(Received for publication, August 1, 1996, and in revised form, November 4, 1996)
From the Institut de Pharmacologie Moleculaire et
Cellulaire, Centre National de la Recherche Scientifique-UPR 411, 660, Route des lucioles, 06560 Valbonne, France and the
Montreal Neurological Institute, Department of
Neurology and Neurosurgery, McGill University, Montreal,
Quebec, Canada H3A 2B4
The binding and internalization of radioiodinated
and fluorescent µ and opioid peptides in mammalian cells were
quantitatively studied by biochemical techniques and directly
visualized by confocal microscopy. The labeled peptides were prepared
by inserting either a 125I-Bolton-Hunter group or a
fluorescent probe into the C-terminal part of 5-aminopentylamide
derivatives of deltorphin-I and [Lys7]dermorphin. The
purified derivatives kept most of their specificity and selectivity
toward
and µ opioid receptors, respectively. Biochemical and
confocal microscopy data showed that both µ and
opioid peptides
were internalized in mammalian cells transfected with the corresponding
opioid receptor according to a receptor-mediated mechanism. The
internalization process was time- and temperature-dependent and was completely blocked by the endocytosis inhibitor phenylarsine oxyde. Internalization of both
and µ ligands occurred from a single large cap at one pole of the cell, indicating that
polymerization of ligand-receptor complexes preceeded internalization.
Finally, green and red fluorescent analogues of deltorphin-I and
[Lys7]dermorphin, respectively, were found to internalize
through partly distinct endocytic pathways in cells co-transfected with µ and
receptors, suggesting that each of these receptors
interacts with distinct proteins mediating intracellular sorting and
trafficking.
The pharmacological, behavioral, and binding properties of µ,
, and
opioid receptors have been extensively studied. By comparison, much less is known about the intracellular routing and
addressing of opioid receptors either before or after agonist binding.
Yet, understanding the cellular regulation of this class of receptors
is of prime importance, since it is at least partly involved in the
mechanisms of tolerance and physical dependence (1). There is a
considerable amount of in vitro pharmacological evidence to
suggest that both µ (2, 3) and
(4-7) opioid receptors may
undergo rapid down-regulation following exposure to agonists. Whether,
and under which condition, such down-regulation involves
internalization of receptor-ligand complexes remains a matter of
debate. Thus, while biochemical studies have reported on either the
occurrence (5, 8) or absence (9) of internalization of the
tritiated enkephalin agonist [3H]D-Ala,
D-Leu-enkephalin in cultured neuroblastoma cells,
morphological studies clearly failed to observe internalization of a
fluorescent derivative of enkephalin in the same cell system (10-12).
More recently, confocal microscopic studies carried out on transfected cells have shown a rapid endocytosis of µ (13, 14) and
(13) antigenic epitope-tagged receptors following exposure to enkephalins, but not to morphine. Whether this endocytosis occurs in conjunction with that of the bound ligand, however, remains unclear. In the present
study, we have reinvestigated the fate of receptor-bound opioid
peptides using newly developed radioactive and fluorescent derivatives
of the selective µ and
opioid agonists dermorphin and
deltorphin.
Dermorphin, isolated from the skin of the frog Phylomedusa
sauvagei (15), was the first natural peptide described as having high affinity and selectivity for the µ opioid receptor. Another unusual property of dermorphin is the D configuration of
the alanine residue in position 2, which is responsible for its strong
resistance to enzymatic degradation and for its good opioid binding
site recognition. The most interesting analogue of this peptide is [Lys7]dermorphin (16), which possesses the highest
affinity for the µ opioid receptor (less than 1010
M) together with the highest µ/
specificity
(10,000-fold more specific for the µ than for the
receptor). Two
other peptides having also a D-alanine in position 2 have
been purified from the skin of another frog, Phylomedusa
bicolor (17). They have the same first three amino acids
(Tyr-D-Ala-Phe) but differ in their C-terminal sequence
(Asp-Val-Val-Gly for deltorphin-I and Glu-Val-Val-Gly for
deltorphin-II). Deltorphin-I was chosen for our study because of its
superior affinity and specificity for the
opioid receptor.
