From the Fraser Laboratories, Departments of Medicine, Neurology, and Neurosurgery and Pharmacology and Therapeutics, McGill University, Royal Victoria Hospital and the Montreal Neurological Institute, Montreal, Quebec H3A 1A1, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have investigated the role of the
cytoplasmic tail (C-tail) of the human somatostatin receptor type 5 (hSSTR5) in regulating receptor coupling to adenylyl cyclase (AC) and
in mediating agonist-dependent desensitization and
internalization responses. Mutant receptors with progressive C-tail
truncation (347,
338,
328,
318), Cys320
Ala substitution (to block palmitoylation), or Tyr304
Ala substitution of a putative NPXXY internalization
motif were stably expressed in Chinese hamster ovary K1 cells. Except for the Tyr304
Ala mutant, which showed no binding, all
other mutant receptors exhibited binding characteristics
(Kd and Bmax) and G protein
coupling comparable with wild type (wt) hSSTR5. The C-tail truncation
mutants displayed progressive reduction in coupling to AC, with the
318 mutant showing complete loss of effector coupling. Agonist
pretreatment of wt hSSTR5 led to uncoupling of AC inhibition, whereas
the desensitization response of the C-tail deletion mutants was
variably impaired. Compared with internalization (66% at 60 min) of wt
hSSTR5, truncation of the C-tail to 318, 328, and 338 residues reduced
receptor internalization to 46, 46, and 23%, respectively, whereas
truncation to 347 residues slightly improved internalization (72%).
Mutation of Cys320
Ala induced a reduction in AC
coupling, desensitization, and internalization. These studies show that
the C-tail of hSSTR5 serves a multifunctional role in mediating
effector coupling, desensitization, and internalization. Whereas
coupling to AC is dependent on the length of the C-tail,
desensitization and internalization require specific structural
domains. Furthermore, internalization is regulated through both
positive and negative molecular signals in the C-tail and can be
dissociated from the signaling and acute desensitization responses of
the receptor.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The neurohormone somatostatin
(SST)1 is synthesized widely
in the body and acts as a potent inhibitor of hormone and exocrine secretion as well as a modulator of neurotransmission and cell proliferation (1). These actions are mediated by a family of five G
protein-coupled receptors (GPCRs) with seven helical transmembrane
segments termed SSTR1-5 (2). All five SSTRs inhibit adenylyl cyclase.
Some of the receptor isotypes also modulate other effectors such as
phosphotyrosine phosphatase, K+ and Ca2+ ion
channels, a Na +/H + exchanger, phospholipase
C, phospholipase A2, and mitogen-activated protein kinase (2). The five
SSTRs display an overlapping pattern of expression throughout the brain
and in peripheral organs (2, 3). SSTR2 is the most widely expressed
isoform (2, 3). SSTR5 is the predominant subtype in the pituitary and
hypothalamus (2-5).
An important property of many GPCRs is their ability to regulate their responsiveness in the presence of continued agonist exposure (6). Such agonist-specific regulation by GPCRs involves a series of discrete cellular steps that include loss of binding affinity and signaling capability due to receptor uncoupling from G proteins (desensitization), receptor internalization, and receptor degradation. Like other GPCRs, SSTRs also appear to be dynamically regulated at the membrane by agonist treatment (2). For instance, during pharmacotherapy with SST analogs in man, the acute effects on pituitary islet and gastric functions subside with continued exposure to the peptide due to the development of tolerance (7). Agonist-dependent internalization of SSTRs occurs in rat pituitary and islet cells and in AtT-20 cells (8-10). In GH4C1 and Rin m5f cells, however, prolonged agonist treatment up-regulates SSTRs (11, 12). These differences may be explained by differential expression of SSTR subtypes since AtT-20 cells express predominantly the SSTR5 subtype, whereas GH4C1 cells are rich in the SSTR1 isotype (13, 14). Furthermore, because pituitary and islet cells or their tumor cell derivatives express multiple SSTR subtypes, it is difficult to determine subtype-selective responses in these systems (4, 5, 19, 20). To circumvent these problems, several recent studies have characterized agonist regulation of individual SSTRs using subtype-selective SST analogs or cell lines stably transfected with SSTR cDNAs (10, 15-18). These studies have shown differential internalization of SSTR2,3,4, and 5 but not of SSTR1 (15-18).
