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Montreal Neurological Institute, McGill University (D.N., M.H., A.B.), Montreal, Quebec, Canada H3A 2B4; Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice-Sophia Antipolis (G.G, J.-P.V., J.M.), Valbonne, France; and the Department of Pharmacology, University of Pennsylvania School of Medicine (T.R.), Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Dr. Alain Beaudet, Department of Neurobiology, McGill University, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. E-mail: MCIN{at}MUSICA.MCGILL.CA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells
transfected with complementary DNA encoding either sst1 or
sst2A receptors both displayed specific, high affinity
binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined
to the cell surface in both cell types, as indicated by the fact that
it was entirely removable by a hypertonic acid wash and assumed a
pericellular distribution in the confocal microscope. At 37 C, the fate
of specifically bound ligand varied markedly according to the type of
receptor transfected. In cells encoding the sst1 receptor,
approximately 20% of specifically bound ligand was recovered in the
acid-resistant (i.e. intracellular) fraction. This
fraction remained clustered at the periphery of the cell, suggesting
that it was being sequestered either within or immediately beneath the
plasma membrane. By contrast, in cells transfected with
sst2A receptors, up to 75% of specifically bound ligand
was recovered inside the cells, where it clustered into small
endosome-like particles. These particles increased in size and moved
toward the nucleus with time, suggestive of receptor-ligand complexes
proceeding down the endocytic pathway. These results demonstrate that
neuropeptides may be processed differently depending on the subtype of
receptor expressed in their target cells and suggest that these
different processing patterns may reflect different modes of
sensitization/desensitization and recycling of the receptors, and
thereby of transmembrane signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent molecular biological studies have demonstrated the existence of five different SRIF receptor subtypes with distinct central and peripheral distributions. These cloned receptors have been termed sst1 (4), sst2 (4), sst3 (5, 6), sst4 (7), and sst5 (8, 9) according to the order in which they were isolated. The sst2 receptor comprises two isoforms generated from the same gene; sst2A, the unspliced form, and sst2B, a 23-amino acid shorter splice variant (10, 11). Sequence and hydropathy analyses have confirmed that all of these SRIF receptor subtypes contain the seven putative transmembrane domains characteristic of other G protein-coupled receptors. The cloned SRIF receptors have a high degree of amino acid and structural homology among themselves; however, it is believed that they may represent a unique neurosecretagogue receptor subfamily, as their sequences differ from those of any other known receptors (12, 13). Pharmacological studies in cells transfected with complementary DNA (cDNA) encoding the various SRIF receptor subtypes have demonstrated that the sst2A/B, sst3, and sst5 receptors correspond to the so-called type 1 subclass previously defined as displaying a relatively high affinity for the biologically stable synthetic SRIF agonists MK678 and octreotide (14, 15). These receptors are desensitizable and are sensitive to GTP and divalent cations (14, 5). The sst1 and sst4 receptors comprise the type 2 subclass, which displays relatively lower affinity for MK678 and octreotide and is not desensitizable (14, 15).
Data from studies in cell culture systems and in tissue preparations have demonstrated, both biochemically and morphologically, that various ligands interacting with G protein-coupled receptors rapidly enter their target cells after binding to cell surface receptors. This process, referred to as receptor-mediated internalization, is believed to involve local clustering of receptors followed by their endocytosis through clathrin-coated pits (for review, see Ref.16). Ligand and/or receptor internalization has been described for a variety of neuropeptides, including gonadotropin- (17), corticotropin- (18), and thyrotropin-releasing hormones (19), as well as substance P (20, 21, 22), cholecystokinin (23, 24), vasopressin (25), angiotensin II (26), vasoactive intestinal peptide (27) glucagon-like peptide-2 (28), gastrin-releasing peptide (29), and neurotensin (30, 31, 32, 33) in cell cultures in vitro and in whole organs in vivo.
