* Department of Biological Chemistry, and Department of Cell Biology and Anatomy, School of Medicine, Johns Hopkins
University, Baltimore, Maryland 21205
While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to "cap" rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.
CHEMOTAXIS is a fascinating phenomenon whereby
motile cells sense and respond directionally to
chemical gradients. This important biological response plays a fundamental role in inflammation, neurogenesis, angiogenesis, and other morphogenetic processes (Behar et al., 1994 A critical, unanswered question concerns the distribution
profile of these receptors. Previous immunohistochemical
studies of randomly oriented cells or cells taken from shaken
suspension have shown chemoattractant receptors to be
uniformly distributed around the cell periphery (Raposo
et al., 1987 Another important question about GPCRs in general
concerns their desensitization pathways. Desensitization is
a series of processes that prevent continuous activation of
the cell during prolonged exposure to agonist, thus protecting it from over stimulation. The commonly recognized
mechanisms of desensitization are: (a) reduction in ligand
binding capacity due to internalization or decreased affinity; (b) decrease in the efficiency of coupling to cognate G
proteins and down-stream effectors; and (c) reduction in
the receptor protein level. In many systems, these processes are promoted by agonist-induced phosphorylation
of the receptors. A number of GPCRs including the Previously it has not been possible to characterize
chemotaxis and desensitization under physiological conditions, since most of the available techniques involve immunohistochemistry or immunofluorescence, which requires
the prefixation of cells. In alternative methods which allow
study of live cells, the receptor still has to be prelabeled
with fluorecent ligands or antibodies. These reactions could cause unwanted alterations in receptor metabolism
or trafficking. Green fluorescence protein (GFP), a stable
fluorescent protein from a Pacific Northwest jellyfish, Aequorea victoria, has been extensively used as a visualization
tag to study a variety of physiological phenomena (Cubitt
et al., 1995 We fused GFP to the COOH terminus of cAR1 and
found that this chimeric protein is indistinguishable from
wild-type cAR1 in all testable biochemical and genetic
properties, including agonist binding, agonist-induced phosphorylation, and phenotypic rescue of cAR1-null cells. By
expressing this construct in a cAR1-null cell line, we could
follow the distribution of receptors during chemotaxis and
desensitization nonintrusively. This represents the first
successful attempt to study a GPCR in unperturbed living cells and to instantaneously visualize a receptor during
stimulus presentation. Our results show for the first time
that chemoattractant receptors remain uniformly distributed
on the surface of cells that have been polarized by chemotactic gradients and also in cells that have been desensitized
by persistent treatment with chemoattractant. This study
demonstrates that GFP fusions with GPCRs may be an effective means to study the localization of these receptors.
Construction of cAR1-GFP Fusion Protein
A mutant GFP sequence (S65T), cloned into the BamHI site of pRSETB
(Invitrogen, Carlsbad, CA), was kindly provided by Dr. Roger Tsien
(University of California, San Diego, CA). This mutant has been shown to
give greater brightness and sustain slower photobleaching than wild-type
GFP (Cubitt et al., 1995 Immunoblotting
Protein samples were solubilized in SDS-sample buffer and resolved by
SDS-PAGE on 10% gels. In the case of ligand-induced receptor phosphorylation, the samples were run on gels with lower cross-linker concentration (Klein et al., 1985 Detergent Resistance of cAR1-GFP
The procedure was essentially as described previously (Xiao and Devreotes, 1997 Ligand-induced Phosphorylation and Electrophoretic
Mobility Shift of cAR1-GFP
cAR1-GFP cells were washed once with development buffer (DB; 10 mM
sodium phosphate, pH 6.2, 1 mM MgCl2, 0.2 mM CaCl2) and developed in
DB suspension at a cell density of 2 × 107/ml for 6 h. Cells were first
shaken at 200 rpm for 30 min in the presence of 4 mM caffeine and then
treated with increasing doses of cAMP in the presence of 10 mM DTT for
15 min (Kim and Devreotes, 1994 Desensitization of Fusion Receptor and
Loss-of-Ligand Binding
The loss-of-ligand binding (LLB) assay was performed essentially as described (Caterina et al., 1995 Confocal Fluorescence Microscopy Analysis
Chemotaxis-competent cAR1-GFP cells were plated onto glass surfaces
and allowed to adhere. They were fixed with 4% paraformaldehyde/0.1% Triton X-100 in PBS for 8 min, rinsed twice with PB, submerged in PB,
and then analyzed with a confocal laser scanning microscope (Noran OZ;
Noran, Middleton, WI) at an excitation wavelength 488 nm from a Krypton-Argon multi-line laser. A barrier filter was used to detect 500-550-nm
emissions. Cells were examined through an inverted microscope (IX-50;
Olympus, New Hyde Park, NY) with a U plan-apo 100×/1.35 NA oil immersion lens. Z-axis images were processed and three-dimensional reconstructions created and rotated with Intervision 1.6 software (Noran).
