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Montreal Neurological Institute, McGill University (T.S., A.C.J., A.B.), Montréal, Québec, Canada H3A 2B4; Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis (P.S., C.D.F., J.-P.V., J.M.), 06560 Valbonne, France; and Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg (H.-J.K.), D-20246 Hamburg, Germany
Address all correspondence and requests for reprints to: Alain Beaudet, M.D., Ph.D., Montreal Neurological Institute, McGill University, 3801 University Street, Montréal, Québec, Canada H3A 2B4. E-mail: mcin{at}musica.mcgill.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The actions of SRIF are mediated through a family of G protein-coupled membrane receptors. The known receptors are encoded by five individual genes, designated sst1-sst5 (10, 11, 12, 13, 14, 15). One of the cloned receptor genes, sst2, gives rise to two splice variants, termed sst2A and sst2B, whereas the other receptor genes lack introns. Although these different receptor subtypes have been linked to a variety of signal transduction pathways (for review, see Ref. 16), their individual roles have yet to be fully elucidated. In the pituitary, sst2 and sst5 appear to play a major role in mediating SRIF’s regulatory influence on the release of pituitary hormones such as GH (17). In the pancreas, sst5 is reportedly a key player in the regulation of insulin release by SRIF (5). All five sst receptor subtypes are present in mammalian brain (18, 19, 20). Whereas sst1–3 appear to be abundantly expressed in brain, the distribution of sst4 and sst5 is much more limited (18, 19, 20, 21, 22, 23, 24, 25, 26).
All five SRIF receptors, with the possible exception of sst4, have been shown to internalize upon ligand activation (27, 28, 29, 30, 31). Internalization of receptor-ligand complexes has been shown to play a predominant role both for resensitization through dephosphorylation (32) and for desensitization through sequestration (33, 34) of G protein-coupled receptors. Furthermore, recent studies on a variety of G protein-coupled receptors, including those of the sst family (35), have suggested that internalization might subserve specific signaling functions either through endosomal signaling (36) or through activation of mitogen-activated protein kinase pathways independent of G protein recruitment (37, 38).
A striking feature of sst receptor internalization is the wide spectrum of maximal capacities and intracellular trafficking patterns reported between the various subtypes (27, 28, 29, 30). For example, in COS-7 cells transfected with the sst1 receptor, SRIF only poorly internalizes and remains clustered beneath the plasma membrane at all times (28). By contrast, in COS-7 cells transfected with sst2A, internalization of fluorescent SRIF is extremely efficient and characterized by a rapid down-regulation of cell surface receptors and the formation of small intracytoplasmic particles reminiscent of endosomes (28). Internalization of immunoreactive sst3 receptors, as studied in transfected HEK-293 (30) and RIN 1046–38 cells (29), is also highly efficient and gives rise to the same type of intracellular labeling pattern as that observed in cells transfected with sst2A. In the case of sst4, major species differences have been reported between rat and human clones. Whereas the native rat sst4 receptor does not internalize following SRIF binding (29, 30), the human sst4 internalizes well (27). Species differences were also observed in the case of sst5; rat sst5 internalizes only in response to stimulation with SRIF-28 and not SRIF-14 (29, 30), whereas human sst5 does internalize when stimulated with D-Trp8-SRIF (39).
The aim of the present study was to further characterize the properties and pattern of SRIF-induced internalization of the sst5 receptor subtype, in an attempt to gain further insight into the regulation of this receptor. We employed biochemical, confocal, and electron microscopic techniques to track sst5 and SRIF through the events following SRIF-induced internalization. We show that a constant population of functional sst5 receptors is maintained at the cell surface at all times through a process of receptor recycling and recruitment of intracellular sst5 proteins. We propose that this mechanism may protect sst5 from long term desensitization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of cell homogenates
Confluent COS-7 cells were washed and scraped into ice-cold
Tris-buffered saline (pH 7.5). Subsequently, the cells were centrifuged
for 5 min in microfuge tubes at 13,000 x g and
resuspended in hypotonic TE buffer (5 mM EDTA and
10 mM Tris-HCl, pH 7.5). Membrane homogenates
were then sonicated, recentrifuged at 13,000 x g for
30 min at 4 C, and resuspended in the same buffer.