Whether SRIF is internalized in a comparable manner after ligand-receptor interaction has been a matter of debate. Several reports indicate that SRIF is internalized in pancreatic (34, 35), hypophyseal (36, 37, 38), and human carcinoid tumor (39) cells after application of exogenous SRIF ligands in vitro. In vivo peptide scintigraphic data also argue in favor of SRIF internalization (40). Other reports, to the contrary, indicate that SRIF is not internalized to any appreciable extent in either GH4C1 pituitary cells (41) or RINm5F insulinoma cells (42). A possible interpretation for these discrepancies is that there exists a differential capacity of the various SRIF receptor subtypes to internalize SRIF and that the observed tissue variations merely reflect differences in the expression of these subtypes. To test this hypothesis, we compared the binding and internalization of iodinated and fluorescent analogs of the metabolically stable SRIF agonist [D-Trp8]SRIF-14 in COS-7 cells transfected with sst1 and sst2A receptor subtypes, taken as representatives of SRIF-2 and SRIF-1 classes of receptors, respectively.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A second series of fluorescent conjugates was prepared as described above, except that NHS-fluorescein was replaced by the NHS ester of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-3-propionic acid (Bodipy FL C3-SE, Molecular Probes, Eugene, OR). Here again, labeled derivatives were purified by HPLC, and elution peaks were subjected to Edman degradation. The amino acid composition was further verified by amino acid analysis, and the mol wt was measured by laser desorption mass spectrometry. A red fluorescent analog of SRIF was also prepared and purified as described above by reacting [D-Trp8]SRIF with the NHS ester of Bodipy 576/589 C3. The binding properties of this analog were undistinguishable from those of the green fluorescent Bodipy FC C3 (results not shown).
Cell culture
COS-7 cells were grown in DMEM containing glutamine and
supplemented with 44 mM NaHCO3, 10% FCS, and
50 mg/liter gentamicin. Twenty-four hours before transfection, the
cells were plated in 100-mm diameter petri dishes at a density of
106 cells/dish. When semiconfluent, cells were transfected
with cDNA encoding either the sst1 or the sst2A
receptor subtype. For this purpose, the PstI-XmnI
cDNA fragment (1.5 kilobases) bearing the coding region for the human
SSTR1 was inserted into the BamHI site of the pCMV-6b
expression vector after addition of BglII linkers. The
XbaI cDNA fragment encoding the mouse SSTR2a was subcloned
into the corresponding site of the pCMV-6c expression vector.
Transfections were carried out by incubating the cells for 30 min at
room temperature with 1 ml Tris-buffered saline containing 1 µg
recombinant plasmid and 0.5 mg/ml diethylaminoethyl-dextran. At the end
of the incubation, the buffer was replaced by culture medium containing
100 µM chloroquine, and the cells were further incubated
at 37 C for at least 3 h. They were then rinsed in TBS, covered
with culture medium, and grown at 37 C for approximately 60 h.
Binding properties of fluo-SRIF derivatives
The capacity of the different HPLC eluates of fluorescent SRIF
to compete with iodinated SRIF binding was tested on membranes prepared
from adult rat cerebral cortex. For membrane preparation, rats were
killed by decapitation, and the brains were rapidly removed. Cerebral
cortex was dissected on ice and placed in a 10-fold volume of 5
mM Tris-HCl supplemented with 2 mM EDTA,
homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) for
20 sec, and centrifuged at 15,000 x g for 15 min at 4
C. The pellet was then resuspended in the same volume of buffer,
recentrifuged for 15 min at 15,000 x g, and frozen
until use.
Binding experiments were carried out by incubating 50 µg
membranes with 0.3 nM 125I-labeled
Tyr0-[D-Trp8]SRIF (2000 Ci/mmol)
in the presence of increasing concentrations of fluorescent peptide
(10-12–10-6 M).
Tyr0-[D-Trp8]SRIF was iodinated
by the chloramine-T method as previously described (43).
