(Received for publication, May 5, 1995; and in revised form, June 12, 1995)
From the
To investigate the role of the carboxyl terminus in the
regulation of the bombesin (BN) receptor, we constructed two chimeric
receptors with carboxyl termini transferred from either m3 muscarinic
cholinergic (m3 ACh) (BMC) or cholecystokinin A (CCK) (BCC)
receptors and expressed them in Chinese hamster ovary cells. Previous
studies showed that agonist treatment caused rapid internalization of
CCK
but not m3 ACh receptors in these cells. In the current
study we conducted separate analyses of ligand and receptor
internalization and analyzed receptor recycling. Ligand internalization
was assessed using acid washing. BN and CCK
receptors
internalized ligand with 80 ± 3 and 85 ± 7% in an
acid-resistant compartment at equilibrium. Ligand internalization of
chimeric receptors generally assumed the properties of the donor
receptors. Thus, BCC receptors internalized ligand to a similar extent
as wild-type CCK
receptors (75 ± 3%), whereas, BMC
receptors showed reduced ligand internalization (38 ± 1%).
Receptor internalization was more directly assessed by determining
agonist-induced loss of surface binding. BN and CCK
receptors were largely internalized (56 ± 8 and 50
± 7%, respectively). BCC receptors were also extensively
internalized (82 ± 3%). In contrast, BMC receptors were
minimally internalized (22 ± 8%). Receptor recycling was
assessed as recovery from agonist induced loss of binding. BN,
CCK
, and BMC receptors showed rapid recycling. In contrast,
BCC receptors did not recycle. These data indicate that
carboxyl-terminal structures determine both internalization of
ligand-receptor complexes and subsequent receptor recycling.
Receptor internalization is a ubiquitous process occurring for virtually all plasma membrane receptors. The endocytotic receptors, such as those for low density lipoprotein and transferrin, are constitutively internalized(1, 2) . In contrast receptors for hormones, neurotransmitters, and growth factors require agonist occupancy for internalization, suggesting the need for an agonist-dependent signal to initiate the internalization process. Tyrosine kinase activity is required for internalization of growth factor receptors that are tyrosine kinases, such as insulin (3) and epidermal growth factor (4) receptors. However, for the family of seven transmembrane G protein-coupled receptors the nature of the internalization signal is unknown. This signal is unlikely to be a common second messenger because receptors having diverse effects on intracellular second messengers are similarly internalized. However, whether the internalization signal involves activation of G proteins is less clear. On one hand, antagonists are not internalized and the internalization of partial agonists correlates with their abilities to activate G proteins(5) . Also, many uncoupled mutant receptors are not internalized, and the degree of uncoupling often correlates with the decrease in internalization(6, 7) . On the other hand, certain mutant receptors with reduced G protein coupling internalize normally(8, 9, 10, 11, 12) . One interpretation of these data is that both internalization and activation of heterotrimeric G proteins require agonist-induced conformational changes in the receptors and that the domains involved in internalization are near but separate from those involved in coupling to G proteins(6, 13) .
The search for internalization domains has occurred at a rapid pace because of the importance of internalization in receptor function and regulation. A large number of domains have been implicated in the internalization of specific G protein-linked receptors(14, 15, 16, 17, 18, 19) . However, these domains have localized to divergent regions of the various receptors and often involve sequences not conserved in other members of the receptor superfamily. Alterations in the carboxyl terminus have been found to influence internalization in the widest variety of receptors. Yet, the role of the carboxyl terminus remains uncertain as it has been found to be either necessary(11, 16, 20) , inhibitory(17, 21, 22, 23) , or not involved in internalization(24) , depending upon the receptor. One complication is that many of the studies have involved deletions, truncations, or multiple substitutions in the carboxyl terminus, such that it has often been difficult to discern whether the experimental alterations have been specific or may have resulted in nonspecific conformational effects.