Binding of
125I-Tyr0-D-Trp8-SRIF
to COS-7 cells
To assess the pharmacological properties of
sst5 receptors in transfected COS-7 cells,
binding experiments were performed on freshly prepared membranes.
Briefly, 25 µg membranes were incubated with concentrations of
[125I]Tyr0-D-Trp8-SRIF
(2000 Ci/mmol; hereafter referred to as
[125I]SRIF) ranging from 0.1–100
nM for 30 min at 25 C in 250 µl Tris-HCl (pH 7.5)
containing 2 mM MgCl2, 0.1% BSA
(Roche Molecular Biochemicals, Laval, Canada), and 0.8
mM 1,10-phenanthroline (Sigma, St. Louis, MO)
to prevent peptide degradation. Binding was terminated by the addition
of 2 ml ice-cold buffer followed by filtration through glass-microfiber
filters (GF/C, Whatman, Clifton, NJ) preincubated in 0.5%
polyethylenimine. After washing twice with 2 ml ice-cold buffer, the
radioactivity retained on the filters was counted in a 
-counter.
Nonspecific binding was measured in the presence of 1 µM
nonlabeled D-Trp8-SRIF
(Sigma). The dissociation constant
(Kd) and maximal binding capacity
(Bmax) were derived from Scatchard analysis of
the data.
For confocal microscopic tracking of internalized ligand, the
pH-insensitive dye Bodipy 576/589 (40) (Molecular Probes, Inc., Eugene, OR) emitting red fluorescence was covalently
conjugated to the degradation-resistant SRIF analog
D-Trp8-SRIF in the 
position (for
more detail on preparation and purification, see Ref. 28). To compare
the affinity of this fluorescent SRIF analog (hereafter referred to as
fluo-SRIF) toward sst5 with those of native
molecular forms of SRIF and other synthetic SRIF analogs, 25 µg
membranes were incubated with 0.3 nM
[125I]SRIF in the presence of increasing
concentrations of SRIF-14, SRIF-28,
D-Trp8-SRIF,
Tyr0-D-Trp8-SRIF
(all from Sigma), or fluo-SRIF ranging from 1
pM to 1 µM. Binding was performed for 30 min
at room temperature in 250 µl Tris-HCl buffer, pH 7.5, containing 2
mM MgCl2, 0.1% BSA, and 0.8
mM 1,10-phenanthroline. The experiment was terminated, and
radioactivity was counted as described above.
To characterize the kinetics of sst5
internalization in transfected COS-7 cells, internalization assays were
carried out on whole cells in 12-mm dishes containing 2 x
105 cells. After discarding the culture medium,
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
MgCl2·6H20, and 25
mM HEPES) containing 0.8 mM phenanthroline,
0.1% D-glucose, and 1% BSA in the presence or absence of
10 µM phenylarsine oxide (PAO; Sigma), a
drug that blocks endocytosis (but also many other cellular functions)
through chemical reaction with sulfhydryl groups (41), or of 25
µM monensin (Sigma), which inhibits the
membrane recycling process by raising the pH within endosomes and
thereby preventing the dissociation of receptor and ligand (41). Some
experiments were carried out in the presence of 70 µM
cycloheximide (Sigma) (41), a protein synthesis inhibitor,
in which case the preincubation period was increased to 2 h.
Equilibration buffer was then replaced with 250 µl binding buffer
containing 0.3 nM [125I]SRIF for
3–40 min (time course) or 0.1–100 nM
[125I]SRIF for 45 min (saturation), both at 37
C. In time-course experiments, cells were then washed twice with pure
equilibration buffer or a hypertonic acid buffer (Earle’s buffer
containing 0.2 M acetic acid and 0.5 M NaCl, pH
4) to strip off surface-bound radioactivity (41). In saturation
experiments, the cells were washed with equilibration buffer. All 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 an excess (1 µM) of
nonlabeled D-Trp8-SRIF.