Sequences of [Lys7]dermorphin and deltorphin-I may be
divided into two parts, the same first three amino acids
(Tyr-D-Ala-Phe) confirming something like an opioid master
key and the last four being responsible for the µ/ selectivity and
affinity. The only site that could be modified without changing the
properties of these peptides is the C-terminal end. We have thus
incorporated radiolabeled and fluorescent groups into C-terminal
extensions of deltorphin-I and dermorphin and used these specific tools
to compare the cellular distribution and fate of specifically labeled µ and
opioid receptors. Radioactive compounds were used to
quantitatively assess the binding and internalization of the opioid
derivatives, while fluorescent compounds were used to study their
distribution in the confocal microscope, an approach that has been
successfully resorted to for a variety of other neuropeptides,
including fluorescent analogues of cholecystokinin (18),
gastrin-releasing peptide (19), neurotensin (20), thyrotropin-releasing
hormone (21, 22), substance P (23, 24), and somatostatin (25).
Preparation of Peptide Precursors
DLT-I 5APA1 and [K7]DRM 5APA were prepared as described previously (26). Briefly, deltorphin-I and dermorphin were assembled by stepwise solid phase synthesis using a t-butoxycarbonyl-benzyl strategy. The aminopentyl group was then grafted on the C-terminal amino acid by aminolysis of the peptide resin with 1,5-diaminopentane.
Preparation of Radioactive Peptides (27)
Bolton-Hunter reagent was iodinated with Na125I and
purified as described previously (28). DLT-I 5APA or [K7]DRM 5APA (40 nmol) was incubated with iodinated Bolton-Hunter reagent (0.5-2 mCi, 2000 Ci/mmol) in 50 µl of borate/phosphate buffer (50 mM/50 mM) for 2 h at pH 8.5. The
incubation mixture was injected on a C18 reverse phase HPLC (Merck,
lichrocart), and the different products were eluted in 0.1%
trifluoroacetic acid, 0.05% triethylamine by a linear gradient of
acetonitrile from 10 to 60% in 70 min. The pure -specific
(
-BH* DLT-I 5APA) and µ-specific (
-BH*
[K7]DRM 5APA) 125I-labeled peptides were diluted in 50 mM Tris-Cl, pH 7.5, containing 0.2% bovine serum albumin
and stored at
20 °C.
Preparation of Fluorescent Peptides
Peptide precursors were reacted with the
N-hydroxysuccinimide esters of Bodipy 503/512 or Bodipy
576/589 (Molecular Probes). NHS-Bodipy (2 µmol in 400 µl of
dimethyl sulfoxide) was incubated with DLT-I 5APA or [K7]DRM 5APA (2 µmol) in a final volume of 1 ml of boric acid (50 mM),
sodium phosphate (50 mM) buffer, pH 8.5, for 3 h at
4 °C. The different derivatives were purified by reverse phase HPLC
on a C18 Ultrosphere ODS column (10 x 250 mm, Beckmann) eluted in 0.1%
trifluoroacetic acid with a linear gradient of acetonitrile from 20 to
60% during 60 min. Fluorescent peaks were tested for their ability to
displace the specific binding of -BH* [K7]DRM 5APA and
-BH* DLT-I 5APA to the µ and
opioid receptors,
respectively (27). Seven fluorescent peaks were found to have a high
binding activity and were further characterized by Edman
degradation.
Preparation of Receptor-encoding Plasmids
Rat µ (MOR) (29) and (DOR) (30) opioid receptor cDNAs
were amplified from rat brain cDNAs by polymerase chain reaction with specific oligonucleotides. Polymerase chain reaction products were
subcloned in pcDNAI. pcDNAI, pcDNAI-MOR, and pcDNAI-DOR
were transfected in COS cells by the DEAE-Dextran method (31).
Binding of Fluorescent Peptides to COS-7 Cell Membranes
Binding properties of fluorescent derivatives were determined by
displacement of -BH* DLT-I 5APA and
-BH*
[K7]DRM 5APA-specific binding to membranes of cells transfected with
pcDNAI-DOR and pcDNAI-MOR, respectively. Forty-eight hours after transfection, cells were rinsed with PBS
, scraped
in Tris/EDTA (5 mM/5 mM, pH 7.5) and
centrifuged at 100,000 × g. Radioactive ligands (0.2 nM) were incubated with transfected cell membranes (5-20
µg of membrane proteins) and increasing concentrations of fluorescent
peptides in 250 µl of 0.2% bovine serum albumin, 50 mM
Tris-Cl, pH 7.5, during 30 min at 25 °C as described previously
(27). Incubations were stopped by filtration on GF/C filter presoaked
in 0.3% polyethyleneimine-Cl, pH 7.5. Filters were rinsed three times
with 3 ml of binding buffer and counted in a
counter.
