM NaCl, 2.7 mM KCl, 1.8 mM
CaCl2, 0.49 mM MgCl2, 0.37 mM NaH2PO4, 5.6 mM
glucose, 25 mM Hepes, pH 7.4) containing 0.1% bovine serum
albumin (BSA) at 37 °C for 30 min. After incubation in the presence
or absence of 10 Tiotidine binding assays involving
intact cells were performed as described previously (14). COS7 cells,
grown in 24-well plates, were assayed at a density of 1 × 105 cells/well. The cells were incubated for 2 h at
37 °C in 200 µl of Hepes-Tyrode's buffer containing 1 nM [3H]tiotidine and various concentrations
of unlabeled tiotidine (15). All samples were analyzed in triplicate.
After incubation, cells were washed three times with ice-cold PBS, then
removed from the wells in 0.1% SDS, and radioactivities were
determined by liquid scintillation counting. Tiotidine bindings in
membranes were assessed as described previously, with some
modifications (2). Membrane fractions (200 µg) from COS7 cells were
incubated with 1 nM [3H]tiotidine and varying
concentrations of unlabeled tiotidine in 25 mM Hepes, 0.1%
BSA, pH 7.4, in a final volume of 200 µl at 37 °C for 2 h.
The binding reaction was terminated by filtration over Whatman GF/C
glass fiber filters, followed by 10-ml washes with ice-cold incubation
buffer. Radioactivity on the filters was determined by liquid
scintillation counting. In both experiments, specific bindings were
calculated by subtraction of the nonspecific bindings determined in the
presence of 10 COS7 cells, plated onto
24-well plates, were assayed at a density of 1 × 105
cells/well, as described previously (14). The cells were incubated for
30 min at 37 °C in 450 µl of Hepes-Tyrode's buffer containing 0.1% BSA and 0.1 mM 3-isobutyl-1-methylxanthine, after
which 50 µl of histamine solution was added to initiate the reaction.
After 10 min of incubation at 37 °C, the reaction was terminated by the addition of 500 µl of 12% trichloroacetic acid. The samples were
centrifuged for 5 min at 3000 × g at 4 °C.
Following extraction of the supernatants three times with diethylether,
cAMP contents in the samples were measured using a radioimmunoassay
(16). Histamine-dependent and
forskolin-dependent cAMP productions were determined by
subtracting basal cAMP productions.
To investigate the role of the cytoplasmic tail of the
histamine H2 receptor in signaling and desensitization, we generated C
terminus-truncated receptor cDNAs. Fig. 1 presents a
schematic representation of the 359-amino acid canine histamine H2
receptor. Consensus sites for post-translational modifications,
N-glycosylation, and palmitoylation are also indicated (17).
We chose to delete 51 and 70 amino acids from the C-terminal tail,
yielding two distinct mutant receptors, termed T308 and
T289, lacking 11 and 13 serine/threonine residues,
respectively. The T289 receptor was truncated at the
beginning of the cytoplasmic C terminus and the T308
receptor just distal to the putative palmitoylation site
(Cys305). All receptor constructs were tagged at their N
termini with a 9-amino acid HA epitope, as shown in Fig. 1. As the
HA-epitope is located in the extracellular region, the cell-surface
receptor amount can be estimated utilizing the antibody against the
HA-epitope, 12CA5. The cDNA constructs were subcloned into an
expression vector, pCAGGS and transfected into COS7 cells. COS7 cells
do not have detectable endogenous histamine H2 receptors, as
demonstrated by the absence of histamine-dependent cAMP
production and the absence of specific [3H]tiotidine
binding (data not shown), and are thus a suitable model system for
these studies. At 48 h after transfection, the cells were
subjected to immunocytochemistry using the anti-HA antibody or the
anti-H2RCT antibody. Expression of the receptors is shown
in Fig. 2. The WT, the HA-wild-type (HA-WT), and the
HA-T308 receptors were distributed in the plasma membranes,
while the HA-T289 receptor, the 70 C-terminal deleted amino
acids of which include Cys305, a consensus site for
palmitoylation, was distributed intracellularly as well as in the
plasma membranes. Whether the different distribution of the
HA-T289 receptor was due to the loss of
palmitoylation or to lack of the proximal amino acid residues from 290 to 307 was not determined. In either case, it can be concluded that the
51 C-terminal amino acid residues of the histamine H2 receptor, a large
part of the C terminus, are not involved in trafficking of the H2
receptor to the plasma membrane. Similar results were obtained when
various amounts of plasmid DNAs were used for transfection of
COS7 cells (data not shown), indicating that the different
distribution of the receptors is not a function of receptor
numbers.