Internalization of -Bodipy
Red-D-Trp8-SRIF in
sst5-transfected COS-7 cells
COS-7 cells transfected with cDNA encoding
sst5 were preincubated for 10 min at 37 C in
Earle’s buffer containing 0.8 mM phenanthroline, 0.9 g/ml
D-glucose, and 0.2% BSA and then incubated for 5–60 min
in the presence of 10 nM fluo-SRIF in the same buffer. To
determine nonspecific binding, 1 µM nonfluorescent
D-Trp8-SRIF was added to the
incubation medium. To evaluate temperature dependence, additional cells
were incubated at 4 C. At the end of the incubation, cells were rinsed
twice for 2 min each time with cold Earle’s buffer and air-dried. To
selectively observe internalized fluo-SRIF, cells were washed twice
with hypertonic acid buffer, pH 4, before rinsing and air-drying. To
determine whether fluo-SRIF internalization was clathrin-mediated,
cells were preincubated for 30 min in equilibration buffer containing
either 20 µM PAO or 0.45 sucrose (two compounds known to
inhibit clathrin-mediated endocytosis) (41). They were then incubated
with fluo-SRIF in the presence of PAO or sucrose, respectively, and
treated as described above. Air-dried cells were examined under a
Carl Zeiss laser scanning microscope attached to an
Axiovert 100 inverted microscope (Carl Zeiss Canada Ltd.,
Con Mills, Canada). Images were acquired as single optical sections
through the center of the cells at 16 scans/frame in the LUTS mode.
After the initial adjustment of laser power and contrast, all images of
cells from 1 experiment were acquired using identical settings of the
confocal unit. The images were processed using Photoshop version 4.0.1
and Illustrator version 7.0 (both from Adobe Systems, Inc., San Jose,
CA) on an IBM-compatible computer.
Pulse-chase experiments: confocal microscopy
To examine the fate of SRIF in parallel with that of
sst5 receptors in the course of ligand-induced
internalization, transfected COS-7 cells were preincubated for 90 min
at 37 C in DMEM containing 70 µM cycloheximide, a protein
synthesis inhibitor, followed by 5- to 10-min equilibration at 4 C in
Earle’s buffer containing 70 µM cycloheximide, 0.8
mM phenanthroline, 0.9 g/ml D-glucose, and
0.2% BSA. Cycloheximide was also included in the incubation medium in
all following steps before the final rinses. After exposure to 10
nM fluo-SRIF for 30 min at 4 C in supplemented Earle’s
buffer, the incubation medium containing the peptide was aspirated and
replaced with supplemented Earle’s buffer at 37 C without the ligand,
and internalization was allowed to proceed for 0, 5, 10, 20, 40, or 60
min, respectively. Finally, the cells were subjected to hypertonic acid
wash followed by air-drying. Additional cells from the same
transfection were incubated with 10 nM nonlabeled
D-Trp8-SRIF in lieu of fluo-SRIF.
These cells were incubated for 30 min at 4 C, chased at 37 C as
described above, rinsed twice in 0.1 M phosphate buffer, pH
7.4 (PB), and fixed with 4% paraformaldehyde (PFA) in the same buffer
at room temperature. Subsequently, the cells were rinsed twice in PB
and twice in 0.1 M Tris-buffered saline, pH 7.4 (TBS). They
were then incubated overnight at 4 C with 0.2 µg/ml affinity-purified
rabbit anti-sst5 antibody (for details on
antibody generation and characterization, see Refs. 26, 30) in TBS
containing 0.5% normal goat serum (NGS) and 0.05% Triton X-100. The
next day, the cells were rinsed twice in TBS and incubated for 40 min
at room temperature with goat antirabbit-Cy3 secondary antibodies
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA) diluted 1:800 in TBS followed by two final rinses in TBS and
mounting in Aquamount (Polyscience, Markham, Canada). As controls,
transfected cells were immunostained under the same conditions, but
without stimulation by D-Trp8-SRIF.
All specimens were examined under a confocal microscope as described
above.