IC50 values were determined by measuring the concentration
of fluorescent peptide that displaced 50% of the bound radioactive
ligand.
Internalization Studies
Cell PreparationForty-eight hours after transfection, cells were rinsed in PBS, detached with a PBS solution containing 0.05% trypsin and 0.53 mM EDTA, and equilibrated with 10% fetal calf serum in Dulbecco's modified Eagle's medium during 2 h at 37 °C. They were then centrifuged at 1000 rpm during 5 min and equilibrated for 15 min at 37 °C in binding buffer (Earle-HEPES buffer, pH 7.4, supplemented with 0.09% glucose and 0.2% of bovine serum albumin) at a final concentration of 50-200,000 cell/ml.
Radioactive Binding ExperimentsCells transfected with
pcDNAI-DOR and pcDNAI-MOR were incubated for 0-60 min at
37 °C with -BH* DLT-I 5APA and
-BH*
[K7]DRM 5APA (0.1-10 nM, 2000 Ci/mmol), respectively, in
the presence or absence of 10 µM of the endocytosis
inhibitor, phenylarsine oxide. The incubation was terminated by adding
3 ml of hypertonic acid buffer (Earle-HEPES, pH 4, acetic acid, 0.4 M NaCl) or of control buffer (Earle-HEPES at neutral pH)
for 2 min, after which the cells were filtered on GF/C filters
presoaked in binding buffer and rinsed three times with 3 ml of binding
buffer. Cell-bound ligand was determined by counting the radioactivity
retained on filters with a
counter. Nonspecific binding was
determined by carrying the incubation in the presence of 10 µM naloxone. Temperature sensitivity was verified by
incubating additional cells for 60 min at 0 °C with 0.2 nM radioactive ligand. Stability of the ligands was
determined by reverse phase HPLC analysis of a fraction of ligand
recovered at the end of the incubation. Fractions collected after HPLC
were counted and compared with initial solutions.
Cells transfected with
pcDNAI-DOR and pcDNAI-MOR were preincubated as described above
and incubated for 15-90 min at 37 °C in binding buffer containing
10 nM -Bodipy 503/512 DLT-1 5APA or
-Bodipy 503/512
[K7]DRM 5APA in the presence (nonspecific binding) or absence (total
binding) of 10 µM naloxone. In some experiments, the
incubation was carried out in the presence of phenylarsine oxide, to
prevent ligand endocytosis. At the end of the incubation, cells were
washed with either hypertonic acid or isotonic neutral buffers as
above, centrifuged at 2000 rpm during 1 min, deposited in 10 µl of
Earle-HEPES on glass microscope slides, air-dried, and examined by
confocal microscopy.
Concomitant labeling of and µ opioid receptors was carried out on
COS cells co-transfected with the pcDNAI-DOR and pcDNAI-MOR plasmids. Co-transfected cells were incubated at 37 °C for 90 min
with a mixture of 10 nM of
-Bodipy 503/512 DLT-I 5APA
(green) and
-Bodipy 576/89 [K7] DRM 5APA (red). At the end of the
incubation, cells were centrifuged at 2000 rpm, deposited on glass
slides, and air-dried for confocal microscopic examination.
Confocal Microscopy
Labeled COS cells were examined under a Leica confocal laser
scanning microscope configured with a Leica Diaplan inverted microscope
equipped with an argon/krypton laser with an output power of 2-50 mV
(Leica, St. Laurent, Canada). Images of cells were acquired as single
midcellular optical sections and averaged over 32 scans/frame. For
double labeling experiments, and µ ligand images were acquired in
the green and red channels, respectively.
The binding and internalization of the
selective opioid agonists deltorphin-I and dermophin were first
assessed quantitatively in COS-7 cells transfected with cDNA
encoding and µ opioid receptors, using 125I-labeled
Bolton-Hunter derivatives of deltorphin-I (
-BH*DLT-I
5APA) and dermorphin (
-BH*[K7]DRM5APA), respectively.