Chemotactic Stimulation of Cells
through Micropipettes
Cells at 2 × 107/ml were shaken in DB suspension with addition of 50 nM
cAMP every 6 min for 6 h to allow the expression of the full complement
of chemotactic proteins (Devreotes et al., 1987 Ligand-induced Desensitization of Cells and
Con A-stimulated Receptor Capping
For desensitization, a concentrated dose of cAMP was applied to the vicinity of the cells adherent to a glass coverslip, and time-lapse fluorescence images were taken at 20-s intervals for a total duration of 5 to 10 min. The final equilibrated concentration of cAMP inside the observation
chamber was >10 For Con A treatment, Con A was added to a final concentration of 20 to 50 µg/ml. Fluorescence images were taken before and after the treatment at intervals of 40 s for a total duration of 5 to 10 min.
The cAR1-GFP Fusion Protein Targets
to the Plasma Membrane and Is Biochemically and
Functionally Indistinguishable from WT cAR1
We expressed the cAR1-GFP fusion construct in a cAR1-null cell line, thereby replacing the endogenous receptor.
Multiple lines of evidence suggested that the fusion protein correctly reported the distribution of the functionally
active chemoattractant receptors. First, we investigated
whether the fusion protein is effectively synthesized and
processed by immunoblotting the whole cell protein extract with purified GFP antibody or cAR1 antibody (Fig. 1
A). Both antibodies detected an identical 65-kD protein,
which is the expected size of the fusion protein. Smaller
proteins corresponding to the size of free GFP (30 kD) or
cAR1 (40 kD) were not observed. There was a faint band
at 85 kD which corresponds to GFP fused to a minor 55-kD form of cAR1 routinely observed in cells expressing WT cAR1. We have speculated that this minor form may
derive from a low level of premature translation from an
upstream initiation site or a post-translational modification. Our previous study has established that WT cAR1 resides on special detergent-resistant microdomains of plasma
membrane (Xiao and Devreotes, 1997
To investigate the ligand-binding affinity and functional
properties of cAR1-GFP, we carried out the cAMP-induced
receptor gel mobility shift assay. Cells were treated with
increasing doses of cAMP to induce different extents of
steady-state receptor phosphorylation. This phosphorylation can be visualized as a mobility shift on SDS-PAGE;
the phosphorylated protein migrates more slowly. For wild-type receptors, the concentration of cAMP at which
half of the molecules are shifted to the lower mobility
form (defined as EC50) is closely correlated with the dissociation constant (Kd) for cAMP binding (Johnson et al.,
1992 To assess whether the fusion receptor is expressed to the
same level as cAR1, we performed [3H]cAMP binding for
both the fusion cell line and a control cell line that expresses cAR1 in the same expression vector. It has been
previously shown that ammonium sulfate converts the receptors into a single high-affinity species with a Kd value
of 5 nM (Johnson et al., 1991 To further test the functionality of the fusion protein, we
plated the cells on non-nutrient agar. Under nutrient starvation, D. discoideum cells start a developmental program
which allows them to form multicellular aggregates that
differentiate into fruiting bodies. Central cells initially start
to secrete cAMP, which causes neighboring cells to move
chemotactically towards the center; in turn these cells secrete cAMP, attracting even more distal cells. This chain
of events culminates in the formation of aggregates containing up to 105 cells. cAR1 is primarily responsible for
mediating this process (Devreotes, 1994
The Chimeric Receptor Is Uniformly Distributed on the
Cell Surface and Remains So during Chemotaxis
Under conventional fluorescence microscopy, the cAR1-GFP
cells displayed intense fluorescence on the cell periphery;
the cell body showed a faint diffuse signal (see below).
Such a profile is expected if the protein is evenly localized
on the surface membrane and further suggests that GFP
does not interfere with the correct targeting of cAR1 to
the plasma membrane. The uniformity of the fluorescence
signal was remarkably preserved during random movement of the cell. Rapidly extending and retracting pseudopods and fine filopods were clearly visible due to the bright
fluorescence, despite the fact that the signals showed little
preferential localization onto these structures or to the
leading or trailing edges of the cell (see below). The peripheral fluorescence might have been anticipated based
on previous immunohistochemical studies, but it was not
possible to observe the receptors on thin cellular projections in those experiments.