Pulse-chase experiments: electron microscopy
To follow the fate of internalized sst5
receptors at the subcellular level, a series of pulse-chase experiments
was carried out as described above. However, for the sake of better
ultrastructural preservation, cycloheximide was excluded from all
incubation buffers, and pulse-labeling with 20 nM
D-Trp8-SRIF was performed at 10 C,
followed by chase periods of 0, 10, and 40 min at 37 C. Subsequent to
this pulse-chase protocol, cells were fixed for 20 min in 2%
acrolein/2% PFA in 0.1 M PB followed by another 20 min in
2% PFA alone. They were then briefly washed in 0.1 M TBS,
exposed for 30 min to a preincubation solution consisting of 3% NGS in
0.1 M TBS, and incubated overnight at 4 C with 0.4 µg/ml
sst5 receptor antibody along with 0.02% Triton
X-100 and 0.5% NGS in 0.1 M TBS. After primary antibody
incubation, the cells were repeatedly rinsed in 0.01 M PBS,
and nonspecific sites were blocked with a mixture of 0.1% gelatin and
0.8% BSA in 0.01 M PBS. Cells were then incubated with a
1:50 dilution of goat antirabbit IgG-gold (AuroProbe One GAR,
Amersham Pharmacia Biotech, Arlington Heights, IL) at room
temperature for 2 h, fixed with 2% glutaraldehyde in 0.01
M PBS, and rinsed in 0.2 M citrate buffer,
after which the reaction was enhanced by incubation with ionic silver
(IntenSE M Silver Enhancement Kit, Amersham Pharmacia Biotech). After a rinse in 0.1 M PB, cells were
postfixed in a solution of 2% osmium tetroxide in 0.1 M PB
in the dark for 10 min, dehydrated in graded concentrations of ethanol,
and embedded in Epon. Ultrathin sections were cut and impregnated with
uranyl acetate and lead citrate according to standard electron
microscopy protocols before examination with a JEOL 100CX transmission
electron microscope (JEOL USA, Inc., Peabody, MA).
For quantification, an average of 20 separate cell profiles were obtained from each experimental condition in every individual experiment. The distribution of immunogold particles was then analyzed using computer-assisted morphometry (Biocom, Les Ulis, France). Specifically, the total numbers of gold particles associated with the plasmalemma and located within the intracellular compartment were counted separately and normalized as a function of membrane length and area of cell sampled, respectively. The distance between each intracellular receptor and the plasmalemma was also measured and distributed into bins at increasing distance from the cell surface. Finally, a frequency distribution of intracellular particles and the ratio between intracellular and membrane-associated receptors were calculated for each experimental condition. Statistical significance was verified using Student’s t test, (two-tailed) for distant measurements and Mann-Whitney or Wilcoxon tests (two tailed) for membrane/cell interior ratios. All calculations and statistical analyses were performed using Excel 5.0 (Microsoft Corp., San Francisco, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
The maximal binding capacity was 92 ± 5 fmol/mg (n = 3; Fig. 1a). Membranes prepared from nontransfected cells were totally devoid
of [125I]SRIF binding (not shown).
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and Table 1). SRIF-28 showed the highest
affinity (IC50 = 0.42 nM) and was 5
times more potent than SRIF-14 in displacing specific binding from
transfected sst5 receptors
(IC50 = 2.1 nM; Fig. 1b and Table 1).
The two synthetic analogs D-Trp8-SRIF
and
Tyr0-D-Trp8-SRIF
displayed identical affinities for sst5 and were
almost as potent as SRIF-28 in competing for binding at this receptor
(IC50 = 0.6 nM; Fig. 1b and Table 1).
The fluorescent analog
-Bodipy-D-Trp8-SRIF also competed
with specific [125I]SRIF binding, but with a
potency similar to that of SRIF-14 (IC50 = 2.3
nM; Fig. 1b and Table 1).
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). At that time, 45% of the radioactivity
associated with the cells was acid wash resistant, i.e. was
no longer surface bound (Fig. 2a). After preincubation and labeling of
the cells in the presence of the endocytosis inhibitor PAO, the amount
of radioactivity specifically associated with the cells at equilibrium
was about 55% of that measured in the absence of treatment (Fig. 2a).
This proportion was therefore the same as the acid-washable
(i.e. cell surface) fraction in experiments carried out in
the absence of PAO. Accordingly, the radioactivity associated with
cells incubated in the presence of PAO was almost entirely washed away
by hypertonic acid buffer treatment, indicating that PAO totally
prevented internalization (Fig. 2a).
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).