These derivatives have been documented to selectively bind to
and µ opioid receptors with respective Kd values of
0.7 and 0.14 nM (27). Binding and internalization kinetics
were established for each of these compounds by incubating whole cells
at 37 °C. As can be seen in Fig. 1, in which total specific binding (open symbols) corresponds to the sum of
acid wash-resistant specific binding (corresponding to internalized ligand; closed symbols) and of acid-washable specific
binding (corresponding to membrane-bound ligand; not shown), total
binding kinetics were very rapid (t1/2 of about 1 min), whereas internalization kinetics were about 10 times slower
(t1/2 = 13 and 10 min for
and µ receptors,
respectively). The main difference between the two opioid systems was
the maximal proportion of internalizable ligand, which represented
approximately 55 and 25% of the total specific binding in cells
transfected with
and µ receptors, respectively. As shown in Fig.
2, this proportion was not very sensitive to ligand
concentration as increasing the concentration of each radioligand by a
factor of 100 (from 0.1 to 10 nM) enhanced maximal
internalization by a factor of less than 2. Thus, independent of ligand
concentration, the internalization process was approximately twice as
efficient for the
as for the µ ligand.
Internalization of both and µ ligands was almost totally
inhibited by blocking endocytosis with phenylarsine oxide or by lowering the temperature of incubation, as reflected by the reduction in size of the acid wash-resistant fraction (Fig. 3).
Somewhat surprisingly, the addition of phenylarsine oxide also markedly reduced the total binding of 125I-labeled deltorphin to
cells transfected with the
receptor (Fig. 3A), but not
that of 125I-labeled dermorphin to cells transfected with
the µ receptor (Fig. 3B). In both systems, lowering the
temperature of incubation to 0 °C resulted in large losses of total
specific binding (35 and 55% of the binding observed at 37° C for
and µ systems, respectively). Finally, neither binding nor
internalization of either 125I-deltorphin or
125I-dermorphin were observed in COS-7 cells transfected
with the noncorresponding opioid receptor.
Biochemical Characterization of Fluorescent Analogues
In
order to visualize at the cellular level the interaction of and µ opioid ligands with their respective receptors, we have synthesized two
different fluorescent derivatives of deltorphin-I and
[Lys7]dermorphin. These two heptapeptides are the most
potent and selective
and µ agonists currently known. The
synthesis was carried out in two steps. First, an aminopentyl group was
grafted on the C-terminal carboxyl function of dermorphin and
deltorphin-I. Second, peptide precursors DLT-I 5APA and [K7]DRM 5APA
were reacted with the N-hydroxysuccinimide esters of green
(Bodipy 503/512) or red (Bodipy 576/589) fluorescent probes. The four
different mixtures resulting from the reaction of the two peptide
precursors with the two fluorescent reagents were fractionated by
reverse phase HPLC. Elution profiles illustrated in Fig.
4 show that it was always possible to separate the
unmodified peptide (N) from its fluorescent derivatives.
Peaks numbered 1-6 in Fig. 4 were selected for further
characterization on the basis of their ability to compete for binding
to the
(peaks 1 and 2) or to the µ (peaks 3-6) opioid receptor.
Amino acid analysis and UV-visible spectra of each fraction (not shown)
indicated that fluorescent peptides 1-6 contained a single fluorescent
group per mol of peptide. The position of the fluorophore into each peptide sequence was determined by Edman degradation (Table
I). The sequences of peaks 1 and 2 were identical to
that of deltorphin-I, indicating that the green (peak 1) and
red (peak 2) Bodipy fluorophores had been incorporated on
the
amine function of the 5-aminopentylamide group in DLT-I 5APA.
In the same way, peaks 4 and 6 were identified as the red and green
-substituted analogues of [K7]DRM 5APA, because their sequences
were indistinguishable from that of [K7]DRM. By contrast, the seventh
cycle of sequencing of fractions 3 and 5 did not give a PTH-Lysine,
indicating that the
-amino group of Lys 7 has been modified.
Therefore, fractions 3 and 5 were identified as the green and red
-labeled analogues of [K7]DRM 5APA.
|
The binding properties of the six fluorescent peptides were then
evaluated by measuring their ability to displace the specific binding
of 125I-labeled analogues of DLT-I 5APA and [K7]DRM 5APA
to the and µ opioid receptors transiently expressed in COS cells.