Very little is currently known about the molecular determinants of the desensitization and internalization responses of the SSTR family. For other GPCRs, the presence in the C-tail of Ser and Thr phosphorylation sites as well as Tyr internalization signals is critically important in these processes (6, 19). In the present study we have characterized by mutagenesis the structural domains in the C-tail of hSSTR5 necessary for agonist-dependent desensitization and internalization. We show that the C-tail of hSSTR5 is critical for internalization, receptor coupling to adenylyl cyclase, and acute desensitization responses. Although internalization and signaling both require the C-tail, they are independent functions of the receptor, which appear to be residue-specific in the case of internalization, or dependent on the length of the C-tail for coupling to adenylyl cyclase.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
SST-28 and
Leu8-D-Trp22-Tyr25
SST-28 (LTT SST-28) were from Bachem (Marina Del Rey, CA). The
fluorescent SST ligand,
-fluoresceinyl-[D-Trp8] SST-14 (fluo-SST)
was from Advanced Bioconcept, Montreal, Quebec. Phenylmethylsulfonyl
fluoride, bacitracin, and GTP
S were from Sigma. Pertussis toxin was
from List Biological Laboratories. Ham's F-12 medium, fetal bovine
serum, and G418 were from Life Technologies, Inc.. Carrier-free
Na125I was obtained from Amersham Pharmacia Biotech.
Rhodamine-conjugated goat antirabbit IgG was from Jackson
Immunoresearch Labs (West Grove, PA). Cyclic AMP radioimmunoassay kits
were obtained from Diagnostic Products Corp. (Los Angeles, CA). All
other reagents were of analytical grade and purchased from various
suppliers.
Construction of Wild Type Cassette sstr5 cDNA and Mutant
sstr5 cDNAs--
A hSSTR5 cassette gene consisting of five
cDNA fragments corresponding to consecutive segments of hSSTR5 was
created as described previously by introducing silent mutations to
generate unique restriction sites to facilitate the manipulation of the
sequence as discrete restriction fragments (20). Using this construct, a series of point mutations in the C-tail of hSSTR5 were created to
investigate the role of the length and of specific residues in the
signaling, desensitization, and internalization properties of the
receptor (Fig. 1). The wt hSSTR5
cytoplasmic tail (C-tail) contains 55 amino acid residues with 7 serine
and threonine residues that could serve as putative phosphorylation
sites. Stop codons were introduced at positions 318, 328, 338, and 347 to produce truncated receptors with variable length C-tail. The 318
truncation contains only 10 amino acid residues, with one serine
residue at position 314. The
328 contains 20 amino acid residues in
the C-tail and incorporates the cAMP-dependent protein
kinase phosphorylation site at Ser325. The
338
truncation contains 30 amino acid residues in the C-tail. The
347
mutant contains 39 amino acid residues and includes the 342QQQEAT347 motif, a putative G protein
receptor kinase phosphorylation site. A palmitoylation site on a
conserved Cys residue present in the C-tail of many GPCRs has been
shown to be important for receptor sequestration. Such a site is
present on Cys320 of hSSTR5 and was mutated to Ala. The Tyr
residue in the NPXnY motif located at the junction
of the VIIth transmembrane (TM) helix and the C-tail acts as an
internalization signal for a number of G protein-coupled receptors.
This motif exists in hSSTR5 as NPVLY and was mutated at the
Tyr304 position to Ala. Desired mutations were introduced
into the MluI-EcoRI fragment of the cassette
construct (20). Point mutations were created using the PCR overlap
extension technique; for the C-tail-truncated mutants, oligonucleotide
primers were used that contain an appropriately placed stop codon (20).