Incubation in the presence of the protein synthesis inhibitor,
cycloheximide, yielded the same result (Fig. 2b). By contrast, the
amount of [125I]SRIF associated with cells in
the presence of PAO, i.e. bound to receptors immobilized at
the cell surface, was saturable, indicating that the lack of saturation
observed in baseline conditions was linked to receptor dynamics (Fig. 2b). In the presence of the receptor recycling inhibitor monensin or of
monensin together with cycloheximide, saturation with
[125I]SRIF was also observed (Fig. 2b). The
maximal binding capacity under these conditions was about twice as high
as that obtained in the presence of PAO (Fig. 2b), indicating that
part, but not all, of the baseline binding could be accounted for by
receptor recycling.
Internalization of fluo-SRIF in
sst5-transfected COS-7 cells
Incubation of transfected COS-7 cells with 10 nM
fluo-SRIF for 40 min at 4 C (data not shown) or 37 C (Figs. 3a and 4) resulted in intense fluorescent
labeling of approximately 20% of the cells. This labeling was
specific, in that it was no longer apparent in the presence of a
100-fold excess (1 µM) of nonradioactive
D-Trp8-SRIF (Fig. 3b). When the
incubation was carried out at 4 C, or at 37 C in the presence of 20
µM PAO or 0.45 M sucrose, the labeling
remained confined to the periphery of the cells (Fig. 3c). This
peripheral labeling was completely abolished by acid wash, confirming
that it was exclusively surface bound (Fig. 3d).
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and 4). Increasing the incubation time from 5 to 60 min resulted in a
continuous increase in fluorescent labeling (Fig. 4, left column). Hypertonic acid
wash revealed that this increase in fluorescent labeling was mainly due
to an accumulation of fluorescent peptide inside the cells (Fig. 4, right column). At all time intervals,
acid-resistant binding took on the form of small fluorescent particles
reminiscent of endosomal organelles (Fig. 4). After 5 min of
internalization, these organelles remained relatively peripheral. After
10 min, they were visible deeper in the cytoplasm, and after 20 min,
fluo-SRIF filled out most of the cytoplasm, sparing the nucleus.
Finally, after 40 (Fig. 4) and 60 (not shown) min, the fluorescent
ligand was concentrated in the center of the cell proximal to the
nucleus.
Parallel tracking of fluo-SRIF and sst5
receptor immunoreactivity in the course of ligand-induced
internalization
In pulse-chase experiments (n = 3), in which the cells were
preincubated at 4 C with or without 10 nM nonlabeled
D-Trp8-SRIF or with 10 nM
fluo-SRIF followed by sst5 immunolabeling or
air-drying, respectively, the fate of the receptor and the ligand in
the course of the internalization process were followed in parallel.
These experiments were carried out after 90 min of preincubation with
70 µM cycloheximide and in the presence of cycloheximide
throughout to suppress receptor neosynthesis during the course of the
experiment. Cells incubated with fluo-SRIF were acid-washed after the
labeling period for selective visualization of internalized ligand.
At time zero, i.e. at the end of the 4 C pulse, no
intracellular ligand fluorescence was evident (Fig. 5, right column).
Immunocytochemistry revealed that at this time point, receptors were
present both at the cell surface and in a prominent intracellular pool
(Fig. 5, left and middle columns). In cells that
were not exposed to
D-Trp8-SRIF, this pattern
of receptor distribution showed little change over time (Fig. 5, left column). By contrast, dramatic movements of both
receptor and ligand were observed in cells incubated with SRIF (Fig. 5, middle and right columns). At short time
intervals (5 and 10 min), internalized fluo-SRIF was confined to the
periphery of the cells. At the same time points,
sst5 immunoreactivity was still visible at the
cell surface and within the intracellular core. After 20 min of chase
at 37 C, internalized fluo-SRIF was visible throughout the cytoplasm,
in the form of small fluorescent vesicles. At the same time point,
sst5 immunofluorescence was still evident at the
cell surface. However, the brightness of the central cytoplasmic pool
of receptor immunofluorescence was greatly reduced, and the whole
cytoplasm was pervaded by punctate receptor immunoreactivity. The most
dramatic differences between ligand and receptor labeling patterns were
observed after long periods of chase (40 and 60 min). At those time
points, internalized fluo-SRIF became progressively more concentrated
in the cell center, near the nucleus. By contrast, receptor
immunoreactivity was entirely confined to the cell periphery.