The two fluorescent analogues of DLT-I 5APA interacted with the
opioid receptor with high affinity (K0.5 = 2 nM) and retained much of their selectivity since their
affinity for the µ receptor was lower by at least two orders of
magnitude (Table II). The four fluorescent derivatives
of [K7]DRM 5APA bound to the µ opioid receptor with affinities in
the nanomolar range. However, their selectivity was not as good as that
of
ligands, since the
/µ ratio of IC50 values
varied only from 4 to 21 (Table II). The fluorescent analogues 4 (
-Bodipy 503/512[K7]DRM 5APA) and 6 (
-Bodipy 576/589[K7]DRM
5APA) were selected for the confocal studies described below because of
their higher selectivity for the µ receptor as compared with
derivatives 3 and 5.
|
Confocal microscopic examination
of COS-7 cells transfected with a cDNA encoding the opioid
receptor and incubated for 15-90 min with 10 nM
-Bodipy
503/512 DLT-15APA revealed selective fluorescent labeling of
approximately 30% of the cells, in keeping with the documented
transfection yield of this cell line (32) (Fig.
5A). This labeling was specific in that it
was no longer detected when the incubation was carried out in the
presence of 10 µM naloxone (Fig. 5D) or with
cells transfected with an empty plasmid (Fig. 5C). It was
selective for
opioid receptor-expressing cells, since cells
transfected with cDNA encoding the µ site were totally devoid of
fluorescent labeling (Fig. 5B).
Similarly, approximately 30% of COS-7 cells transfected with a
cDNA encoding the µ opioid receptor and incubated with the µ-selective ligand -Bodipy 503/512 [K7]DRM 5APA exhibited
intense fluorescent labeling (Fig. 6A). Here
again, this labeling was specific in that it was displaced by naloxone
(Fig. 6D) and absent from cells transfected with an empty
plasmid (Fig. 6C). It was also selective for µ receptor-transfected cells as it was no longer apparent in cells
transfected with cDNA encoding the
opioid receptor (Fig.
6B).
At all time intervals examined, the bulk of DLT-I 5APA fluorescent
labeling was intracellular, as attested by its intracytoplasmic distribution in single optical sections passing through the core of the
cells (Figs. 5A and 7A) and by its resistance to
hypertonic acid wash (Fig. 7C).
Internalization of the fluorescent agonist was totally prevented by
addition of the endocytosis inhibitor, phenylarsine oxide, in which
case the bound fluorescent molecules remained clustered at the
periphery of the cells (Fig. 7B). This excentric labeling
pattern corresponded to cell surface labeling as it completely
disappeared after hypertonic acid wash (Fig. 7D).
Similarly, a sizeable fraction of -Bodipy 503/512 labeling of µ receptor-expressing cells was found to be acid wash-resistant, i.e. intracellular, at all times examined (Figs.
8, A and C). In keeping with our
biochemical results, this fraction was smaller overall than in the case
of
labeling. As with the
ligand, incubation in the presence of
phenylarsine oxide prevented internalization of the µ opioid agonist
and resulted in an acid-washable (Fig. 8D), cell surface
clustering of the bound fluorescence (Fig. 8B).
The intracellular distribution of -Bodipy 503/512 DLT-I 5APA and
-Bodipy 503/512 [K7]DRM 5APA, specifically bound to
opioid- and µ opioid-transfected cells, respectively, varied markedly as a
function of time. After 15 min of incubation, both fluorescent markers
were clustered at one pole of the cell, onto and/or immediately beneath
the plasma membrane (Fig. 9, A and
B). At 30 min, they were detected in the form of small
fluorescent particles excentrically clustered in the cytoplasm of the
cells (Fig. 9, C and D). By 60 min,
and µ opioid labeling remained highly punctate, but entirely filled the
cytoplasm of the cells, sparing the nucleus (Fig. 9, E and
F). However, by that time, the dermorphin-labeled fluorescent particles were on average larger and less numerous than the
deltorphin-labeled ones and stood out less clearly against a greater
intracytoplasmic background labeling.
In order to compare the distributional pattern of and µ opioid
agonists internalized within the same cells, COS-7 cells were
co-transfected with the pcDNAI-DOR and pcDNAI-MOR plasmids and
co-incubated with the green fluorescent analogue
-Bodipy 503/512
DLT-I 5APA and the red fluorescent analogue
-Bodipy 576/589 [K7]DRM 5APA for 90 min at 37 °C. As shown in Fig.