Mutated DNA fragments were used to replace the corresponding wild type
restriction fragment in the cassette construct in the expression vector
pTEJ8. The structure of the cassette construct and the mutated
cDNAs was confirmed by sequence analysis (University Core DNA
Service, University of Calgary, Calgary, Alberta, Canada). CHO-K1 cells
were transfected with cDNAs for wild type or mutant receptors by
the Lipofectin method, and stable G418- resistant nonclonally selected
cells were prepared for study.
|
Binding Assays--
CHO-K1 cells expressing wild type and mutant
hSSTR5 were cultured to ~70% confluency in D-75 flasks in Ham's
F-12 medium containing 10% fetal calf serum and 700 µg/ml G418. The
cells were washed and pelleted by centrifugation, and membranes were
prepared by homogenization. Binding studies were carried out for 30 min
at 37 °C with 20-40 µg of membrane protein and
125I-LTT SST-28 radioligand as described previously (13,
20). To determine G protein coupling of the expressed receptors, the effect of treatment with 104 M GTP
S for 30 min on 125I-LTT SST-28 binding to membranes from cells
expressing wild type and mutant SSTRs was evaluated. In addition,
binding was analyzed after pretreatment of membranes with pertussis
toxin (100 ng/ml) for 2 h at 37 °C to determine pertussis toxin
sensitivity of the receptor-bound G proteins.
Receptor Coupling to Adenylyl Cyclase--
Transfected cells
were plated in Falcon 6-well dishes (2 × 105
cells/well) and used two days later at ~70% confluency. Receptor coupling to adenylyl cyclase was investigated by measuring the dose-dependent inhibitory effects of SST-28 on
forskolin-stimulated cAMP accumulation. Cells were exposed to 1 µM forskolin with or without SST-28
(106-10
10 M) for 30 min at
37 °C, scraped in 1 ml of ice-cold 0.1 N HCl, and
assayed for cAMP by radioimmunoassay. To study
agonist-dependent desensitization of adenylyl cyclase
response, cells were preincubated for 1 h at 37 °C in binding
buffer with or without 100 nM SST-28. The cells were then
washed twice with cold binding buffer to remove unbound SST-28.
Receptor-bound SST-28 was then stripped by incubation for 10 min at
37 °C in Hanks' buffered saline acidified to pH 5.0 with 20 mM sodium acetate (acid wash). The cells were washed twice
and analyzed along with control cells for receptor coupling to adenylyl
cyclase.
Internalization Experiments-- Cultured CHO-K1 cells expressing wild type and mutant SSTRs were incubated overnight at 4 °C in binding buffer with 125I-LTT SST-28 (200,000 cpm) with or without 100 nM SST-28 (15). Cells were washed three times with ice-cold HEPES binding buffer containing 5% Ficoll to remove unbound ligand and then warmed to 37 °C for different times (0, 15, 30, and 60 min) to initiate internalization. Surface-bound radioligand was removed by treatment for 10 min with acid wash solution. Internalized radioligand was measured as acid-resistant counts in 0.1 N NaOH extracts of acid-washed cells. Internalization of receptor-bound ligand was also assessed using fluo-SST. This peptide binds with high affinity (IC50 4.9 nM) to SSTRs in rat cortical homogenate and has been reported to undergo agonist-dependent internalization in COS-7 cells transfected with SSTR2A (16). CHO-K1 cells expressing wild type and mutant hSSTR5 receptors were grown to ~70% confluency. On the day of the experiment, the culture medium was removed, and the cells were washed and incubated in 1 ml of binding buffer containing 10 nM fluo-SST at 4 °C for 1-2 h. To examine internalization, sister cultures were incubated with fluo-SST under identical conditions for 45 min at 37 °C. At the end of each incubation, media were removed, and the cells were washed, mounted in immunofluor, and viewed under a Zeiss LSM 410 inverted confocal microscope (5, 20). All images were archived on a Bernoulli multidisc and printed on Kodak XLS8300 high resolution printer.