We next used the micropipette assay to study distribution of the cAR1-GFP receptor during chemotaxis (Fig. 3,
1-8). cAR1-GFP cells were able to move chemotactically
towards the source of cAMP in a fashion that is indistinguishable from cells expressing wild-type cAR1. We detected little change in receptor localization profiles under
any conditions. The fluorescence signals remained uniform on the cell surface and projections, even though the
cells were undergoing rapid morphological changes associated with chemotaxis. Even in highly polarized cells, there
was no increase of signal at the migrating front. Specifically, in Fig. 3, 1-4, which represents one complete sequence, cell "a" initially extended two pseudopods (1, arrowhead). The one pointed in the wrong direction was
quickly suppressed and taken over by the correct one,
which persisted throughout the process (Fig. 3, 2, arrowhead). In Fig. 3, 3, the cell extended a pseudopod in the
wrong direction (arrowhead), which was again competed
out by the right one. Cell "b" provided another example of
this behavior. Little apparent change in receptor distribution accompanied these contortions.
We frequently switched the position of the pipette to induce formation of novel chemotactic fronts (Fig. 4, 1-8).
Cells initially moved towards the right asterisk (Fig. 4, 1 and 2). Upon repositioning of the micropipette to the bottom left asterisk (Fig. 4, 3-8), cells extended pseudopods
from the old migrating front towards the new spot. This
was followed by a dramatic shape change that bent the
cells in the direction of the new gradient. The cells reoriented themselves, and thus the old front served as the new front. In other instances, a new front would form de novo
from the side of the cell proximal to the new cAMP
source, and the old front would disappear. In either case
little change in the receptor localization profile was noticed.
To quantitatively assess the uniformity of the signals
around the cell periphery, we directly counted the pixel
numbers of fluorescent signals from various regions or
converted the digitized signal to a false color image. We
set the color range in such a way that signal intensity variations of >10% would cause a different color to appear. In
most cases, the signal was uniform along the circumference and varied randomly by <10-20%. In some instances there was a decrease in intensity of ~20 to 30% on the extending fronts, especially on the most recently projected
pseudopods.
Confocal Image Analysis of cAR1-GFP Distribution
on Cells
To further elucidate the details of receptor distribution on
various regions of the cell membrane, we carried out a
Z-axis confocal analysis (Fig. 5 A). It was necessary to
lightly fix the cells since they are extremely mobile. Sections of 0.5 µm in thickness were taken from the bottom of
the cell close to the substratum to the upper surface at a
distance of 1.0 µm per section. The cell under study was a
typically flat and adhesive cell with many pseudopods and
fine filopods. The bottom section (Fig. 5 A, 1) showed fluorescence in the interior of the profile, which was likely
due to the upward invaginations of its basal membrane. The latter frames of Fig. 5 A displayed mostly peripheral
fluorescence and little internal signal, indicating that
cAR1 is highly localized on the surface. Pseudopods and
filopods seemed most apparent in the lower sections. The
upper surface of the cell seemed to be quite uneven: one
portion of the cell (near bottom of the frame) rose up
higher than the other portion. After Fig. 5 A, 3, only cross-sections of this portion were clearly present. The receptors seem to be more or less evenly distributed on the plasma
membrane, although some small, randomly localized regions do contain more signals than neighboring domains.
A three-dimensional reconstruction was created for another cell (Fig. 5 B). Rotation of the image along the x axis
provides further spatial information of the receptor. The
first image is the view from bottom to top with the top projecting into the plane of the paper. After a 150° rotation,
the last image displays the view from top to bottom. The
receptors again appear to be uniformly distributed on the
flat part of the cell surface, as well as along the numerous
folds, creases, and projections found on the surface.