To
examine the effect of C-terminal truncation on ligand binding, intact
COS7 cells on 24-well plates were subjected to binding of tiotidine
(15), an H2 receptor antagonist, at 48 h post-transfection. The
affinities of the WT and truncated receptors for tiotidine were
determined by Scatchard plot analysis. Kd values for
tiotidine of the WT and T308 receptors were comparable
(Table I). In addition, HA-tagging did not affect the
affinity of these receptors for tiotidine (Table I). However,
irrespective of the HA-tagging, the T289 receptor showed no
tiotidine binding. The third and fifth transmembrane regions are
reportedly important for ligand binding of the H2 receptor (18). This
finding might be accounted for by a conformational change in the
T289 receptor induced by the deletion of amino acid
residues from 290 to 307. Since tiotidine is membrane-permeable and is
capable of binding to receptors distributed intracellularly, the
inability of the T289 mutant to bind to tiotidine is not
due to the difference in the intracellular distribution of the receptor
shown in Fig. 2.
Table I.
Dissociation constants (Kd) for tiotidine of wild-type and
truncated H2 receptors
Third Department of Internal Medicine and
Second Department of Biochemistry,
Health Service Center, University of Tokyo,
Hongo, Tokyo 113, the ¶ Laboratory of Molecular and Cellular
Morphology,
5
M histamine desensitized cAMP productions via WT and
T308 receptors to similar extents. Incubation of cells
expressing WT receptors with 10
5 M histamine
reduced cell-surface anti-HA antibody binding by approximately 30% (by
30 min, t1/2 ~ 15 min), but did not affect the Bmax of tiotidine in membrane fractions,
which represents total receptor amounts, suggesting that WT receptors
were internalized from the cell surface. In contrast, no
internalization was observed with T308 receptors following
histamine treatment. A mutant with a deletion of the 30 C-terminal
amino acids, termed T329, was functional but was as potent
as the WT in terms of cAMP production. Apart from being desensitized by
histamine, the internalization of the receptor was indistinguishable
from that of the WT. Internalization was observed in the
T320 but not in T313 mutant, narrowing
the region involved in internalization to that between
Glu314 and Asn320 (ETSLRSN). Of these
seven residues, either Thr315, Ser316, or both,
were replaced with Ala. Thr315 and Ser316 are
conserved among species. The mutation at Thr315 (but not
that at Ser316) abolished internalization. Taken together,
these results demonstrate that Thr315 is involved in
agonist-induced internalization. Furthermore, the finding that
T308 receptors were desensitized in the absence of
internalization suggests that internalization and desensitization are
meditated by independent mechanisms.
2-adrenergic,
1B-adrenergic, lutropin/choriogonadotropin, platelet-activating factor, and neurokinin-2 receptors resulted in
impairment of homologous desensitization (8-12). Thus, the C termini
of GPCRs have functional importances, which may differ among receptors.
The present study was designed to analyze the role of this important
portion of the histamine H2 receptor in signaling, desensitization, and
agonist-induced internalization. To this end, we constructed H2
receptor cDNAs, devoid of either the 71 or the 51 amino acids at
the C terminus, and expressed these cDNAs in COS7 cells. Herein, we
present evidence that the 51 C-terminal amino acids of the histamine H2
receptor are important for agonist-induced internalization, but not for
either signaling or homologous desensitization of cAMP response, which
occurs via this receptor. In addition, these amino acids exert
inhibitory effects on cAMP production via the H2 receptor.