Immunocytochemistry--
Confocal immunofluorescence studies
were performed to confirm cell surface expression of the
Tyr304 Ala mutant in live unfixed transfected cells
using a rabbit polyclonal antibody directed against the amino-terminal
4-11 peptide sequence of hSSTR5 as described previously (5, 20).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Binding Affinity--
Table I shows
the results of membrane binding analyses of CHO-K1 cells transfected
with wt hSSTR5 cassette cDNA and the hSSTR5 C-tail mutant
cDNAs. By saturation analysis, wt hSSTR5 displayed high affinity
binding with a Kd of 0.31 nM and a
Bmax of 162 ± 42 fmol/mg of protein. The
328,
338, and the
347 C-tail truncation mutants as well as the
Cys320
Ala mutants displayed binding affinities of
0.21-0.47 nM, comparable with that of wt hSSTR5. The
318 truncation mutant also displayed high affinity ligand binding
(Kd 0.89 nM), which, however, was 3-fold
lower than that of the wild type receptor. Bmax
of the four C-tail deletion mutants ranged between 247 and 352 fmol/mg of protein, 1.5-2-fold higher than that of wt hSSTR5; the
Bmax of the Cys320
Ala mutant
(179 ± 51 fmol/mg of protein) was comparable with that of the
wild type receptor. No specific binding was observed in membranes
prepared from cells transfected with the Tyr304
Ala
mutant. The mutant protein, however, was expressed on the cell membrane
as determined by immunocytochemistry using live unfixed cells. Using
the hSSTR5 primary antibody, nonpermeabilized CHO-K1 cells expressing
wt hSSTR5 showed rhodamine immunofluorescence localized to the cell
surface (not shown). Cells transfected with the Tyr304
Ala mutant also exhibited surface immunofluorescence with this antibody, indicating that the mutant receptor was properly targeted to
the plasma membrane. No specific immunofluorescence was detected in
nontransfected CHO-K1 cells or in transfected cells probed with
preimmune serum or antigen-absorbed primary antibody. To exclude any
breach of the plasma membrane and labeling of cytosolic structures
beneath the plasmalemna during incubation with primary antibody,
parallel immunocytochemistry was performed with antibody to vimentin,
an intracellular protein. Under these conditions, vimentin
immunoreactivity was detected only in cells permeabilized with 0.2%
Triton X-100 but not in intact CHO-K1 cells. These findings suggest
that the loss of binding of the Tyr304
Ala mutant is
not due to a failure of the mutant receptor to be localized to the
plasma membrane but rather reflects an important structural requirement
of the Tyr residue in maintaining a high affinity ligand binding
conformation. Loss of agonist binding by this mutant precluded further
analysis of the role of the Tyr304 residue as an
internalization signal.
|
G Protein Coupling--
To determine whether the hSSTR5 C-tail
mutants were coupled to G proteins, the effect of 104
M GTP
S on membrane binding was assessed in cells
expressing wild type and mutant hSSTR5 receptors (Fig.
2). Pretreatment with GTP
S reduced
125I-LTT SST-28 binding of wt hSSTR5 to 67 ± 2% that
of control. The four C-tail truncation mutants as well as the
Cys320
Ala mutant also displayed significant loss of
radioligand binding of 50-70% that of control, comparable with that
of the wild type receptor. This suggests that the mutant receptors are
capable of associating with G proteins. Pretreatment of membranes with pertussis toxin also led to a significant 40-50% reduction of 125I-LTT SST-28 binding to the wild type and the C-tail
mutant SSTRs, suggesting that the mutant receptors associate with
pertussis toxin-sensitive G proteins.
|
Coupling to Adenylyl Cyclase and Desensitization
Responses--
Fig. 3 depicts the
results of coupling of the C-tail mutants to adenylyl cyclase. Basal
cAMP level in cells expressing the mutant receptors was comparable with
that in cells transfected with wt hSSTR5. Compared with the wild type
receptor, which showed a maximum of 70 ± 6% inhibition by SST-28
of forskolin-stimulated cAMP accumulation, the C-tail deletion mutants
displayed a progressive loss of the ability to inhibit
forskolin-stimulated cAMP from 69.8 ± 2% for the 347 mutant
to 63 ± 3.8% for the
338 mutant to 60 ± 3.1% for the
328 mutant. The Cys320
Ala mutant showed only
57 ± 3.4% maximum inhibition of forskolin-stimulated cAMP; the
318 showed complete loss of coupling to adenylyl cyclase.