During Persistent Stimulation with Chemoattractant
Leading to Cell Desensitization, cAR1-GFP Undergoes
a Decrease in Affinity and Remains on the Cell Surface
We next compared the properties of the wild-type and fusion receptors during agonist-induced desensitization. As
shown previously, agonist occupancy leads to phosphorylation of cAR1. The phosphorylated receptors then display diminished ligand binding capacity due to a decreased
affinity (LLB; Caterina et al., 1995
To visualize the possible changes in receptor distribution during persistent treatment with chemoattractant, we
gently applied a dose of cAMP to cells adherent on a glass
surface; the addition caused a uniform increase of cAMP
concentration to ~10 Although the above result showed that under a conventional microscope, receptors do not display gross rearrangement after desensitization, it was possibile that a
small percentage of receptors was internalized to certain
interior compartments or plasma membrane-proximal vesicle structures that may not be obvious under our test conditions. To address these issues and further investigate the
desensitization phenomenon, we carried out two-dimensional confocal analysis of persistently stimulated cells
(Fig. 6 C). This cell had been completely desensitized by
treatment with 10 Con A Is Able to Cause the Patching and
Capping of cAR1-GFP
The multivalent lectin Con A induces cross-linking of cell
surface glycoproteins, resulting in the patching and eventual "capping" of these surface membrane proteins (Patton et al., 1990
Since the Con A capping process has been postulated to
be mediated through the cytoskeletal structures (de Priester
et al., 1990 We were able to study the details of the cellular distribution of a chemoattractant receptor during chemotaxis and
under constant ligand stimulation in real time within motile cells by the use of a GFP fusion protein. Our study represents the first application of the GFP fusion technology
towards the study of a GPCR. In randomly moving cells,
the receptor is evenly distributed throughout the plasma
membrane; and during chemotaxis, the distribution undergoes minimal change. If challenged with an uniform increase in cAMP, which is sufficient to induce receptors
phosphorylation, a decrease in affinity, and cellular desensitization, the localization profile remains essentially unaltered. The receptor is not absolutely static on cell surface
since Con A induces its dramatic redistribution into a polar cap. Thus, while both coronin, a cytoplasmic actin-associated protein, and filamentous actin (F-actin) accumulate
at the migration front during chemotaxis, the chemoattractant receptor showed no overall rearrangement in distribution. This indicates that local populations of receptors
are sufficient for the signal transduction processes required to redistribute the cytoskeletal proteins and that
global mobilization is unnecessary. Often cells display a strong degree of polarization upon gradient reversals. Our
results demonstrate that this gradient-induced polarization is not due to receptor redistribution.
We observed that the frequently extended and retracted
pseudopods and filopods in the chemotactic cells are uniformly covered with chemoattractant receptors (Figs. 3
and 4, and data not shown). These observations support a
"pilot pseudopod" model of chemotaxis (Gerisch et al.,
1975 In certain instances (Fig. 3), we noticed slightly reduced
signals on some of the migration front pseudopods. This
may be simply due to a transient thinning of the cell front
caused by pseudopod protrusion. Alternatively, the protruded, streched out membrane may initially have a lower
density of receptors that is quickly replenished by receptors flowing in from adjacent zones. Further confocal studies of chemotaxing cells (at the migration front) will be required to resolve this issue. It appears that generation of a
pseudopod corresponds to simple protrusion of a portion
of cell plasma membrane rather than deposit of new materials, and when pseudopods retract, the extended membrane directly falls back into surrounding membranes.
We fused GFP to the COOH terminus of cAR1, and this
apparently did not interfere with either signal sequence
recognition or correct translocation of its multiple membrane-spanning domains. Furthermore, the fusion receptor retains most of the biochemical properties and physiological functions of cAR1. This suggests that the COOH
terminus of a GPCR can be highly tolerant of genetic manipulation. Besides allowing real time imaging, the GFP
fusion technique also avoids fixation-induced artifacts inherent in conventional immunohistochemical techniques.
The distribution of the receptor did not depend on its
expression level. Our observations were obtained in a cell
line that overexpresses the fusion protein by less than onefold compared with optimally developed wild-type cells. A
similar level of overexpression has been observed for wild-type cAR1 in the same expression vector. However, individual cells displayed fluorescent intensities that varied
about fivefold (Fig. 4). Previous immunoperoxidase (HRP) labeling study of cAR1 also showed a wide range of labeling intensities among different cells (Xiao and Devreotes,
1997 The uniform distribution of the chemoattractant receptors on the plasma membrane may be actively maintained.
D. discoideum cells are vigorous phagocytes, similar to
macrophages; the entire cell membrane turns over within
45 min, mainly through endocytosis and vesicular trafficking. Several mechanisms may preserve the uniform distribution of the receptor in the face of this dynamic membrane turnover. An active sorting program may restrict
cAR1 from the internalizing vesicles; or a retrieval system
may quickly return cAR1 back to plasma membrane. Earlier studies showed that cAR1 can be quantitatively recovered from detergent lysates of cells as bilayered membrane fragments. These domains are highly enriched in
sterol but poor in phospholipids (Xiao and Devreotes, 1997 The apparently static distribution of receptor on cell
surface should not be confused with an absolute immobility. Rather, this only means that there is no net movement
of receptor within the membrane or insertion and removal
from the membrane. Individual receptor molecules may
be very laterally mobile and involved in active intra-membrane protein trafficking. It will be interesting to compare
the lateral mobility of the receptor with that of other membrane proteins that do display visible translocations,
and to compare mobilities of receptors at front and rear
ends of the same chemotacting cell. It will be equally intriguing to compare the mobility of receptors in resting
cells with that of desensitized cells. We are currently attempting to address these issues through fluorescence
photobleaching recovery techniques.