Monoiodo-[125I]Tyr0-[D-Trp8]SRIF
([125I]SRIF) was purified by HPLC on a C18
column (Lichrosorb, Merck, Clevenot, France) to exclude any possible
contamination resulting from chloramine-T-induced destruction of the
tryptophan side-chain. The iodinated peptide was eluted in 0.05%
triethylamine-0.1% trifluoroacetic acid with a linear gradient of
acetonitrile from 10–50% for 56 min. (elution time, 37 min). Binding
was carried out for 45 min at room temperature in 250 µl 50
mM Tris-HCl (pH 7.5) containing 2 mM
MgCl2, 0.1% BSA, and 1 mM 1,10-phenanthroline
to prevent peptide degradation. Experiments were terminated by the
addition of 2 ml ice-cold buffer followed by filtration through glass
microfiber filters preincubated in 0.5% polyethylenimine (GF/C,
Whatman, Clifton, NJ). Filters were then washed twice with ice-cold
buffer, and the radioactivity retained in them was counted with a
-counter. Non specific binding was measured in the presence of 1
µM nonradioactive
[D-Trp8]SRIF.
Binding of [125I]SRIF to transfected
COS-7 cells
To compare the affinity and efficiency of
[125I]SRIF binding to COS-7 cells transfected with
the sst1 and sst2A subtypes, the first set of
binding assays was carried out on membranes freshly prepared from
transfected COS-7 cells. For this purpose, the cells were scraped off
the culture dishes with PBS and centrifuged at 5000 x
g for 5 min, and the pellets were homogenized by incubation
in a hypotonic 5 mM Tris-HCl buffer (pH 7.5) containing 5
µg/ml deoxyribonuclease I for 30 min at 0 C. Membrane homogenates
were then recovered by centrifugation at 15,000 x g
for 30 min at 4 C. Binding assays were carried out as described above
by incubating 10 µg membranes with concentrations of
[125I]SRIF ranging between 20 pM and 1
nM for 30 min at room temperature.
To demonstrate [125I]SRIF internalization, a second set
of binding experiments was carried out on whole cells at 37 C. The
culture medium was discarded from 12-mm diameter dishes containing
2 x 105 transfected COS-7 cells, and the cells were
equilibrated for 10 min at 37 C in Earle’s buffer (140 mM
NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.9
mM MgCl2, and 25 mM HEPES, pH 7.4)
supplemented with 0.1% glucose and 1% BSA in the presence or absence
of 10 µM of the endocytosis inhibitor, phenylarsine oxide
(PAO). Equilibration buffer was then replaced by 250 µl binding
buffer containing 0.3 nM [125I]SRIF in the
presence of 1 mM 1,10-phenanthroline for intervals ranging
between 3–60 min. At the end of the incubation, the cells were washed
twice for 2 min each time with 1 ml pure equilibration buffer with a
hypertonic acid (pH 4) buffer consisting of 0.2 M acetic
acid and 0.5 M NaCl in Earle-Tris-HEPES buffer to strip off
surface-bound radioactivity (30). Cells were harvested with 1 ml 0.1
M NaOH, and associated radioactivity was counted in a
-counter. Nonspecific binding was measured in the presence of 1
µM unlabeled [D-Trp8]SRIF.
Binding of fluo-SRIF to transfected COS-7 cells
Dissociated COS-7 cells transfected with cDNA encoding
sst1 or sst2A receptors, nontransfected cells,
and cells transfected with neurotensin in lieu of sst receptor cDNA,
were preincubated for 10 min at 37 C in Earle’s buffer containing
0.2% BSA and 0.1% glucose in the presence or absence of 10
µM PAO. They were then incubated for 45 min at 37 C with
5 nM of either
-fluoresceinyl-[D-Trp8]SRIF-14 (FTC-SRIF)
or -Bodipy-[D-Trp8]SRIF-14 (Bodipy-SRIF),
both generically referred to hereafter as fluo-SRIF, in the same
buffer, again supplemented or not with PAO. For determination of
nonspecific labeling, 1 µM
[D-Trp8]SRIF was added to the incubation
medium. To examine temperature dependence, additional cells were
incubated in parallel at 4 C. Incubation was terminated by
centrifugation at 500 x g for 10 min, followed by a
2-min rinse in Earle’s buffer at 4 C. Cells were then recentrifuged
for 5 min at 500 x g, and the pellets were suspended
in 20 µl fresh buffer, deposited on glass microscope slides,
air-dried, and examined under the confocal microscope.