10, A and B, and at higher
magnification in Fig. 10, A
and B
, images
acquired through distinct red and green excitation/emission channels
showed labeling patterns comparable with those obtained in singly
transfected cells confirming the efficiency of the co-transfection
procedure. Superimposition of the two images (Figs. 10, C
and C
) showed only partial overlap of the red and green
fluorescent clusters, indicating that the µ and
fluorescent
ligands were sequestered partly in the same and partly in distinct
compartments. This partial overlap could not be attributed to a
bleed-through of one of the fluorophores into the other channel since
Bodipy 576/589 and Bodipy 503/512 derivatives gave no signal in the
green and red channels, respectively. Interestingly, the bulk of
distinct green and red particles were small and predominated at the
periphery of the cell (Fig. 10C). By contrast,
double-labeled endosome-like particles (in yellow, Fig. 10C)
were larger and mainly concentrated within the cytoplasmic core.
In the present study, the binding and internalization of µ and
opioid peptides in mammalian cells were quantitatively studied by
means of biochemical techniques and directly visualized by confocal
microscopy. The radiolabeled and fluorescent analogues developed for
this purpose were synthesized by inserting either an
125I-labeled Bolton-Hunter group or a fluorescent probe
into the C-terminal part of 5-aminopentylamide derivatives of
deltorphin-I and dermorphin. Elongation of both peptide sequences by a
C-terminal extension bearing a primary amine proved essential for
introducing reporter groups into both dermorphin-I and deltorphin
without a dramatic loss in binding properties. Indeed, all other
labeling approaches tried by us, including direct iodination of
Tyr1 or acylation of the N-terminal amine function with
Bolton-Hunter or fluorescent groups, led to dermorphin and deltorphin-I
derivatives of low binding activity toward µ and
opioid
receptors. Although both radiolabeled (27) and fluorescent (Table II)
analogues synthesized in the present work had a somewhat lower affinity than their native counterpart, they retained sufficient biological activity and specificity for biochemical analysis and confocal imaging
of µ and
opioid receptors in transfected cells. The reduced
affinity observed after radioactive or fluorescent tagging probably
resulted from the steric hindrance of the markers, since insertion of a
Bolton-Hunter group into the
- or
-amine functions of
deltorphin-I and dermorphin resulted in smaller losses of affinity than
the incorporation of the more bulky Bodipy fluorophores. Replacement of
the red or green Bodipy probes by larger fluorophores like fluorescein
resulted in further decreases in affinity of the corresponding
deltorphin-I and dermorphin analogues for their specific receptors
(results not shown).
Biochemical and confocal microscopic data showed that both µ and opioid ligands were internalized in mammalian cells according to a
time- and temperature-dependent process. This
internalization was clearly receptor-mediated, since it was no longer
observed in nontransfected cells or in cells transfected with a
receptor not specifically recognized by the ligand. Furthermore, it was completely prevented by incubation with the non selective opioid antagonist naloxone. The kinetics of internalization of µ and
opioid ligands were considerably slower than those of ligand binding
(t1/2 of 10 and 13 versus 1 min for µ and
ligands, respectively), but within the same range as reported
for bradikinin receptor (33) and vasopressin V2 receptor (34) complexes
(t1/2 = 9 and 13 min, respectively). Slightly faster
receptor-mediated internalization kinetics have been described for
peptides bound to vasopressin V1 (35, 36), angiotensin II 1a and 1b
(37), substance P (23), gastrin-releasing peptide (38), and neurotensin
(39, 40) receptors, with t1/2 values ranging between
3 and 5 min. The similarity between the internalization kinetics of
these different neuropeptide-receptor complexes suggests that they may
be controlled by a common rate-limiting step.