An alternative to deletion and substitution
experiments is the creation of receptor chimeras. This strategy offers
substantial advantages for analysis of structure and function in that
the expected outcome is the alteration or addition, rather than the
loss of function(25) . We chose this approach to analyze the
role of the carboxyl terminus in the internalization of the bombesin
receptor. Bombesin receptors rapidly internalize ligand both in
natively expressing cells, such as pancreatic acinar (26) and
Swiss 3T3 (27) cells, as well as in transfected cell models,
such as Balb C 3T3 cells(7, 11) . In addition,
truncation or major alterations of the bombesin receptor carboxyl
terminus were previously shown to block internalization in Balb C 3T3
cells(11) . As sources of donor carboxyl termini, we chose the
m3 muscarinic cholinergic receptor (m3 ACh) ()and the
cholecystokinin A (CCK
) receptor, as these receptors have
different internalization characteristics. CHO cells show rapid
agonist-induced internalization of CCK
(28) but not
m3 ACh receptors(29) . Also, CCK
receptors are
internalized via both clathrin-coated and -uncoated vesicles (28) whereas bombesin receptors are internalized exclusively
via clathrin-coated vesicles(30) . As a working hypothesis we
predicted that internalization of chimeric receptors would reflect the
properties of the carboxyl-terminal donor receptors. Thus, a
bombesin/m3 ACh chimeric receptor would show impaired internalization.
In contrast, a bombesin/CCK
chimeric receptor would
internalize to the same extent as wild-type BN or CCK
receptors. Ligand internalization, receptor internalization, and
receptor recycling were separately analyzed. The data supported the
working hypothesis and thus confirmed the previously proposed
importance of carboxyl-terminal domains in receptor internalization. In
addition, important differences were noted between internalization of
ligand and receptor that reflected differences in the methodologies.
Furthermore, we found that the carboxyl terminus influenced receptor
recycling independently of internalization, thus indicating an
additional role of the carboxyl terminus in receptor regulation.
[I-Tyr
]Bombesin (81.4
TBq/mmol) and [
I-BH]CCK
(81.4
TBq/mmol) were obtained from DuPont NEN. myo-[
H]Inositol (19.1 Ci/mmol) was
obtained from Amersham Corp. Analytical grade Dowex 1-X8 (AG1-X8,
100-200 mesh) and Bio-Rad protein assay reagent were obtained
from Bio-Rad. Restriction endonucleases were purchased from Life
Technologies, Inc.. Taq polymerase was obtained from Promega
(Madison, WI). Oligonucleotides were synthesized by an Applied
Biosystems 380B DNA synthesizer. Bombesin and CCK8 were obtained from
Bachem (Torrance, CA). Trichloroacetic acid was obtained from J. T.
Baker. Soybean trypsin inhibitor type I-S, bovine serum albumin,
fraction V, and all non-specified reagents were obtained from Sigma.
Dulbecco's modified Eagle's medium, fetal bovine serum,
penicillin, streptomycin, G418, Lipofectin reagent, and amphotericin B
were obtained from Life Technologies, Inc. Tissue culture plastic-ware
(24- and 6-well plates and 10-cm Petri dishes) were obtained from
Costar (Cambridge, MA). CHO-K1 cells were obtained from the American
Type Culture Collection (Rockville, MD).
All receptors were subcloned into pTEJ-8 for expression in cell lines.
For determination of receptor
internalization, we measured the loss of surface binding after agonist
treatment. The analysis of agonist-induced loss of binding was
conducted on cells plated in 24-well dishes at 2-3
10
cells/ml the day before the assay. Incubations were
performed at 37 °C with 100 nM of bombesin (for BN and BN
chimeric receptor) or CCK
(for CCK
receptor).
After specified periods of time, the cells were washed with 1 ml of
ice-cold PBS and incubated in 1 ml of a low pH ligand removal solution
(90 mM NaCl, 50 mM sodium citrate, 0.2 mM Na
HPO
, 0.1% bovine serum albumin, pH 5) (36) for 10 min at 4 °C. Cells were then washed with PBS at
4 °C. This procedure removed >90% of receptor-associated ligand
and rebinding was not impaired by this treatment (data not shown). The
acid wash solution utilized for analysis of ligand internalization was
not suitable for this assay as the cells did not rebind labeled agonist
after the acid wash treatment (data not shown). Cells were then
incubated in binding buffer (HR buffer) with
I-bombesin
or
I-CCK
(10 pM) overnight at 4
°C. Nonspecific binding was determined in the presence of 100
nM bombesin or CCK
. Incubation was terminated by
washing the cells with 1 ml of ice-cold PBS twice. The cells were then
removed from the plate with 0.1 N NaOH, and cell-associated
radioactivity was counted in a
-counter.