|
|
Internalization of Receptor Bound 125I-LTT
SST-28--
Internalization of 125I-LTT SST-28 ligand was
studied in stably transfected CHO-K1 cells initially treated with
ligand for 12 h at 4 °C to allow for equilibrium binding but to
limit internalization (Fig. 5). Switching
from 4 to 37 °C led to a rapid time-dependent internalization of radioligand that, in the case of wt hSSTR5, reached
a maximum of 66 ± 2% at 60 min (Fig. 5). Truncation of the
C-tail to 318 and 328 residues produced moderate decreases in receptor
internalization to 46% at 60 min. Truncation to 338 residues led to a
dramatic loss of radioligand internalization of only 23 ± 3% at
60 min. In contrast, truncation to 347 residues improved the efficiency
of internalization even more than that of the wild type receptor
(72 ± 3% compared with 66%). This suggests the presence of a
positive internalization signal between residues 338 and 347 and a
negative signal between 347 and 364 residues and 328 and 338 residues.
Mutation of the Cys320 palmitoylation site reduced ligand
internalization to 42 ± 3% at 1 h. The extent of the loss
of internalization of this mutant was comparable with that of the
318 mutant, which also lacked the Cys320 residue and
suggests an important function of the palmitoylation anchor in
producing the impaired internalization of both these mutant
receptors.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Role of C-Tail in Ligand Binding and in Coupling to G Protein and
Adenylyl Cyclase--
Progressive deletion of the C-tail of hSSTR5 had
no effect on high affinity ligand binding, indicating that the C-tail,
like that of other GPCRs, does not influence receptor targeting or binding conformation (21). Mutation of the Tyr304 residue,
however, produced a receptor protein that was correctly targeted to the
plasma membrane but which showed complete loss of binding, suggesting a
critical role of Tyr304 in ligand binding through either
direct hydrophobic interaction with SST ligand or through an allosteric
change in the receptor binding conformation. The C-tail as well as the
second and third intracellular loops of several GPCRs have been
implicated in G protein interaction (6, 22). Radioligand binding by all
of the C-tail mutants of hSSTR5 was inhibited to the same degree as the
wild type receptor by GTPS and pertussis toxin, indicating that the
mutant receptors are capable of associating with pertussis toxin-sensitive G proteins and that the C-tail of hSSTR5 is not required for this interaction. Interestingly, despite the ability to
associate with G proteins, the four C-tail deletion mutants as well as
the Cys320
Ala mutant displayed reduced efficiency for
adenylyl cyclase coupling. This was most pronounced in the case of the
318 mutant, which showed a complete loss of the ability to inhibit
adenylyl cyclase. Whether this mutant can signal through other effector pathways remains to be seen. There are two other examples of
dissociated G protein and effector coupling by C-tail mutants of GPCRs.
The first is the C-tail-truncated postaglandin EP3 receptor, which retains the ability to associate with Gi2 but which shows
no forskolin-induced inhibition of cAMP acccumulation, identical to the
318 hSSTR5 mutant (23). The second is an Ala
Glu substitution in
the distal third intracellular loop of the gastrin-releasing peptide receptor, which abrogates phospholipase C coupling while retaining full
efficacy for G protein interaction (24). In contrast to the C-tail of
hSSTR5, which is required for inhibitory regulation of adenylyl
cyclase, the naturally occurring SSTR2B splice variant with a shorter
C-tail length than SSTR2A is more efficiently coupled to adenylyl
cyclase (25).
Role of C-Tail in Mediating Acute Desensitization--
We found
that hSSTR5 stably expressed in CHO-K1 cells was desensitized by
agonist pretreatment. Phosphorylation of the rat SSTR2A receptor
primarily on serine residues and of the rat SSTR3 receptor on both
serine and threonine residues in the C-tail has been reported to be
crucial for desensitization and internalization of these two subtypes
(17, 18). hSSTR5 features three serine (Ser314,
Ser325, Ser361) and four threonine
(Thr333, Thr347, Thr351,
Thr360) residues in the C-tail (Fig. 1). The
Ser325 and Thr360 sites fit the consensus
sequence for phosphorylation by protein kinase A and protein kinase C,
respectively, and the Thr347 position qualifies as a
putative G protein-coupled receptor kinase phosphorylation site (26).