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). Quantitatively, however, pronounced
changes in the distribution of gold particles were observed between
membrane and cytoplasm in cells exposed to the agonist. At 0 min, the
ratio between plasma membrane-associated receptors and intracellular
gold particles was small both in nonstimulated cells and in cells
exposed to D-Trp8-SRIF, indicating
that the bulk of immunoreactive receptors was located intracellularly
(Fig. 7). At 10 min, the ratio between
surface receptors and intracellular immunoreactivity remained almost
unchanged in cells unexposed to the agonist (Fig. 7). By contrast, in
stimulated cells there was a marked increase in this ratio, reflecting
a net movement of receptors from the cytoplasm to the membrane compared
with that at 0 min (Fig. 7; P 
0.01, by Mann-Whitney
U test, two-tailed) and with nonstimulated cells at 10 min (Fig. 8;
P 0.01, by Wilcoxon two-sample test, two-tailed).
After 40 min of chase at 37 C, the ratio between cell
surface-associated and intracellular receptors still remained stable in
nonstimulated cells and returned to baseline in cells exposed to the
agonist (Fig. 7).
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, arrows). Only occasionally
were stray particles found free floating in the cytoplasm. A few
immunolabeled receptors were observed in close apposition to the
nuclear envelope, but never inside the nucleus proper. No gold
particles were detected in lysosomes. There were, however, marked
differences in terms of intracellular receptor distribution depending
on whether the cells were stimulated with SRIF. In cells exposed to
SRIF, gold particles were detected more frequently in clathrin-coated
pits at the cell membrane as well as in coated vesicles and endosomal
organelles in the subplasmalemmal zone than in nonexposed cells (Fig. 8). In fact, quantitative data showed that
whereas under baseline conditions the relative frequency of
intracellular gold particles in bins of increasing distance from the
plasma membrane was very similar in stimulated and nonstimulated cells,
after 10 min of chase at 37 C, the mean distance of immunoreactive
receptors from the membrane was significantly decreased (from 2–3 to
0.25–1 µM; P < 0.01 compared
with 0 min) in cells exposed to the ligand, but not in those that were
not stimulated by SRIF, indicating a SRIF-induced mobilization of
receptors from the cell center to the periphery (Fig. 9). After 40 min of chase, the frequency
distribution of gold particles remained virtually unchanged in cells
that were not exposed to
D-Trp8-SRIF, whereas in
stimulated cells, two peaks of receptor density were apparent, one at
0.25–0.5 µm from the membrane and another at 2–3
µM (significantly different from 0 min,
P 0.01; not significantly different from 10 min,
P = 0.06; Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pharmacological characteristics of recombinant rat
sst5 in our experiments conformed to those
obtained by others in various transfection systems (42; for review, see
Ref. 43). Thus, as in previous studies investigating both rat and human
sst5 in transfected CHO-K1 or COS-7 cells (13, 42, 43), SRIF-28 was approximately 5 times as potent as SRIF-14 in
displacing radioiodinated
Tyr0-D-Trp8-SRIF
from the sst5 binding site. The synthetic analogs
D-Trp8-SRIF and
Tyr0-D-Trp8-SRIF
showed identical affinities for the receptor and were almost as potent
as SRIF-28 (IC50 = 0.6 nM). In CHO-K1
cells, D-Trp8-SRIF exhibited an even
higher potency in displacing [125I]CGP23996
from the sst5-binding site and was more potent in
doing so than SRIF-28 itself (IC50 = 0.002
nM) (42). Finally, the fluorescent ligand employed in our
study, -Bodipy Red-D-Trp8-SRIF,
was virtually equipotent with SRIF-14 in competing for
[125I]D-Trp8-SRIF
binding at the sst5 site, validating its
usefulness as a high affinity marker of sst5
receptors.
Previous studies have demonstrated that both human (27, 39) and rat (29, 30) sst5 are internalized in the presence of SRIF in a variety of cell lines. In HEK cells transfected with cDNA encoding rat sst5 (30) as well as in RIN 1046–38 insulinoma cells transfected with tagged rat sst5 (29), this process was ligand specific, in that internalization was observed after exposure to SRIF-28, but not to SRIF-14. The present results indicate that in COS-7 cells expressing the rat sst5 receptor, as in CHO-K1 cells expressing the human sst5 receptor (39), D-Trp8-SRIF-14 is able to efficiently induce sst5 internalization. Whether this property is dependent upon the cell lines used or reflects specific properties of this SRIF analog, which could be linked to its near equipotency with SRIF-28 at the sst5 binding site, remains to be determined.