Materials
5 M histamine at 37 °C for
the indicated times, the cells were fixed with PBS containing 3%
paraformaldehyde, washed again with PBS, and incubated with 5% skim
milk/PBS for 1 h at room temperature. They were incubated with 200 µl of 12CA5 (5 µg/ml) in PBS for 2 h at room temperature,
washed again with PBS, and incubated for another 1 h with 200 µl
of 125I-labeled goat anti-mouse IgG (1:200 dilution) at
room temperature. The wells were then washed twice with 5% skim
milk/PBS, twice with PBS, and three times with 0.05% Tween 20/PBS.
Bound 125I-labeled goat anti-mouse IgG was solubilized in
1% SDS, and radioactivities were determined in a
-counter. Specific
bindings were determined by subtraction of the nonspecific bindings
observed in parental COS7 cells.
4 M cimetidine. No specific
binding was observed in either parental COS7 cells or COS7 cells
transfected with the expression vector alone.
Expression of Wild-type and C Terminus-truncated Histamine H2
Receptors
Fig. 1.
Schematic representation of the canine
histamine H2 receptor. The sites of truncation for the deletion
mutants, T308 and T289, are indicated by
arrows. Serine and threonine residues in the C terminus are
shown in boldface type. The palmitoylation and membrane
anchorage of Cys305 are presumed. Consensus sequences of
N-glycosylation sites, Asn4,
Asn162 and Asn168, are also presented.
[View Larger Version of this Image (15K GIF file)]
Fig. 2.
Immunocytochemistry of the WT and truncated
receptors expressed in COS7 cells. COS7 cells were transfected
with each plasmid DNA. At 48 h post-transfection COS7 cells were
fixed in PBS with 3% paraformaldehyde. Thin sections were prepared and immunostained with anti-HA antibody (a, b,
c) or anti-H2RCT antibody (d).
a, HA-T289; b, HA-T308;
c, HA-WT; d, WT. Scale bar, 10 µm.
[View Larger Version of this Image (52K GIF file)]
Kd
nM
WT
23.2 ± 1.1
T308
25.2 ± 2.7
T289
No binding
HA-WT
25.6 ± 0.8
HA-T308
22.1
± 1.5
HA-T289
No
binding
To examine the role of the C terminus in H2 receptor signaling, we measured histamine-dependent cAMP productions via the WT and truncated receptors in COS7 cells. cAMP productions mediated by these receptors can be compared only when the amounts of cell-surface receptors are equivalent. Iida-Klein et al. (19) reported that in a COS7 transfection system using the DEAE-dextran method the amount of plasmid DNA used for transfection determined the number of cell-surface parathyroid hormone/parathyroid hormone-related protein receptors expressed per cell but not the percentage of transfected cells. We have obtained similar results, in that the amounts of plasmid DNA affected the number of H2 receptors expressed per cell and the percentage of transfected cells were constant even when the amount of plasmid DNA used was not equivalent (Table II). We transfected various amounts of plasmid DNA for HA-WT, HA-T308, and HA-T289 receptors and measured the number of receptors expressed per cell. As shown in Fig. 3A, the amounts of cell-surface receptors were dependent on the amounts of plasmid DNA used for transfection and, in addition, differed markedly among the WT and mutant receptors even when equivalent amounts (molar) of plasmid DNAs were used for transfection. The number of cell-surface HA-WT receptors expressed was larger than that of HA-T308 receptors when the equivalent amounts of plasmid DNAs were used for transfection. It is essential to study the functions of these receptors at similar expression level. Therefore, we measured histamine-dependent cAMP productions via the WT and mutant receptors at each transfection levels. This allowed comparison of the functions of these receptors. As shown in Fig. 3B, histamine-dependent cAMP productions were observed in HA-WT and HA-T308 cells but not in HA-T289 cells. Histamine-dependent productions of cAMP observed in HA-WT and HA-T308 were inhibited by cimetidine but not by diphenhydramine (data not shown). Interestingly, as compared with HA-WT receptors, HA-T308 receptors produced more cAMP than the WT receptor (Fig. 3B) if the numbers of cell-surface receptors per cell were equivalent. Therefore, the 51 C-terminal amino acids are not only essential for, but rather may actually inhibit, cAMP production mediated by the H2 receptor. It is also possible that inhibitory effects on the H2 receptor are exerted via the C-terminal region. Although less marked than those in HA-WT and HA-T308 cells, specific cell-surface anti-HA antibody bindings were also observed in HA-T289 cells (Fig. 3A). Thus, the absence of histamine-dependent cAMP production via T289 receptors was not due to a lack of cell-surface T289 receptors, but rather to the inability of the receptor itself to couple to Gs. It is not clear whether this is due to the absence of the amino acid residues from 289 to 307, or to a conformational change induced by the deletion. Furthermore, the possible contributions of these amino acid residues to cAMP production could not be examined.