To selectively visualize internalized fluorescent molecules, transfected COS-7 cells were grown as a monolayer on 12-mm polylysine-treated glass coverslips to 80% confluence in 18-mm petri dishes, incubated with fluo-SRIF as described above, but washed for 2 min with hypertonic acid buffer, before air drying and confocal microscopic examination.
Confocal microscopy
Labeled COS-7 were examined under a Leica confocal laser
scanning microscope (CLSM) configured with a Leica Diaplan inverted
microscope equipped with an argon/krypton laser with an output power of
2–50 mV and a VME bus MC 68020/68881 computer system coupled to an
optical disc for image storage (Leica, St. Laurent, Canada). All
image-generating and processing operations were performed with the
Leica CLSM software package. Micrographs were taken from the image
monitor using a Focus Imagecorder (Foster City, CA). Images were
acquired as single optical sections taken through the middle of the
cells and averaged over 32 scans/frame. For all types of acquisitions,
the gain and black levels were set manually to optimize the dynamic
range of the image while ensuring that no region was completely
suppressed (intensity = 0) or completely saturated (intensity
= 255).
Quantitative data on the number of intracellular fluorescent particles, the area of particles, and the mean distance of each particle from the center of the cell were obtained using the Leica CLSM software. Results were expressed as the mean ± SEM of measurements in 5–11 labeled cells from 3 different experiments. Statistical analyses were performed using a one-way ANOVA, followed by a regression curve analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The first of these
peaks (peak N; elution time, 35 min) was found by Edman degradation to
correspond to native [D-Trp8]SRIF. The second
fraction (peak 1; elution time, 45 min) was resistant to
phenylisothiocyanate and was thereby ascribed to
-fluoresceinyl-[D-Trp8]SRIF. The third
fraction (peak 2, elution time, 47 min) had the expected sequence of
[D-Trp8]SRIF, except for the fourth
phenylthiohydanthoin amino acid, which corresponded to none of
the 20 natural amino acids. This derivative was, therefore, taken
to correspond to
4-fluoresceinyl-[D-Trp8]SRIF. Similarly,
the fourth fraction (peak 3; elution time, 50 min) was identified as
9-fluoresceinyl-[D-Trp8]SRIF. The three
fluoreceinyl peptides were further characterized by amino acid
analysis and mass spectroscopy, which confirmed that the -,
4-, 9-fluoresceinyl derivatives were
isomers (same mass and same amino acid content) and that each
fluorescent peptide contained a single fluoresceinyl group per
mol peptide.
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-, 4- and 9-Bodipy derivatives of
[D-Trp8]SRIF were purified by HPLC and
chemically characterized as described above for the corresponding
fluorescein-labeled analogs. Complete purification of the three
Bodipy-labeled peptides was easily achieved as the observed elution
times of each HPLC fractions were clearly different (35, 44, 58,
and 63 min for native SRIF and its -, 4-, and
9-Bodipy analogs, respectively).
Pharmacological characterization of
fluorescent [D-Trp8]SRIF
derivatives
When tested for its ability to compete with specific binding of
[125I]SRIF to membranes of rat cerebral cortex,
-fluoresceinyl-[D-Trp8]SRIF was 16 times
less potent than unlabeled [D-Trp8]SRIF (Fig. 2A and Table 1).