The Con A capping test showed that the uniformity of
receptor distribution is only relative, and its gross rearrangement can take place during capping. Cytoskeletal
components, such as myosin and actin, have been shown
to be required for this capping process. We speculate that
cAR1 is associated with cytoskeletal proteins that underlie
the plasma membrane or with other surface glycoproteins. cAR1 itself does not appear to be glycosylated. The
chemoattractant-induced phosphorylation of cAR1 does
not interfere with its response to Con A. Hence, if the capping is due to the interaction of receptor with another
component, this interaction is not perturbed during desensitization.
There have been many studies on the fate of desensitized GPCRs after persistent stimulation (Chuang et al.,
1996 Two previous studies have proposed that desensitized
GPCR remains on the cell surface in special plasma membrane microdomains. The CCK (Cholecystokinin) receptor, a GPCR involved in the hormonal regulation of zymogen secretion from pancreatic acinar cells after ingestion
of a meal, appears to desensitize through a process termed
"insulation," whereby the full complement of receptor remains on the plasma membrane but with greatly reduced
lateral mobility (Roettger et al., 1995b In our case, both basal and desensitized forms of cAR1
display a diffuse pattern of localization on plasma membrane through the current GFP fluorescence study and
previous immunogold electron microscopy studies (data
not shown). Adapted receptors remain on surface membranes; this may provide the cells with greater flexibility
and sensitivity. No de novo synthesis or recruitment of internal receptors is necessary; deadaptation can be accomplished on the plasma membrane.
; Szekanecz et al., 1994
; Hogan and Foster, 1996
). With the identification of cAR1, the cAMP
chemoattractant receptor of Dictyostelium discoideum (Klein
et al., 1985
), it became clear that receptors mediating
chemotaxis belong to the G protein-coupled receptor
(GPCR)1 superfamily (Strader et al., 1995
). Since then
~20 functionally related "chemokine" receptors have been
identified that mediate the chemotactic responses of neutrophils, macrophages, and lymphocytes. The features of
chemotaxis and the spectrum of the biochemical responses
triggered by chemoattractants are remarkably conserved between these evolutionarily distant cell types (Chen et
al., 1996
). The recent discovery that certain chemokine receptors act as the coreceptor for HIV virus infection has
raised new interest in this class of receptors (Wells et al.,
1996
; Baiocchi et al., 1997
).
; Wang et al., 1988
; Trogadis et al., 1995). However, since chemotaxis is a dynamic process, the receptors
must be instantaneously visualized in cells undergoing directional migration. Within a few minutes of being placed
in a gradient, cells become polarized; and often, they will
turn when the gradient direction is reversed. While the
trailing and lateral edges of the cells do remain sensitive to
chemoattractant, higher concentrations are required to
elicit new pseudopods in these regions (Swanson and Taylor, 1982
; Fisher et al., 1989
). This gradient-induced polarization might be mediated by a redistribution of receptors.
That is, the altered sensitivities may be due to a reversible
accumulation of receptors at the anterior end. Recent studies have shown that coronin, a cytoplasmic actin-associated protein that is enriched at the cortical sites of moving D. discoideum cells, does transiently accumulate at the
leading edge of chemotaxing cells (Gerisch et al., 1995
;
Maniak et al., 1995
). We sought to determine whether
cAR1, which is responsible for triggering the events that
lead to actin and coronin translocation, displays a similar
dynamic localization profile.
-adrenergic receptor (for review see Perkins et al., 1991
), interleukin 8 receptor (Prado et al., 1996
), thrombin receptor
(Chen et al., 1995
), muscarinic receptor (Maloteaux and
Hermans, 1994
), and angiotensin receptor (Hunyady et al.,
1994
) have been observed to be internalized in response to
agonist treatment. By immunofluoresence, we have observed that ligand occupancy of cAR1 can lead to its clustering or internalization (Wang et al., 1988
). However, we
have also obtained conflicting biochemical evidence indicating that cAR1 remains on the plasma membrane even
after prolonged cAMP stimulation (Caterina et al., 1995
).