|
Desensitization of WT and T308 Receptor-mediated cAMP Responses
The histamine H2 receptor reportedly shows homologous
desensitization of the cAMP response following preincubation with
histamine. We examined whether the C-terminal tail of the H2 receptor
is involved in desensitization of the cAMP response, which occurs via
this receptor. Since COS7 cells expressing the HA-T289
mutant did not mediate cAMP production in response to histamine (Fig.
3B), this mutant was omitted from the experiment. COS7 cells were transfected with plasmid DNAs containing HA-WT (0.4 µg/10-cm plate) or HA-T308 (1.0 µg/10-cm plate) receptors.
Transfection with these DNA amounts resulted in similar expression
levels of the HA-WT and HA-T308 receptors (Fig.
3A). Preincubation of transfected COS7 cells with
105 M histamine for 30 min led to reduced
cAMP production in both the HA-WT and HA-T308 cells as
compared with untreated cells (Fig. 4A),
indicating desensitization, while forskolin-dependent cAMP
production was slightly increased (Fig. 4B). It is
noteworthy that the transfection efficiency of the COS7 cell system is
approximately 20% at best (Table II). Furthermore, the results shown
in Fig. 4B do not necessarily mean that
forskolin-dependent cAMP production in COS7 cells
expressing the receptors was not altered. Therefore, we performed
similar experiments in CHO cells stably expressing HA-WT or
HA-T308 receptors and found that
histamine-dependent cAMP production via these receptors was
reduced by preincubation with 10
5 M
histamine, while forskolin-dependent cAMP production was
not (data not shown). Similar results were obtained following a 15-min preincubation with 10
5 M histamine (data not
shown). Taken together, these results suggest that the 51 amino acids
of the C terminus of the histamine H2 receptor are not essential for
desensitization of the receptor-mediated cAMP response.
Effect of C-terminal Truncation on Agonist-induced Internalization of the H2 Receptor
A number of GPCRs have been reported to be
internalized into compartments inaccessible to agonists acting at the
plasma membrane. Agonist-induced internalizations of several other
GPCRs have been detected by measuring the acid-resistant forms of
radiolabeled agonists (7, 20-23). However, this experiment cannot be
performed for the H2 receptor because of the high levels of nonspecific uptake of histamine by cells. To circumvent this difficulty, we utilized the HA-tag, which is located at the N-terminal extracellular region, to quantify the cell-surface H2 receptor amount. Specific cell-surface bindings of anti-HA antibody were observed in COS7 cells
expressing HA-WT and HA-T308 receptors (Fig.
3A), but not in parental or mock transfected COS7 cells
(data not shown). We examined the effect of incubation with
105 M histamine on the cell-surface H2
receptor amount. As shown in Fig. 5A, in COS7
cells expressing HA-WT receptors the amount of cell-surface anti-HA
antibody binding decreased by approximately 30% with a 30-min
incubation with 10
5 M histamine and the
t1/2 was approximately 15 min. However, neither the binding maximum (Bmax) of tiotidine in
membrane fractions (Fig. 5B), which represents the total
receptor amount, nor the Kd for tiotidine (data not
shown) was decreased. These findings indicate that HA-WT receptors were
internalized from the cell surface. Immunocytochemical examination
after a 30-min incubation with histamine revealed the HA-WT receptors
to be on the plasma membrane, possibly indicating that the receptors
were in coated pits or caveola. In contrast, incubation of COS7 cells expressing HA-T308 receptors with histamine affected
neither cell-surface anti-HA antibody binding (Fig. 5A) nor
the Bmax of tiotidine (Fig. 5B), indicating that the 51 C-terminal amino acids of the H2 receptor play a
role in agonist-induced internalization of the receptor.