4-Fluoresceinyl-[D-Trp8]SRIF
also competed with specific [125I]SRIF binding, but with
an apparent affinity 2–3 times lower than that of the
-fluoresceinyl peptide (Fig. 2A and Table 1). By contrast, the
9-analog competed extremely poorly with
[125I]SRIF binding (Fig. 2A and Table 1).
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-Bodipy-[D-Trp8]SRIF competed for
[125I]SRIF binding with the highest affinity of all of
the fluorescent ligands tested (Fig. 2B
and Table 1). This affinity,
however, was still 4 times lower than that of the unlabeled peptide.
The 4-Bodipy derivative competed for [125I]SRIF
binding with an affinity comparable to that of the -fluoresceinyl
compound. Its solubility, however, was considerably less than that of
the -fluoresceinated derivative, which precluded its use in
biological assays. Finally, as with fluorescein, Bodipy labeling of
[D-Trp8]SRIF in 9 completely abolished the
capacity of the peptide to compete with [125I]SRIF
binding (Fig. 2B and Table 1).
-Fluoresceinyl-[D-Trp8]SRIF and
-Bodipy-[D-Trp8]SRIF were used
interchangeably in the imaging studies described below and are referred
to in the text under the generic term fluo-SRIF.
Binding of [125I]SRIF to COS-7 cells
transfected with cDNA encoding sst1 or
sst2A receptor subtypes
[125I]SRIF bound specifically to membrane
homogenates from COS-7 cells transfected with cDNA encoding either
sst1 or sst2A receptor subtypes, with
Kd values of 0.68 and 0.28 nM, respectively. In
both types of cells, the overall maximal binding capacity was on the
order of 1 pmol/mg whole cell protein, which, based on an average
protein content of 50 µg for 2 x 105 cells,
corresponds to 1.5 x 105 sites/cell.
[125I]SRIF also bound specifically to whole
sst1-expressing cells incubated at 37 C. As shown in Fig. 3A, this specific binding reached a plateau between
40–60 min. Hypertonic acid washes of the bound radioactivity at
various time points indicated that the bulk of specifically bound
radioactivity remained extracellular (i.e. acid washable) at
all times (Fig. 3A). Experiments in which the cells were pretreated
with the endocytosis inhibitor PAO before the incubation with the
radioactive ligand yielded a saturation curve comparable to that of the
acid-washable fraction, confirming that the greatest proportion of the
ligand had remained surface bound (Fig. 3A). Accordingly, the amount of
the intracellular (i.e. acid-resistant) fraction remained
low throughout, accounting for less than 20% of the specifically bound
radioactivity after 45 min of incubation (Fig. 4). The
radioactivity associated with cells preincubated with PAO was entirely
acid washable, confirming the efficacy of PAO in preventing
internalization of [125I]SRIF into the cells (Fig. 3A).
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). However, in contrast to what was observed in
sst1-expressing cells, an important fraction of the
specifically bound radioactivity remained acid resistant throughout.
This fraction of internalized radioactivity increased rapidly with time
(t1/2 = 8–10 min) to reach a plateau at 40 min, at which
time it accounted for almost 75% of the total amount of radioactivity
specifically associated with the cells (Fig. 4). Consequently, the
acid-washable fraction of the bound radioactivity followed a reverse
trend, as did the saturation profile observed in the presence of PAO
(Fig. 3B). Finally, virtually all the radioactivity specifically bound
in the presence of PAO was acid wash-sensitive, confirming the efficacy
of PAO in blocking the internalization of [125I]SRIF
(Fig. 3B).
Confocal microscopic visualization of fluo-SRIF binding to and
internalization in transfected COS-7 cells
After incubation of COS-7 cells transfected with cDNA encoding
either sst1 or sst2A receptor subtype in the
presence of 5 nM of fluo-SRIF for 45 min at 4 or 37 C,
25–30% of the cells exhibited intense fluorescent labeling
(Fig. 5). This labeling was specific, in that it was no
longer apparent when the incubation was carried out in the presence of
1 µM unlabeled [D-Trp8]SRIF
(Fig. 6, C and D) or in cells either nontransfected or
transfected with an irrelevant receptor (not shown). After incubation
at 4 C, the labeling was confined to the cell surface, as demonstrated
by confocal imaging of midcellular planes (Fig. 6, A and B) and
confirmed by its total abolition after hypertonic acid wash (Fig. 6, E
and F).