). Only in a few cases were GFP successfully
fused with integral membrane protein (Marshall et al., 1995
;
Hampton et al., 1996
). A GFP fusion with odr-10, a presumptive seven-transmembrane domain olfactory receptor
in Caenorhabditis elegans has been previously constructed
(Sengupta et al., 1996
). In that study, the fusion was mainly
used in a fixed whole animal fluorescence assay to determine
organ localization of the protein. No detailed biochemical
study or study on the cellular level was carried out.
Materials and Methods
). The multiple linker site was removed by digesting the plasmid with HindIII to XhoI and blunt-end ligating the new ends
after Klenow enzyme treatment. The entire coding sequence of cAR1, including the 5
ribosome-binding site was PCR amplified and cloned into
the remaining EcoRI site in front of the GFP sequence. This cAR1-GFP
fusion sequence was then released by BamHI digestion and cloned into
the BglII site of pJK1, a D. discoideum extra-chromosomal expression
vector (Kim and Devreotes, 1994
). Transformants with the correct orientation were selected by PCR. DNA was purified and transformed into RI9 cells (car1
/car3
cells), and these cells served as the cells under study
(cAR1-GFP cells).
) to effectively resolve the two forms of receptor
(unmodified vs. phosphorylated). cAR1-GFP was detected by immunoblotting with anti-GFP antibody (Clontech, Palo Alto, CA) or cAR1 antiserum (R4 serum; Klein et al., 1985
).
). Briefly, cells expressing cAR1-GFP were resuspended to 1 × 108 cells/ml, and Chaps was added to a final concentration of 1.5%. Extraction was allowed to proceed for 5 min on ice. Lysates were separated
into soluble and pellet (detergent-resistant membrane) fractions by centrifugation at 15,000 g for 10 min. The fractionation profile of cAR1-GFP
was monitored by immunoblotting.
). Samples were resolved on 10% low-bisacrylamide PAGE, transferred to PVDF membranes, and immunoblotted with cAR1 antiserum.
). Briefly, cells were washed, resuspended in
DB, and divided into two equal aliquots. One received 10
5 M cAMP with
10 mM DTT while the other received DTT alone. After treatment for 20 min, cells were diluted into 10-fold excess of ice-cold phosphate buffer
(PB; DB without the MgCl2 and CaCl2) and pelleted by centrifugation at
4° (Sorvall SS34, 2,000 rpm, 3 min). After three washes in cold PB, cells
were resuspended to 108/ml. Binding of 16 nM [3H]cAMP was then performed by a sedimentation assay.
). Cells (at 106 cells/ml)
were washed, dissociated by gentle vortexing, and transferred onto a glass
coverslip. After cell adhesion, the coverslip was mounted on the bottom of
an observation chamber (5.5 × 4.0 cm) and covered with 2 ml DB to prevent drying and allow chemotaxis to take place. Micropipettes (Gerisch
and Keller, 1981
) filled with 10
4 M cAMP were used to stimulate the
chemotaxis of these cells. Cells were observed on an inverted microscope
(Axiovert 135 TV; Zeiss, Inc., Thornwood, NY) equipped with a water
condenser and a phase-contrast 40 or 100× oil-immersion plan neofluar
objective lens. Their movement was recorded using a cooled CCD camera
(PXL; Photometrics, Tucson, AZ) controlled by IPLab-Spectrum software (Signal Analytics, Vienna, VA). Fluorescence microscopy was performed on the same microscope with an HBO 100-W mercury lamp and a
100× oil neoflur objective lens. To avoid UV injury to the cells, the light
intensity was reduced by neutral density filters, and usually 20-50 ms-
exposure images were taken at 10- to 15-s intervals for a total duration of
5 to 10 min.
5 M, sufficient to induce receptor desensitization and
LLB (Caterina et al., 1995
).
Results
). Crude subcellular
fractionation indicated that both the major and minor GFP fusion proteins, like WT cAR1, mostly associated with
this detergent-resistant subdomain (Fig. 1 B). Further fractionation of cellular lysates on sucrose density gradients
showed that both bands were quantitatively localized to the
plasma membrane microdomains (data not shown). All
these observations indicate that cAR1-GFP is a stable fusion
protein that is correctly targeted to the proper location.