Figure 1:
Schematic representations of the amino
acid composition of BN, BCC, and BMC receptor carboxyl termini. A, diagram showing the location of potentially phosphorylated
residues. B, diagram indicating conserved sequences (boxes), potential phosphorylation sites (bold), and
consensus protein kinase C sequences (underlined) between BN,
CCK, and m3 ACh receptors. Chimeric receptors were produced
by a PCR based strategy. All constructs were confirmed by
sequencing.
To obtain CHO cell lines with stable expression of chimeric bombesin
receptors (BMC and BCC), cells transfected with the receptor cDNAs
cloned into the pTEJ-8 expression vector were selected with G418.
Stable expression of receptors was confirmed by I-bombesin binding. Clones with approximately equal
levels of binding were chosen and further characterized. Competition
binding experiments with
I-bombesin and increasing
concentrations of unlabeled bombesin were performed at 4 °C in
order to determine if replacing the carboxyl terminus of the BN
receptor with the analogous portion of either m3 ACh or CCK
receptors altered the affinity of the receptor for agonist.
Computer aided non-linear least-squares analysis of bombesin
competition binding curves fit the behavior of a single class of
binding sites for each cell line. The two BN chimeric receptors had
affinities (K
) for bombesin similar to
the wild-type BN receptor (Table1). The receptor number (B
) of the selected clonal cell lines bearing
chimeric receptors was the same (BMC) or somewhat lower (BCC) than that
of the clone bearing the wild-type BN receptor (Table1).
The
ability of the BN chimeric receptors to mediate bombesin-induced
stimulation of polyphosphoinositide (PPI) metabolism was examined in
the stable cell lines. Both BMC and BCC receptors induced pronounced
PPI hydrolysis responses indicating that the substitution of foreign
carboxyl termini did not greatly affect the ability of the receptors to
activate G proteins. The response of BMC bearing cells was comparable
in magnitude to that of the wild-type BN receptor bearing cells,
whereas the BCC bearing cells had a somewhat lesser response (Fig.2). Activation of mutant receptors also elicited increases
in intracellular Ca mobilization when analyzed using
fura-2 as an indicator (data not shown).
Figure 2:
Effects of bombesin on total PPI
hydrolysis in CHO cells bearing bombesin receptors. Confluent cells
were incubated with 1.5 µCi/ml myo-[2-H]inositol for 24 h after which
they were exposed to the specified concentrations of bombesin for 30
min. Isolation of [
H]inositol phosphates was
performed as detailed under ``Experimental Procedures.'' Data
are expressed as the fold increase over basal PPI release and
represents the mean ± S.E. of three separate experiments, with
each value measured in triplicate in each
experiment.
Figure 3:
Time course of specific binding and
internalization of [I-Tyr
]bombesin
in CHO cells bearing wild-type BN and chimeric BCC and BMC receptors.
Cells were incubated with 10 pM
[
I-Tyr
]bombesin at 37 °C for
the indicated times, then the cells were washed at 4 °C.
Surface-bound (acid removable, open symbols) and intracellular
(acid-resistant, closed symbols) radioactivity were determined
as described under ``Experimental Procedures.'' Nonspecific
binding, defined as binding in the presence of 100 nM bombesin, was determine similarly, and values have been subtracted
from each point. Each point is given as percent of total tracer
specifically bound and is from a representative of three to five
separate experiments.
Figure 4:
Ligand and receptor internalization by
wild-type and chimeric receptors. For analysis of ligand
internalization CHO cells bearing BN, BCC, BMC, and CCK receptors were incubated with 10 pM
[
I-Tyr
]bombesin or
I-CCK
at 37 °C for 90 min then the cells
were washed at 4 °C. Internalized (acid-resistant) radioactivity
was determined. Nonspecific binding, defined as binding in the presence
of 100 nM bombesin or CCK
, was determined
similarly, and values have been subtracted. Data shown are expressed as
the percentage of total tracer specifically bound and represent the
mean ± S.E. of three separate experiments. For analysis of
receptor internalization, cells were treated with 100 nM agonist (bombesin or CCK
) for 30 min at 37 °C then
placed on ice, washed with PBS and low pH buffer, and binding of
I-bombesin or
I-CCK
was
conducted overnight at 4 °C. Receptor internalization is calculated
as the reduction in the percentage of the surface binding in cells not
treated with agonist and data represent means ± S.E. for three
to seven experiments.