The internalization process observed in sst5-transfected COS-7 cells is rapid, reaching a plateau within 20 min (t1/2 = 10 min). These kinetics are comparable to those of SRIF internalization via the rat sst2A receptor in the same cell type (28). With 45% of total specifically bound radioactivity, however, internalization via sst5 does not reach the same capacity as sst2A-mediated endocytosis (85%; 28). In CHO-K1 cells transfected with individual human sst subtypes, on the other hand, sst5 internalized more efficiently than sst2A (66%; 27).
As in the case of sst2A (28), fluo-SRIF internalization via sst5 was entirely blocked by incubation with the endocytosis inhibitor PAO or in hypertonic sucrose, indicating that it is dependent on the formation of clathrin-coated pits (41). Immunoelectron microscopic experiments confirmed that this was indeed the case, by demonstrating SRIF-induced mobilization of sst5 in clathrin-coated pits and vesicles. Molecular studies previously demonstrated the presence of both positive and negative sequence motifs for internalization in the intracellular C-terminal domain of the cloned human and rat sst5 receptors (30, 39), indicating a pivotal role for the C-terminus in mediating internalization. Moreover, a variant of the consensus sequence NPXXY for binding of G protein-coupled receptors to the clathrin-adaptor molecule ß-arrestin (for review, see Refs. 34, 41) is present in position 300–304 (in fact, NPLLY) at the C-terminal end of the cloned rat receptor (13) in exactly the same position as in the human sst5 molecule (39). Although direct evidence is still missing, these data suggest that sst5 might, like other G protein-coupled receptors, interact with clathrin through binding to ß-arrestin.
Combined confocal microscopic tracking of fluo-SRIF and sst5 receptors in cells pretreated with the protein synthesis inhibitor cycloheximide allowed us to determine the fate of receptor-ligand complexes after ligand-induced internalization. Five to 10 min after transfer of fluo-SRIF-labeled COS-7 cells from 4 to 37 C, internalized ligand was confined to the periphery of the cells in the form of small fluorescent particles. By 20 min after the onset of internalization, these endosome-like particles pervaded most of the cytoplasm. Finally, after longer time intervals (40 and 60 min), the ligand had accumulated inside the cytoplasm and concentrated in the center of the cells in close proximity to the nucleus. This course of events is similar to that observed in the same type of cells transfected with cDNA encoding the rat sst2A receptor (28). By contrast, cells transfected with the sst1 receptor barely sequester fluo-SRIF (28). A pattern of endocytic events similar to that seen here was also observed in COS-7 cells transfected with the NT1 neurotensin receptor (44), in CHO cells transfected with the cholecystokinin receptor (45), and in Kirsten murine sarcoma virus-transformed rat kidney (KNRK) cells ectopically expressing the substance P receptor (46). In all of these cell systems, the fluorescent ligand was targeted to a perinuclear compartment after prolonged internalization periods. However, whereas in NT1-transfected cells this juxtanuclear compartment was interpreted as belonging to the trans-Golgi network by virtue of its immunoreactivity for syntaxin-6 (44), in CHO and KNRK cells, it was found to correspond to lysosomes (45, 46). Conceivably, the fate of a metabolically protected ligand such as D-Trp8-SRIF, which was used in our experiments, may differ from that of native SRIF, in that the latter may be more readily targeted to lysosomes and degraded than the former. However, recent studies on CHO cells expressing the sst2 receptor subtype have shown iodinated native SRIF-14 to be internalized, recycled to the extracellular medium, and reinternalized several times (47), suggesting that even native peptide may be targeted to recycling compartments. Clearly, further experiments using native ligands are needed to determine the fate of SRIF internalized via the sst5 receptor subtype under physiological conditions.