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). This pericellular ring was no longer apparent in
cells incubated in the presence of an excess of nonfluorescent ligand
or in cells transfected with an irrelevant receptor (not shown). At
short time intervals (1–5 min), the labeling was almost completely
abolished by a hypertonic acid wash, indicating that the bulk of the
labeling was extracellular (Fig. 7B). The remaining bound fluorescent
molecules were typically clustered at the periphery of the cells (Fig. 7B). At longer time intervals (10–60 min), a greater proportion of the
original labeling was still detected after acid wash (Fig. 7C).
However, it remained confined to the periphery of the cells, in a
pattern undistinguishable from that observed before acid wash in cells
incubated at 4 C (Fig. 6A) or in which receptor internalization was
prevented by treatment with PAO (Fig. 7D).
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This labeling was not observed in cells incubated in the presence of an
excess of nonfluorescent [D-Trp8]SRIF (Fig. 8B) or in COS-7 cells either nontransfected or transfected with a
plasmid encoding the neurotensin receptor in lieu of the somatostatin
receptor (not shown). Stripping of cell surface labeling with a
hypertonic acid wash variably reduced (depending on the duration of
incubation), but never abolished, the fluorescent labeling, indicating
that part of the labeling was intracellular (Fig. 8C). Accordingly, in
cells incubated in the presence of PAO, bound fluo-SRIF molecules
remained clustered on the surface of the cells (Fig. 8D) in a pattern
comparable to that seen after incubation at 4 C or in cells transfected
with cDNA encoding the sst1 receptor.
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). The size and
intracytoplasmic distribution of these fluorescent particles varied
significantly with the duration of the incubation (Fig. 9 and Table 2). At short incubation times (1–5 min), the particles
were small (1.16 ± 0.24 µm2 in area) and sparsely
distributed at the periphery of the cells (Fig. 9A). Their mean surface
progressively increased with time [F(1, 31) = 15.8; t =
2.88; P < 0.01] to reach 2.74 ± 0.44
µm2 at 45 min (Table 2). Conversely, their mean distance
significantly decreased with time [F(1, 31) = 15.1; t =
4.28; P < 0.001] as they invaded the entire cytoplasm
and converged toward the nucleus (Table 2 and Fig. 9, B and C). There
also was a trend toward a time-dependent decrease in the mean number of
intracytoplasmic fluorescent particles, which did not reach statistical
significance (Table 2). The nucleus remained devoid of labeling at all
times examined.
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Discussion |
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To achieve confocal microscopic visualization of SRIF-receptor
interactions, we resorted to a series of fluorescent derivatives of the
SRIF analog [D-Trp8]SRIF-14. This analog was
chosen for its resistance to metabolic degradation and high affinity
for all SRIF receptor subtypes (15, 44). Furthermore, this ligand had
been used successfully in iodinated form for autoradiographic detection
of SRIF receptors (45), suggesting that it might prove equally useful
for their confocal receptor imaging. We originally synthesized six
different fluorescent compounds, each differing in the type of
fluorophore (fluorescein or Bodipy) and the position of the fluorescent
group on one of the three amine functions of the peptide sequence.