Figure 5:
Agonist-induced receptor internalization
in cells bearing BN, BCC, BMC, or CCK receptors. Cells were
treated with 100 nM agonist (bombesin or CCK
) for
various times at 37 °C, then placed on ice, washed with PBS and low
pH buffer, and binding of
I-bombesin or
I-CCK
was conducted overnight at 4°. The
reduction in surface binding after agonist treatment is presented as a
percentage of the binding in cells not treated with agonist, and data
represent means ± S.E. for three to seven
experiments.
Because a reduction in surface binding might involve alterations in receptor affinity, we conducted agonist binding competition studies before and after agonist treatment (Table1). No significant changes were noted in receptor affinities. Reductions of receptor numbers entirely accounted for the observed changes in total specific binding.
To investigate whether the loss of binding reflected internalization via clathrin-coated vesicles, the effects of high sucrose concentrations known to block this pathway (39) were examined. Sucrose treatment completely blocked internalization of wild-type BN receptors (92 ± 2% inhibition, n = 3). In contrast, BCC receptor internalization was only partially inhibited (72 ± 4% inhibition, n = 3).
Figure 6:
Recovery of surface binding to cells
bearing BN, BCC, BMC, or CCK receptors after
agonist-induced receptor internalization. Cells treated with 100 nM agonist (bombesin or CCK
) for 30 min at 37 °C were
washed with PBS and low pH buffer, then allowed to recover for various
times at 37 °C before being placed on ice, and binding of
I-bombesin or
I-CCK
was
conducted overnight at 4 °C. Binding to surface receptors is
presented as a percentage of the binding to cells not treated with
agonist, and data represent means ± S.E. for four to nine
experiments.
The majority of G protein-coupled receptors internalize, and therefore internalization might be expected to involve a common mechanism. However, thus far, generalized domains and mechanisms for receptor internalization have been difficult to ascertain. Several complications have hindered the search for common structural features related to receptor internalization. These include: 1) receptor conformation can be indirectly influenced by structural manipulations; 2) internalization occurs via multiple mechanisms; 3) methodologies for investigating receptor internalization vary between receptor classes and are not strictly comparable; and 4) receptor internalization is cell type-dependent. The current study has investigated the role of the carboxyl terminus in bombesin receptor internalization and has attempted to address these issues.
In the current study, we utilized
a chimeric approach. Previous studies have demonstrated that mutation
or truncation of sequences in the third cytoplasmic
loop(6, 24, 40, 41, 42) ,
the second cytoplasmic loop(6) , and the carboxyl
terminus(15, 16, 17, 18, 20, 21, 29, 43) reduced
internalization of specific receptors. However, interpretation of
mutation studies must take into account the possibility of secondary
conformational effects since receptors exist as three-dimensional
entities. Thus, mutations of specific domains that are not themselves
directly required for internalization may interfere with conformational
changes in domains that are required for internalization. Secondary
conformational effects are also possible in chimeric receptors.
However, the advantage of the chimeric approach is that the predicted
outcome is the retention or acquisition of function, rather than its
loss, and it is unlikely that a function would be nonspecifically
acquired. In the current study, substitution of the carboxyl terminus
from the CCK receptor produced a chimeric receptor with a
fully intact ability to internalize. It has previously been shown that
truncation of the bombesin receptor carboxyl terminus blocked ligand
internalization (11) . Thus, the restoration of the ability to
internalize achieved by addition of the CCK
carboxyl
terminus strongly supports the hypothesis that this domain is an
important determinant of this function. Furthermore, substitution with
the carboxyl terminus from the m3 ACh receptor, which itself is not
rapidly internalized in these cells(29) , produced a receptor
with reduced internalization properties. Thus, the internalization
characteristics of the chimeric receptors were specific, generally
reflected the characteristics of the donor receptors, and strongly
indicated a role of the carboxyl terminus in receptor internalization.