Effects of Truncation of 15 and 30 C-terminal Amino Acids on H2 Receptor Signaling, Desensitization, and Internalization
To
define more clearly the region involved in internalization, two mutant
receptors, T329 and T344, with deletion of 30 and 15 C-terminal amino acids, respectively, were generated. These
mutants were also tagged with the HA-epitope at their N termini and
were expressed in COS7 cells. Both receptors were capable of binding
tiotidine with affinities similar to that of the WT receptor (data not
shown). In contrast to the finding in the HA-T308 mutant
receptor, cAMP productions via both receptors were comparable to those
mediated by the HA-WT receptor provided that the amounts of
cell-surface receptors per cell were equivalent (Fig.
6A). These observations indicate that these
30 amino acids of the C terminus are not involved in inhibition of cAMP
production. In addition, cAMP productions via these receptors were
desensitized by 105 M histamine preincubation
(data not shown). Finally, upon histamine exposure, HA-T329
and HA-T344 receptors showed internalization
indistinguishable from that of the HA-WT receptor (Fig.
6B).
Identification of the Amino Acid Residues Involved in Internalization of the H2 Receptor
The results obtained indicate that the region responsible for agonist-induced internalization and inhibition of cAMP production resides between amino acid residues 308 and 328. To identify the amino acid residues involved in agonist-induced internalization, we generated additional mutants, HA-T313 and HA-T320, with deletions of 39 and 46 C-terminal amino acids, respectively. Interestingly, agonist-induced internalization was observed in the HA-T320, but not in the HA-T313 receptor (data not shown). Thus, the amino acid residues involved in agonist-induced internalization of the histamine H2 receptor are likely to reside between Glu314 and Asn320 (ETSLRSN). Of these seven amino acid residues, assuming that the hypothesis that serine and/or threonine phosphorylation is involved in agonist-induced internalization of the H2 receptor is correct, the likely candidates are Thr315 and Ser316. This is because these two residues are conserved among species (Table III). To determine whether these amino acid residues are involved in agonist-induced internalization, we generated three mutant receptors, HA-A315, HA-A316, and HA-A315,316, in which either Thr315, Ser316, or both were replaced with Ala. As shown in Fig. 7, the HA-A316 receptor internalization was indistinguishable from that of the WT, whereas those of the HA-A315,316 and HA-A315 were not. This finding clearly demonstrates that Thr315 is involved in agonist-induced internalization of the histamine H2 receptor and that the inabilities of the HA-T308 and HA-T313 mutants to internalize were not due to nonspecific effects induced by the truncations.
|
The histamine H2 receptor plays a central role in acid production in gastric parietal cells via production of cAMP (24). Inappropriate gastric acid production can lead to the generation or exacerbation of peptic ulcer disorders. Tight control of signaling via the H2 receptor is thus vital for normal gastric function. It has been accepted that the histamine H2 receptor undergoes rapid desensitization of the cAMP response upon exposure to an agonist (1-5). Although homologous desensitization of agonist-induced signaling via GPCRs is an essentially ubiquitous phenomenon, the functional roles of the C termini vary among receptors. In a number of GPCRs, agonist-induced serine/threonine phosphorylation of the C terminus has been implicated in receptor desensitization (8-10), while in others the C terminus has been suggested to be involved in agonist-induced internalization, but not in desensitization (20, 25, 26). To understand the as yet unidentified roles of the C terminus in the histamine H2 receptor, we generated two mutant receptors, T289 and T308, which were truncated at the beginning of the cytoplasmic C-terminal portion and just distal to putative palmitoylation site Cys305, respectively. The WT and mutant receptors were tagged at their N termini with the HA epitope and expressed in COS7 cells.