Structure-function activity studies and conformation analysis of SRIF
analogs had previously indicated that the sequence required for the
biological activity of SRIF consists in the ß-turn fragment
Phe-Trp-Lys-Thr corresponding to the residue 7–10 of SRIF-14 (46). In
agreement with these data, we found that acetylation of the amine
function on the side-chain of Lys9 with either fluorescein
or Bodipy lead to fluorescent [D-Trp8]SRIF
analogs that were almost completely devoid of binding activity in rat
cortical homogenates. By contrast, fluorescent analogs substituted on
the -amino group or on the side-chain of Lys4 retained
their capacity to specifically bind to brain SRIF receptors, in
conformity with previous data which had shown that deletion of
Ala1 and Lys4 in the SRIF sequence did not
abolish SRIF’s biological activity (47, 48).
Admittedly, the affinity of both -fluoresceinyl and -Bodipy
compounds was somewhat reduced compared to that of native
[D-Trp8]SRIF-14, which may presumably be
accounted for by steric hindrance of the fluorophors. Nonetheless, both
of the compounds (heretofore referred to as fluo-SRIF) retained
sufficient biological activity for confocal imaging of sst1
and sst2 receptors in transfected cells.
Membrane binding assays on COS-7 cells transfected with cDNA coding for sst1 or sst2A receptor subtypes confirmed the expression of specific, high affinity 125I-labeled [D-Trp8]SRIF-binding sites by both types of cells. As previously documented in stably transfected cells, COS-7 cells transfected with the sst2A subtype exhibited a greater affinity for the D-Trp8 derivative than those transfected with the sst1, although the differential observed in our study was not as high as that reported by others (15, 44).
After incubation of whole sst1- and sst2A-transfected cells at 4 C, specific [125I]SRIF or fluo-SRIF binding remained confined to the cell surface, as indicated by the fact that it was entirely acid washable and assumed a pericellular distribution in the confocal microscope. By contrast, after incubation at 37 C, a fraction of the bound radioactive/fluorescent ligand became resistant to hypertonic acid washes. However, both the amount and intracellular distribution of sequestered radioactive/fluorescent ligand were distinct for each cell type.
In cells transfected with cDNA encoding the sst1 receptor, the maximal amount of acid-resistant radioactivity ranged between 20–25% of the specifically bound [125I]SRIF. Comparable effects of hypertonic acid washes had previously been observed for [125I]SRIF binding to RINm5F insulinoma cells (42), pancreatic acinar cells (35), and GH4C1 pituitary cells (41). However, in the case of both RINm5F and GH4C1 cells, this acid-resistant fraction was temperature independent, which led the researchers to conclude against the occurrence of [125I]SRIF internalization in these cells. By contrast, in both the present study and that of Viguerie et al. (35), the build up of the acid-resistant fraction was temperature dependent, suggesting that it might reflect internalization of the radioactivity within the cells. In keeping with this interpretation, pretreatment of the transfected COS-7 cells with the endocytosis inhibitor PAO prevented the formation of an acid-resistant compartment. However, in contrast to the subcellular fractionation results of Viguerie et al. (35), which showed an accumulation of internalized [125I]SRIF into microsomal and nuclear fractions of pancreatic acinar cells, our confocal microscopic observations on sst1-transfected cells suggest that the acid-resistant fraction remains sequestered into or immediately beneath the plasma membrane. This distribution is reminiscent of the insulation effect described for fluorescent CCK on the surface of pancreatic acinar cells, where the ligand was shown to become immobilized and, hence, acid wash resistant, in a time and temperature-dependent manner within the cell membrane (23). Possible mechanisms for this sequestration include the trapping of bound fluorescent molecules within caveolae, a process previously invoked to account for signal transduction of endothelin in transfected COS-7 cells (49) as well as for part of the endocytic processing of fluorescent cholecystokinin in Chinese hamster ovary cells (24).
COS-7 cells transfected with cDNA encoding the sst2A receptor and incubated at 37 C with [125I]SRIF exhibited a rapid, time-dependent increase in the acid-resistant fraction of specifically bound radioactivity. This increase was prevented by coincubation with PAO, indicating that it reflected progressive endocytosis of surface-bound ligand. Both the t1/2 of the internalization process (10 min) and the maximal proportion of bound radioactivity internalized (75%) are concordant with figures reported for other sst2 receptor agonists (39) as well as for other peptide ligands acting through the neurotensin (51, 52), NK1 (20), gastrin (29), cholecystokinin (23, 24), or TRH (19) receptors.