In the current study, internalization of wild-type bombesin
receptors into CHO cells was completely blocked by hypertonic sucrose
treatment, suggesting that internalization occurred exclusively via
clathrin-coated vesicles, as has been previously reported in other
cells(30) . However, internalization of the bombesin/CCK chimera was not completely blocked by sucrose treatment,
suggesting the participation of other non-clathrin mediated
internalization pathways. Previously it was shown that the CCK
receptor is internalized into CHO cells via both coated and
uncoated vesicles and that the latter pathway is not inhibited by
hypertonic media(28) . Therefore, the carboxyl terminus of the
CCK
receptor may have transferred the ability to interact
with a clathrin-independent pathway to the chimeric receptor.
Morphological investigations will be needed to verify this hypothesis.
It is unclear how these morphologically distinct pathways correspond to
known regulatory mechanisms such as sequestration and down-regulation.
Both sequestration and down-regulation involve receptor internalization
but appear to involve separate mechanims as they can be independently
affected by structural
manipulations(8, 9, 15, 18) .
Moreover, it is unknown whether the mechanisms used for the
internalization of peptide ligands are the same as those used for
receptor sequestration or down-regulation, or whether they represent
yet an additional internalization pathway. The existence of multiple
internalization pathways interacting with separate structural domains
may provide a partial explanation for the difficulty in generalizing
the results obtained from studies on internalization of specific
receptors.
In the current study both ligand and receptor internalization were independently measured. In the case of the bombesin/m3 ACh receptor, alterations in receptor internalization fit the predictions based on the characteristics of the carboxyl-terminal donor. Previously we found that m3 ACh receptor sequestration was not increased by agonist in CHO cells(29) . Accordingly, internalization of the bombesin/m3 ACh receptor chimera was nearly blocked. Ligand internalization characteristics are not known for m3 ACh receptors. Unfortunately, the methods and terminology used to describe internalization for peptide binding receptors are different compared to adrenergic and muscarinic receptors. ``Sequestration'' originally referred to the loss of binding of hydrophilic ligands while the binding of hydrophobic ligands remained unchanged in studies of adrenergic and muscarinic receptors (44, 45) . For peptide binding receptors what is sometimes referred to as sequestration is derived from measurements of ligand internalization using acid washing because hydrophobic ligands are generally not available. However, several important differences exist between ligand internalization and receptor sequestration. First, sequestration is concentration dependent and requires concentrations of agonist that activate second messenger pathways. In contrast, ligand internalization occurs with tracer concentrations of labeled ligand that are below the threshold of activating second messengers. Second, because tracer concentrations of ligand only occupy a small fraction of receptors, the internalization of a large proportion of tracer is accomplished by the internalization of only a small proportion of receptors. Third, the accumulation of intracellular ligand is influenced by a large number of factors other than the rate of receptor internalization, including the rates of ligand degradation and release, and receptor recycling. Thus, ligand internalization measured by acid washing does not accurately reflect the process of receptor sequestration. In contrast, agonist-induced loss of binding closely matches the classic sequestration assays as both follow receptors rather than ligand, occur in response to activating concentrations of agonist, and are not affected by ligand degradation. Therefore, comparison between agonist-induced loss of binding and sequestration is more appropriate than between acid washing and sequestration.
In the
current study, receptor recycling was estimated from recovery of
surface binding after agonist-induced loss. Recovery measured in this
assay reflected recycling rather than de novo receptor
synthesis since the time course was rapid and recovery was not blocked
by protein synthesis inhibitors. Rapid recycling of wild-type bombesin
and CCK receptors was observed, as was previously reported (28, 30) . In contrast, the bombesin/CCK
chimeric receptor did not recycle. Interestingly, the proportion
of internalized ligand exceeded the proportion of internalized
receptors for bombesin and CCK
receptors while these
parameters were equal in the non-recycling chimeric receptor. One
likely explanation is that receptor recycling occurred more rapidly
than the efflux of internalized ligand, such that ligand normally
accumulated relative to receptors. Thus, for the recycling-deficient
bombesin/CCK
chimeric receptor, ligand and receptor
internalization occurred to the same extent. Also, for the
internalization-deficient bombesin/m3 ACh chimeric receptor, receptor
recycling provides an explanation for the unexpectedly high extent of
ligand accumulation.