Despite the fact that formation of receptor-ligand complexes was
clearly critical for the initiation of ligand internalization, the
percentage of internalized ligand molecules was in no way proportional
to the degree of receptor occupancy. Indeed, cells transfected with the
receptor and incubated with concentrations of
125I-labeled deltorphin-I ranging from 0.025 to 10 nM internalized between 40 and 70% of the bound
radiolabeled peptide, whereas receptor occupancy varied from 3.5 to
94%. The disproportion was even greater in the case of the µ opioid
ligand, which proportionally internalized about 2-fold less than its
counterpart. Variable internalization capacities have been reported
for other peptidergic systems. Ratios of internalized to bound ligand
ranging between 50 and 80% have been reported for substance P (23),
vasopressin (34, 35), angiotensin II (37, 41), or neurotensin (39, 40)
and of 100% for gastrin-releasing peptide (38) and bradykinin (33).
The low proportion (20-35%) of 125I-labeled dermorphin
internalized by COS cells transfected with the µ opioid receptor
observed in the present work is rather unusual. The only other example
that we are aware of is that of the sst1 somatostatin receptor
expressed in COS cells, which internalizes only 20-25% of
specifically bound 125I-labeled somatostatin 14 (25). The
mechanism that maintains an equilibrium between internalized and
noninternalized peptide-receptor complexes is unknown.
Internalization of both µ and opioid ligands in transfected COS-7
cells was totally prevented in the presence of the endocytosis inhibitor, phenylarsine oxide (PAO), indicating that the
internalization process is endocytic in nature. Accordingly,
internalized ligand molecules were seen by confocal microscopy to be
concentrated within small, endosome-like organelles. Current models of
receptor-mediated internalization call for internalization of
receptor-ligand complexes into clathrin-coated pits, followed by their
mobilization into early and then late endosomes (42). These endosomes
eventually fuse into multivesicular bodies and lysosomes, while
dissociated receptors are either recycled back to the membrane or
degraded (42). The progressive shift in size and intracellular
mobilization of fluorescent organelles observed in the present study
are consistent with this pattern. Although the present approach did not
allow a direct visualization of receptors themselves, the similarity of
the punctate labeling seen here after internalization of fluorescent dermorphin with that observed by immunohistochemistry in cells expressing an epitope-tagged µ opioid receptor (14) and exposed to
the selective µ agonist
Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol strongly suggests
that the internalization involves ligand-receptor complexes. A similar
mechanism has been invoked to account for the internalization of
tritiated D-Ala2,
D-[Leu6]enkephalin in neuroblastoma cells (5,
8) and may play a role in the agonist-induced down-regulation of
opioid receptors documented in several cell lines (6, 7, 43). Our
results are at odds, however, with the reported lack of internalization of a rhodamine-tagged Met-enkephalin derivative in cultured
neuroblastoma cells (10-12). This discrepancy may be due to
differences in receptor behavior dependent on the ligand utilized. Mu
opioid receptors were indeed shown to internalize following exposure to
Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol, but not to
morphine (14).
In addition to blocking receptor-mediated internalization, phenylarsine
oxide markedly decreased total specific binding of the
125I-labeled deltorphin-I analogue to cells transfected
with the receptor without affecting that of the dermorphin analogue
to µ sites. This decrease cannot be imputed to direct effects of the
drug on the plasma membrane, as it was no longer observed when the
binding experiments were performed on COS-7 cell membranes, as opposed
to whole cells (not shown). It is therefore likely that the loss of
specific deltorphin binding observed in whole cells is the result of
cellular traffic blockade by PAO, implying that µ and
opioid
receptors are differentially distributed within the cells at steady
state. Specifically, our results suggest that µ receptors are
predominantly localized on the cell membrane, since they are freely
accessible to radioligand even in the presence of PAO, whereas a
significant fraction (65%) of
receptors are located inside the
cell, within vesicular structures that can no longer be recruited to
the membrane in the presence of PAO. The ability of PAO to inhibit not
only endocytosis but also exocytosis has been documented in various
reports dealing with trafficking properties of glucose transporters
(44) and transferrin receptors (45) or with secretory mechanisms of the
RBL-2H3 mast cell line (46). Furthermore, such differential
distribution of µ and
receptors in COS cells would be consistent
with the results of light and electron microscopic localization studies
in the central nervous system, which have shown µ opioid receptors to
be predominantly associated with the plasmalemma (14, 47, 48) and
receptors to be almost equally distributed between the plasma membrane
and intracellular stores (48-52). Comparable differences in the
subcellular distribution of two homologous G-protein-coupled receptors
have been observed previously within the family of adrenergic
receptors. Thus, the
2- and
2-C10-adrenergic receptors expressed in transfected fibroblasts are mainly localized in the plasma membrane at steady state, whereas the
2-C4-adrenergic receptor is found
both in the plasma membrane and in a population of intracellular
vesicles (53).