One of the primary observations made in our present study was that the T308 receptor with a truncation of 51 amino acids located distal to the putative palmitoylation site was desensitized upon histamine exposure. It is hypothesized that serine/threonine phosphorylation is involved in agonist-induced desensitization of GPCRs. The T308 mutant, which is missing 11 serine/threonine residues present in the C terminus of the histamine H2 receptor, did not show altered agonist-induced uncoupling of the H2 receptor from adenylyl cyclase. Therefore, it is likely that the serine/threonine residues, which may be phosphorylated as part of the desensitization process, reside in other intracellular regions or the proximal C-terminal residues, namely Thr290 and Thr 297. Possible contributions of this proximal carboxyl region could not be examined because T289 receptors, which are devoid of all C-terminal amino acid residues, were nonfunctional (Table I, Fig. 3B). It is noteworthy that the lack of T289 receptor activity is not due to the absence of cell-surface expression but rather to the receptor itself being devoid of functional activity (Fig. 3).
Second, the WT receptor was internalized upon histamine exposure while
the T308 mutant was not. In addition, the T329
mutant with a deletion of 30 amino acids was internalized. Thus, the
region involved in internalization of the histamine H2 receptor is
present within the area including amino acid residues from 308 to 328. The observation that the T320, but not the T313
receptor, was internalized further confined the amino acid residues involved in agonist-induced internalization. Finally, the finding that
internalization occurred in the HA-A316 receptor, but in
neither the HA-A315 nor the HA-A315,316
receptor, identified the amino acid residue involved in agonist-induced internalization of the H2 receptor as Thr315. Our
preliminary observations indicated that both the HA-WT and the
HA-T308 receptors were phosphorylated after histamine
stimulation (data not shown). Thus, although we can speculate that
phosphorylation does actually play a part in desensitization, we cannot
determine whether it is involved in agonist-induced internalization of
the H2 receptor. Recently, Barak et al. (27) reported that a
tyrosine residue highly conserved in G-protein-coupled receptors and in the NPXXY motif is involved in sequestration of the
2-adrenergic receptor. This tyrosine residue is
conserved in the histamine H2 receptor (Tyr288) (17, 28,
29). The T308 receptor contains this tyrosine, such that
the tyrosine residue Tyr288 appears to be necessary for,
but is not by itself sufficient to produce, internalization of the
histamine H2 receptor. Furthermore, desensitization of the cAMP
response occurring via the T308 receptor, which was not
internalized, indicates that internalization per se is not
essential for agonist-induced desensitization of the cAMP response and
that desensitization and internalization are mediated by independent
mechanisms.
Third, we have shown the T308 receptor to be more active than the WT receptor in terms of adenylyl cyclase stimulation. In contrast, the T329 and the T344 receptors showed essentially the same levels of activity as the WT receptor. Thus, the 51 C-terminal amino acids of the histamine H2 receptor contain sequences that suppress the interaction of this receptor with G-proteins, and these sequences apparently lie within the region from amino acids 308 to 329. When these sequences are removed a more productive interaction takes place, and hormonal responsiveness upon truncation is enhanced. As desensitization still took place in the T308 receptor, this inhibitory effect is unlikely to be related to desensitization.
In conclusion, we have shown in COS7 cells that the C terminus of the histamine H2 receptor is involved in agonist-induced internalization, but not in desensitization. Furthermore, the C terminus is likely to exert an inhibitory effect on cAMP production via the receptor. Since these experiments were performed using fibroblast cell lines transfected with histamine H2 receptor cDNAs, the results presented show that the processes involved in histamine-induced uncoupling of the histamine H2 receptor are not specific to gastric parietal cells. However, the extent of internalization observed in this system is minimal compared with those of other receptors. We cannot rule out the possibility that some other cofactor, which is not present in COS7, might function in histamine H2 receptor internalization. Future studies must be designed to identify the specific amino acid residues involved in agonist-induced desensitization.