The lack of recycling of the chimeric
bombesin/CCK receptor observed in the current study was not
predicted from the behavior of the wild-type CCK
receptor,
which has been previously reported to recycle in CHO
cells(28) . Thus, the lack of recycling of the chimeric
receptor may represent a nonspecific interference with normal receptor
trafficking mechanisms. Nevertheless, this observation indicates that
receptor internalization and recycling mechanisms are separate and both
are influenced by the receptor carboxyl terminus. Recent studies on the
epidermal growth factor receptor have indicated that internalization
and recycling are separately regulated and involve separate
signals(46) . For the epidermal growth factor receptor it
appears that the default pathway is recycling and that lysosomal
targeting of receptors is due to specific and saturable endosomal
retention. Also, epidermal growth factor receptors require occupancy
for endosomal retention such that the nature of the ligand, especially
its rate of dissociation, influence recycling(47) . Whether
these characteristics are also important for G protein-linked receptors
is unknown. The current study provides the first indication of a role
for specific receptor domains in coupling to this important regulatory
phenomenon. Further experiments will be necessary to define more
precisely the structures and mechanisms involved in endosomal retention
of bombesin and other G protein-linked receptors.
Receptor internalization is cell type-specific. For example, muscarinic cholinergic (mACh) receptors are down-regulated but not sequestered in CHO cells (29) and JEG-3 cells (43) and are sequestered but not down-regulated in HEK293 cells(42) . Cell-specific differences in internalization have also been reported for luteinizing hormone/chorionic gonadotropin receptors(17) . Thus, the fact that previous studies on receptor internalization have been conducted in a variety of cell models increases the probability that different pathways of internalization may have been investigated. In the current study a comparison was made between the internalization of chimeric receptors and wild-type receptors in the same cell model, in order to avoid these complications.
Many previous studies of receptor
internalization have focused on the role of potential sites of
phosphorylation. Recently, evidence has been obtained for G
protein-coupled receptor kinase (GRK2) involvement in internalization
of mACh receptors(48) . In the current study the number of
potential carboxyl-terminal phosphorylation sites varied from 16 for Bn
(8 Ser, 7 Thr, 1 Tyr) to 10 for CCK (5 Ser, 3 Thr, 2 Tyr),
and five for m3 ACh (1 Ser, 3 Thy, 1 Tyr). While the m3 ACh carboxyl
terminus possessed the least potential phosphorylation sites and was
the least internalized, no definitive conclusions can be drawn from
these observations. Because phosphorylation of tyrosine residues is
important in the internalization of growth factor receptors, tyrosines
have been targeted as potentially important in signaling
internalization(12, 15, 43) . In the current
study all receptor carboxyl termini possessed at least 1 tyrosine and
no obvious pattern or relationship between tyrosines and
internalization or recycling could be discerned. Other investigations
have scrutinized the potential role of protein kinase C in
agonist-induced receptor internalization. Specifically, an involvement
of protein kinase C was suggested in bombesin receptor internalization
as elimination of a carboxyl-terminal consensus site reduced (11) and preincubation of cells with activators of protein
kinase C increased bombesin internalization(7) . In the current
study all receptor carboxyl termini possessed potential protein kinase
C phosphorylation sites and no obvious relationship between numbers or
location of potential sites with receptor trafficking was apparent.
In summary, we have shown that substitution of the carboxyl terminus
of the bombesin receptor with termini from either CCK or m3
ACh receptors had profound effects on receptor internalization and
recycling. In general the chimeric receptors acquired the
internalization characteristics of the carboxyl-terminal donor
receptors. The CCK
receptor carboxyl terminus allowed rapid
ligand internalization, and it may have conveyed to the bombesin
receptor chimera the ability to internalize via a non-clathrin-mediated
pathway. The bombesin/m3 ACh chimeric receptor internalized poorly.
Additionally the bombesin/CCK
receptor was deficient in its
ability to recycle, demonstrating that internalization and recycling
pathways are separable. In conclusion, these results indicate that
separate carboxyl-terminal domains determine receptor interactions with
distinct cellular trafficking mechanisms.