The third intracellular loop of this receptor displays three additional
sites for phosphorylation by second messenger-activated kinases. The
ability of the 347 mutant to be desensitized by agonist to the same
degree as the wild type receptor suggests that the Thr351,
Thr360, and Ser361 sites in the distal C-tail
play a minimal role in the desensitization response. In contrast, the
resistance of the
338 mutant to desensitization suggests an
important role of Thr 347 in the putative G protein
receptor kinase phosphorylation site. This role, however, cannot be
absolute since the
328 mutant, which also lacks the
Thr347 residue, underwent significant desensitization. A
conserved cysteine residue 11-12 amino acids downstream from the 7th
TM is found in the C-tail of most GPCRs and serves as a palmitoylation
membrane anchor for a fourth intracellular loop. Palmitoylation induces differential changes in G protein coupling, desensitization,
intracellular trafficking, and internalization of different GPCRs (27,
28). In the case of hSSTR5, the Cys320
Ala mutant
displayed poor ability to uncouple from adenylyl cyclase, indicating an
important role of C-tail palmitoylation in the desensitization response
of this receptor. Overall, these studies suggest that the C-tail plays
a prominent role in agonist-induced desensitization of hSSTR5 through
both specific motifs, which may serve as sites for phosphorylation, as
well as through conformational changes in the C-tail of the
agonist-occupied receptor, which may determine its substrate
specificity for phosphorylation.
Role of C-Tail in Mediating Receptor Internalization--
The
C-tail segment of hSSTR5 is not only critical for receptor coupling to
adenylyl cyclase and in mediating acute desensitization responses but
also plays an important role in regulating agonist-induced receptor
internalization. This is in agreement with previous studies that have
shown that the C-tail of many other GPCRs, e.g. receptors for angiotensin IIIA (29), 2 adrenergic (30),
m2 muscarinic (31), luteinizing hormone/human chorionic
gonadotrophin (32), parathyroid hormone (33), thyrotrophin releasing
hormone (34), neurotensin (35), and cholecystokinin (36), is also
involved in internalization. Our results indicate that mutant receptors with variable length C-tails are differentially internalized. Truncation of the C-tail at positions 318 and 328 attenuated receptor internalization only partially from 66 to 46% at 60 min. This suggests
that the C-tail distal to position 318, which contains multiple
phosphorylation sites including the putative G protein receptor kinase
site on Thr347, although important, is not a critical
determinant of endocytosis. Furthermore, the comparable rates of
internalization of the
328 mutant, which contains the putative
protein kinase A site at Ser325, and the
318 mutant,
which does not, excludes a role of the protein kinase A site in
agonist-induced hSSTR5 internalization. Truncation of the C-tail to 338 residues led to a dramatic loss of internalization. This mutant has 10 more residues than
328 that appear to contain potent negative
endocytic signals. The
347 deletion mutant internalized slightly
more than the wild type receptor, suggesting that the nine-amino acid
residue stretch between positions 338 and 347 harbors a positive
internalization signal, likely on Thr347 in the putative G
protein receptor kinase phosphorylation site. Furthermore, the ability
of the
347 mutant to internalize more than the wild type receptor
argues for a second negative endocytic signal in the extreme C-tail
segment distal to residue 347. Negative endocytic signals have been
postulated in the case of the luteinizing hormone/human chorionic
gonadotrophin and parathyroid hormone/parathyroid hormone-related
protein receptors (32, 33). The EVQ sequence in the membrane-proximal
C-tail, which is highly conserved across members of the parathyroid
hormone/secretin receptor family has been identified as a negative
endocytic signal for this receptor subclass. Point mutations in the
328-338 and 347-363 segment of hSSTR5 C-tail will help to determine
whether there are similar structural motifs in this receptor capable of
acting as negative endocytic regulators. The palmitoylation-defective
hSSTR5 mutant showed reduced internalization comparable with that
reported for the thyrotrophin-releasing hormone (34) and vasopressin V2
(37) receptors but different from the palmitoylation-deficient
luteinizing hormone/human chorionic gonadotrophin receptor, which
displays enhanced internalization (38). Tyrosine-based internalization signals on NPXnY-type motifs are common to many
classes of membrane receptors (39). In the case of GPCRs, a conserved NPXXY sequence at the interface between the VIIth TM and the
C-tail serves as an endocytic signal for some receptors (19). In other GPCRs such as the gastrin-releasing peptide and the angiotensin II
receptors, however, an identical NPXXY motif has no effect on receptor sequestration, arguing against a general role for this
sequence (40, 41). This motif is found in all members of the SSTR
family, but since its mutation in the case of hSSTR5 abolished high
affinity ligand binding, it will be difficult to characterize its
function as an endocytic signal.