The pattern of sst5 internalization, as visualized here by receptor immunofluorescence, differed sharply from that of its fluorescent ligand. At time zero, sst5 immunoreactivity was concentrated at the level of the plasma membrane as well as in a very prominent cytoplasmic pool in the core of the cell. The existence of such a pool of intracellular sst5, even after prolonged preincubation with cycloheximide, was previously reported by others in different cell lines (29) and presumably largely corresponds to trans-Golgi network/Golgi stores. After exposure to the ligand, there was very little change in the intensity of cell surface labeling. However, the intracellular cytoplasmic pool became progressively less prominent as most of the immunoreactivity accumulated at the periphery of the cell. The lack of an apparent decrease in cell surface labeling was unlike what had previously been reported for either sst2A (35) or sst3 (29, 30) receptors expressed either naturally or ectopically in other cell lines. It also differed from the confocal microscopic pattern of ligand-induced internalization reported for other G protein-coupled receptors, such as the neurokinin-1 receptor (46), the ß-adrenergic receptor (48), or the TRH receptor (49), all cases for which a significant decrease in cell surface labeling was observed in the initial stages of internalization. The question arose as to whether the preservation of cell surface receptors observed here in the case of sst5 could be attributed to receptor recycling and/or to a mobilization of receptors from intracellular stores to the cell surface.
To determine the extent to which receptor recycling contributed to the preservation of cell surface labeling, internalization assays were carried out in the presence and the absence of the recycling inhibitor monensin. In the absence of monensin, [125I]SRIF association with sst5-transfected COS-7 cells did not reach equilibrium, even at concentrations of the ligand as high as 1 µM. This lack of saturability was clearly a consequence of the internalization process, as in the presence of the endocytosis inhibitor PAO, saturation was reached. In the presence of monensin, association of [125I]SRIF with whole cells was also saturable and amounted to roughly 40% of the binding observed at equimolar concentrations of the ligand in the absence of the drug. These results demonstrate that receptor recycling accounts for part of the maintenance of cell surface receptors in the course of internalization, but that other mechanisms, such as targeting of receptors from an intracellular reserve pool to the membrane, must also be invoked.
To further investigate this idea, we carried out an immunoelectron microscopic study of sst5 after pulse-chase stimulation with SRIF. These experiments confirmed that sst5 receptors were present at the plasma membrane at all times, even after ligand exposure. In fact, they revealed that there was even an overshoot in receptor targeting to the membrane at 10 min of incubation, as reflected by the ratio between cell surface-associated and intracellular receptors, which rose from approximately 0.2 at 0 min to almost 0.9 after 10 min of incubation with the ligand. This effect was accompanied by a redistribution of receptor molecules inside the cytoplasm, which, from a peak distance of 2–3 µm from the plasma membrane at time zero, shifted to a peak distance of 0.25–0.5 µm at 10 min, suggesting a massive recruitment of reserve receptors to the plasmalemma. At 40 min, the intracellular receptor distribution had started to return to normal, as reflected by the presence of two peaks, one still close to the membrane (as seen at 10 min), but the second deeper inside the cell.
These joint recycling and recruitment mechanisms, providing for continuous availability of functional receptors at the cell surface even during massive and prolonged ligand exposure, have profound implications for receptor function, in that they might effectively protect the receptor from long term desensitization. It is interesting to recall, in this context, that the rapidly desensitizing receptor subtype sst2A (28, 45, 50, 51) acts together with sst5 to regulate GH release from the anterior pituitary (17). It may be that in cells such as these, and perhaps also in regions of the brain in which sst2A and sst5 coexist (21, 22, 26), sst5 actually provides for a prolongation of SRIF signaling after sst2A has been down-regulated.
In conclusion, the present study demonstrates that SRIF is rapidly and efficiently internalized via the sst5 receptor in a heterologous expression system. It shows that this endocytosis is mediated through a clathrin-dependent mechanism. A major finding is that in response to agonist stimulation and endocytosis, internalized sst5 receptors are rapidly recycled back to the cell surface. At the same time, spare receptors from an intracytoplasmic reserve pool are addressed to the plasma membrane. The combination of these two processes provides for the continuous presence of sst5 receptors at the cell surface and, therefore, accounts for the lack of saturability of SRIF binding to intact cells expressing sst5.
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Received June 16, 1999.
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),
D-Trp8-SRIF (•),
Tyr0-D-Trp8-SRIF (
), SRIF-28
(
), and SRIF-14 (
) as competitors. For IC50 values,
see Table 1