A striking feature of both µ and labeling patterns was their
clustering into a single large cap at one pole of the cell. Although
comparable clustering patterns have been observed after ligand binding
to insulin (54) and muscarinic cholinergic (55) receptors, most
hormone- or neuropeptide-receptor (18, 19, 23, 39) complexes have a
tendency to aggregate into small, distinct patches distributed all over
the cell membrane. In fact, earlier fluorescence studies have reported
rhodamine-tagged enkephalins to form multiple small clusters at the
surface of neuroblastoma cells (10). It may be that the extent of
receptor clustering is controlled by the relative rates of ligand
binding and internalization and that it varies from on type of cell to
the other. When the rate of internalization is equal to (or faster
than) the binding step, as observed for example in the case of
neurotensin-receptor complexes (39), internalization begins as soon as
small aggregates are formed, preventing the formation of large caps. By
contrast, if the internalization proceeds at a much slower pace than
ligand binding, as is the case for opioid receptors, polymerization of the complexes is allowed to proceed for longer times leading to the
formation of large macromolecular aggregates. In any event, our kinetic
results clearly show that internalization of both
and µ ligands
occurs only from a single polar cap, corroborating the idea that
polymerization of the ligand-receptor complexes is a necessary step for
internalization.
A major result of the present work is the demonstration that µ and
receptors co-expressed in the same cells internalize through partly
distinct endocytic pathways. Thus, during the early phase of
internalization, µ and
receptor-ligand complexes appeared to be
mainly localized within mutually exclusive endosomal populations, as
there was little overlap between the small, peripheral red and green
fluorescent particles that likely correspond to early endosomes.
Co-localization of the two fluorophores occurred only in larger
organelles that were observed deeper in the core of the cells and
probably correspond to late endosomes or lysosomes (56). To our
knowledge, the present results provide the first direct evidence that
different receptor subtypes co-expressed in the same cell may be sorted
via different endocytic vesicles. A selective internalization mechanism
has also been described for M
2-10H- and
2-adrenergic receptors coexpressed in K293 cells (53).
However, in that case, only the
2 receptor was
internalized in response to cell stimulation by norepinephrine whereas
the M
2-10H receptor remained in the plasma membrane.
Interestingly, internalization vesicles of the
2
receptor were found to be distinct from those containing the
M
2-10H receptor, a third homologous receptor
co-expressed in the same cells, but which is already present in
intracellular vesicles before any agonist stimulation (53). The
subtype-specific receptor sorting documented in the present study
suggests that individual receptors interact selectively with cellular
proteins mediating intracellular sorting and trafficking. The selective
sorting of internalized receptors could contribute to homologous
desensitization and reactivation processes (57).
In conclusion, we have shown that µ and opioid peptides are
internalized into transfected COS-7 cells as a consequence of their
selective interaction with µ and
opioid receptors, respectively. For each ligand, the internalization is likely to include the following
steps: (i) ligand binding, (ii) partial and selective aggregation of
homologous ligand-receptor complexes, (iii) interaction of aggregates
with cellular proteins that mediate intracellular trafficking, and (iv)
formation of vesicles and internalization. In this scheme, the signal
of selectivity that ultimately leads to sorting of ligand-receptor
complexes into specific vesicles would be given by the binding of the
agonist to its specific receptor. The resulting conformational change
would trigger the homologous aggregation of agonist-receptor complexes.
A recent study on the mechanical origin of receptor-mediated
endocytosis shows that membrane complex clustering can provide the
stimulus for a local membrane motion toward the cytosol (58). However,
it cannot be excluded that the sorting signal is given by selective
interactions between the agonist-receptor complex and cellular proteins
involved in the sequestration process. In addition to interacting with proteins involved in the formation of clathrin-coated (59) or non-clathrin-coated (60) vesicles, G-protein-coupled receptors have
been shown to bind to proteins of the cytoskeleton (61, 62). It will be
important to identify each one of these proteins and to determine
whether their interaction with various receptors can be modulated by
agonist-induced conformational changes of these receptors.
We thank F. Aguila for excellent artwork and J. Kervella for expert secretarial assistance.