Relationship Between Receptor Signaling and
Internalization--
An important question concerning GPCRs is the
relationship between receptor signaling and internalization. Although
there has been some controversy in the past, many recent studies have suggested that the two events can be readily dissociated (24, 29, 36,
42, 43). For instance, receptor mutations that inhibit G protein
coupling or signaling do not prevent endocytosis (24, 29, 36, 42, 43).
The addition of second messengers such as phorbol 12-myristate
13-acetate, Ca2+, and cAMP fails to stimulate
internalization of mutant thyrotrophin-releasing hormone receptors or
antagonist-blocked luteinizing hormone/human chorionic gonadotrophin
receptors that cannot activate phospholipase C or adenylyl cyclase (34,
36). A cholecystokinin antagonist has been reported to induce receptor
internalization independent of G protein coupling, signaling events,
and receptor phosphorylation (36). The human muscarinic receptor
subtype 1 has been shown to undergo internalization by interaction with
antibody against an epitope tagged to the amino terminus, independent
of exogenous ligand or second messenger activation (44). The
dissociated effects of the hSSTR5 C-tail mutants on adenylyl cyclase
coupling and internalization lend further support to these arguments.
For instance, there was no correlation between the progressive loss of
the ability of the C-tail deletion mutants of hSSTR5 to inhibit adenylyl cyclase and receptor internalization, which was both inhibited
or accelerated. In particular, the 318 mutant, which was rendered
inert with respect to its ability to inhibit adenylyl cyclase,
nonetheless exhibited reduced internalization. Although activation of
second messenger systems may exert a secondary influence on the
internalization process, the collective findings from all of these
studies suggest that internalization is an intrinsic property of most
receptors, dependent on specific conformational changes rather than
receptor signaling capability.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. D. Laird for the vimentin antibody and M. Correia for secretarial help.
![]() |
FOOTNOTES |
---|
* This work was supported by Medical Research Council of Canada Grant MT-10411 and grants from the National Institutes of Health, the U. S. Department of Defense, and the National Cancer Institute of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to this work.
§ Supported by a fellowship from the Fonds De La Recherche En Sante Du Quebec.
¶ Recipient of studentship support from the Royal Victoria Hospital Research Institute.
A Distinguished Scientist of the Canadian Medical Research
Council. To whom correspondence should be addressed: Royal Victoria Hospital, Room M3-15, 687 Pine Avenue West, Montreal, Quebec H3A 1A1,
Canada. Tel: 514-842-1231 (ext. 5042); Fax: 514-849-3681; E-mail:
patel{at}rvhmed.lan.mcgill.ca.
The abbreviations used are:
SST, somatostatin; LTT SST-28, Leu8-D-Trp22-Tyr25
SST-28fluo-SST, -fluoresceinyl-[D-Trp8]
SST-14SSTR, somatostatin receptorwt hSSTR5, wild type human
somatostatin receptor type 5GPCR, G protein-coupled receptorTM, transmembrane domainC-tail, cytoplasmic carboxyl-terminal segmentPCR, polymerase chain reactionCHO, Chinese hamster ovaryGTP
S, guanosine 5'-O-(thiotriphosphate).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|