Fig. 1.
cAR1-GFP is functionally indistinguishable from wild-type cAR1. (A) cAR1-GFP fusion protein as detected by immunoblotting with GFP and cAR1 antisera. Proteins were solubilized in SDS-sample buffer, resolved on 10% SDS-PAGE, and
transferred onto PVDF membranes. Duplicate samples were
loaded and analyzed in parallel. In the left lane, the fusion protein was detected with affinity-purified anti-GFP antibodies. In
the right lane, it was detected with anti-cAR1 antiserum. (B)
Both the major and minor forms of cAR1-GFP are localized to a
detergent-resistant plasma membrane subdomain. Whole cells
(lane 1) were extracted with 1.5% CHAPS at 4°C and lysate separated into soluble (lane 2) and detergent-resistant membrane
(lane 3) fractions by centrifugation, as described in Materials and
Methods. Protein samples were resolved by SDS-PAGE, transferred to membranes, and fusion protein detected by anti-cAR1 antibody. (C) Agonist-induced phosphorylation of cAR1-GFP
and gel mobility shift assay. cAR1-GFP cells were treated with
increasing doses of cAMP for 15 min to induce the phosphorylation of the COOH terminus of cAR1. cAMP doses: 1, 0 nM; 2, 5 nM; 3, 20 nM; 4, 50 nM; 5, 100 nM; 6, 200 nM; 7, 500 nM; 8, 1 µM;
and 9, 5 µM. The unmodified or phosphorylated forms of the fusion receptor were detected with cAR1 antiserum. (D) cAR1-GFP cells express the same total surface cAMP-binding sites as
wild-type cAR1 cells. cAR1-GFP and cAR1 expressing cells were
washed in PB and resuspended to 1 × 108 cells/ml density in ice-cold PB. 60 µl cells were used in the ammonium sulfate-binding
assay (Materials and Methods). The specific binding for both cell
lines in cpm were compared and later translated into the number
of binding sites per cell (see text).
[View Larger Versions of these Images (34 + 27 + 62 + 31K GIF file)]
). As shown in Fig. 1 C, the fusion protein showed a
similar mobility shift, with an EC50 between 20 and 50 nM.
Densitometric analysis gave a value of 27 nM, which is essentially identical to the wild-type value (30 nM; Kim and
Devreotes, 1994
). This observation indicates that cAR1-GFP has the same cAMP-binding properties as cAR1 and
couples with equal efficiency to the receptor kinase.
). Therefore, binding obtained at 20 nM [3H]cAMP represents the total number of
surface cAMP binding sites. As shown in Fig. 1 D, the
cAR1-GFP cells express about the same number of sites as
the WT cAR1 cells (3.5 × 105 sites/cell). This value is ~70%
more than that of optimally differentiated AX3 cells (2 × 105 sites/cell) in which cAR1 is expressed from its endogenous promotor.
). As shown in Fig. 2,
cAR1 null cells cannot initiate this pathway and hence remain unaggregated (3), and wild-type cAR1 rescues the
program such that normal fruiting bodies are eventually
formed (2). The cAR1-GFP-expressing cell line displayed a normal developmental phenotype, reaching each stage
of the program at the same time as the WT cAR1 cells
(data not shown). Fig. 2, 1 shows its final fruiting body
stage. These observations indicate that cAR1-GFP and
WT cAR1 are functionally interchangeable.
Fig. 2.
cAR1-GFP rescues the developmental defect of cAR1-null cells. The cAR1 null cell line was transformed with 1, cAR1-GFP construct; 2, wild-type cAR1; and 3, empty vector. Transformed cells were washed with DB and developed on a 35-mm
agar plate (1 × 107 cells/plate). After 36 h, pictures were taken of
each plate for the formation of fruiting bodies.
[View Larger Version of this Image (38K GIF file)]
Fig. 3.
Distribution of cAR1-GFP during chemotaxis. All figures shown were fluorescence images captured in a 50-100-millisecond exposure; phase contrast images were unnecessary since
cell shapes were obvious from the peripheral fluorescence of
cAR1-GFP. There was a 10-15 second interval between consecutive frames. Frames 1-4 show the complete sequence of one
chemotaxis event, frames 5-8 showed another. Positions of the
micropipette were indicated by "*". Arrowheads indicate pseudopods. Bar, 5 µm.
[View Larger Version of this Image (124K GIF file)]
Fig. 4.
Distribution of cAR1-GFP during cell turning after
chemoattractant source movement. Sequence shows cells turning
in response to cAMP source movement. Frame 3 is a phase-contrast image to highlight the micropipette position shift. In frames
1 and 2, asterisks indicate the original cAMP source; in all other
frames, the asterisk denotes the new position. Pseudopod positions were indicated with arrowheads. Time interval is 15 s. Bar, 5 µm.