Confocal microscopy confirmed that specifically bound SRIF ligand was taken up inside sst2-transfected cells and demonstrated that the internalization proceeded through the formation of small intracytoplasmic organelles. These endosome-like particles increased in size with time, presumably due to their conglomeration and/or fusion given the trend toward their concurrent decrease in number. They also proceeded toward the center of the cell, where they showed a tendency to cluster along the nuclear membrane. This pattern of internalization is congruent with what has now been described for a variety of peptide ligands both in vitro and in vivo and shown to correspond, at least in some systems, to sequestration of receptor-ligand complexes into clathrin-coated pits, leading to their transfer into early endosomes, late endosomes, multivesicular bodies, and lysosomes (16). It is also consistent with the results of subcellular fractionation studies, which demonstrated sequestration of [125I]SRIF into coated vesicles (36) as well as with electron microscopic data that showed labeling of vesicular as well as lysosomal elements after in vivo or in vitro labeling of pituitary cells with [125I]SRIF (37, 38) or gold-conjugated SRIF (35, 52). Whether, as demonstrated for other peptides (19, 29, 53), this internalization process results in a recycling of the receptors and/or provides for their down-regulation remains to be established.
Previous immunohistochemical (54, 55) and autoradiographic (56, 57)
studies have demonstrated striking differences in the steady state
subcellular distribution of different peptide receptor subtypes
expressed by the same type of cells. The present findings further
indicate that there may be major differences in the way peptide
receptor subtypes are sorted. Subtype-specific differences in receptor
regulation profiles in response to agonist exposure have previously
been reported for - and ß-adrenergic receptors (58, 59), and it
is, therefore, possible that subtype-specific sorting constitutes a
general feature of the G protein-coupled receptor superfamily. The
precise difference in the amino acid sequence between the
sst1 and sst2A receptors that underlies their
different pattern of ligand internalization is unknown. It is likely,
however, to be imparted by variations in amino acid sequences between
their respective C terminals, which differ by more than 51%. This
hypothesis appears all the more likely because C terminal amino acid
sequences have been reported to be critical for ligand internalization
mediated by neurotensin (60) TRH (61), gastrin-releasing peptide (62),
and ß2-adrenergic (63) receptors. Additional molecular
biological studies are needed to substantiate this hypothesis.
The subtype-specific differences in the kinetic and distributional profiles of SRIF internalization observed here in COS-7 cells may explain the differences in the efficiency of SRIF internalization previously reported among various cell lines. They may also account for the difference in the progression of endocytosed [125I]SRIF from endocytic vesicles to downstream vesicular structures observed between A/B- and D-type pancreatic islet cells (34). The physiological significance of this differential internalization pattern remains a matter of speculation. The low efficiency, intra- or subplasmalemmal sequestration of SRIF mediated by the sst1 receptor may reflect rapid recycling of the receptor and maintenance of cell surface receptor availability. By contrast, the high efficiency internalization of SRIF mediated by the sst2A receptor may underlie rapid receptor down-regulation and, consequently, high receptor turnover. It is interesting in this context that the sst1 receptor, which internalizes poorly, does not desensitize (14), whereas the sst2 receptor, which exhibits a high efficiency internalization, desensitizes rapidly (14, 15). Clearly, elucidation of the mechanisms underlying these subtype-specific differences will prove critical for understanding their role in the regulation of SRIF signal transduction.
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Received May 14, 1996.
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,
,
,
, binding in the presence of PAO; 
) or sst2A (
)
receptor subtypes: internalization profiles. Experiments were the same
as described in Fig. 3
30%) of the cells
have internalized the fluorescent ligand (arrows) in
conformity with reported transfection yields in this cell line (64).
Scale bar = 20 µm.