[View Larger Version of this Image (101K GIF file)]
Fig. 5.
(A) Z-axis two-dimensional confocal fluorescence image scanning analysis of cAR1-GFP cells. Cells adhering to a
glass coverslip were fixed in 4% paraformaldehyde/0.1% Triton
X-100 and subjected to confocal analysis. Section thickness: 0.5 µm; frame interval: 1.0 µm. (B) Three-dimensional reconstruction image of cAR1-GFP cell fluorescence. All three-dimensional
images were created from a Z-axis scanning series with the Intervision 1.6 software program. The reconstructed image was rotated around the X-axis for 150° starting from the bottom to top
image (top of cell projecting into paper plane). Images were captured every 30°. Final image shows top to bottom view. Bars, 10 µm.
[View Larger Version of this Image (80K GIF file)]
). As shown in Fig. 6 A,
cells expressing either type of receptor display ~80-90%
apparent LLB after cAMP pretreatment, in good agreement with previous studies. This demonstrates, as expected from its capacity to be normally phosphorylated,
that cAR1-GFP desensitizes as efficiently as the wild-type
receptor and confirms the physiological relevance of using
the fusion construct to study this phenomenon.
Fig. 6.
(A) Cells expressing cAR1-GFP receptors display normal desensitization properties. Wild-type cAR1 cells and cAR1-GFP cells were washed in PB and treated with either buffer (control) or 105 M cAMP in the presence of 10 mM DTT for 15 min
to induce LLB. After extensive washing, the cells were assayed
for their cAMP binding (16 nM [3H]cAMP) in PB (Materials and
Methods). The binding was expressed as a percentage of the
binding obtained from buffer-treated control cells. (B) Distribution of desensitized cAR1-GFP analyzed through time-lapse fluorescence imaging. All frames are fluorescence images. Frame 1 showed the cell in random movement. cAMP was applied 5 s before frame 2 was taken. Time interval is 1 min between 2 and 4;
90 s between 4 and 6. (C) Z-axis confocal analysis of receptor distribution on desensitized cells. Cells were pretreated with excess
cAMP for 5 to 10 min to induce desensitization and then quickly
fixed with 4% paraformaldehyde/0.08% Triton X-100. Z-axis sections were taken from the bottom of the cell (substrate adhesion side) to the top surface. Section thickness: 0.5 µm; frame distance: 1.0 µm. Bars, 10 µm.
[View Larger Versions of these Images (76 + 30K GIF file)]
5 M (the effects of phosphodiesterase are negligible here since there are very few cells
plated and they are in a large volume of buffer). The cells
were monitored by time-lapse imaging before and after
cAMP addition (Fig. 6 B). Fig. 6 B, 1 shows three randomly moving cells with different fluorescence intensities.
Note the rapidly extending and retracting pseudopods. All
of the green fluorescence signals were primarily localized
to the cell surface. (Certain internal speckled light spots
were visible, but careful study revealed that these were
yellow and red autofluorescence signals, perhaps due to
mitochondrially bound NADH [Arbin, 1979]. This condition seemed especially pronounced for inactive or apoptotic cells [note the two nonfluorescent and totally inert
cells in the same field].) cAMP was applied 5 s before Fig.
6 B, 2. From Fig. 6 B, 2 to 6, ~5 min elapsed. The cells displayed a general trend of rounding up throughout the process, which is a phenomenon usually associated with cell
adaptation induced by prolonged application of uniform
stimulus. The initial pseudopods of the cells were quickly
withdrawn after the ligand application, but in the case of
the dimmer cell (Fig. 6 B, upper right cell), new ones were subsequently produced at random orientations before
they were eventually suppressed. Visual monitoring of this
process suggested little rearragement (internalization, degradation, local depletion, or concentration) of the receptor. Quantitative measurements further proved that the
fluorescence signals remained essentially unchanged on the
cell surface during this response to persistent stimulation.
5 M cAMP for 7 min and then quickly
fixed. In contrast to the previous confocal profile (Fig. 5
A), the size of the cells was smaller in the lower sections
and gradually increased in the upper sections, consistent
with the rounded shape of desensitized cells. Otherwise, this profile is similar to the previous one. The bottom
frame displayed the most internal fluorescence, indicating
upward projections of its basal membranes, and all the upper frames showed essentially uniform peripheral signals
with little internal fluorescence. This result strongly corroborates our earlier observations. Desensitization does
not lead to receptor rearrangement.
). Since cAR1-GFP maintained a uniform
distribution on cell surface under all physiological conditions, we tested whether the Con A treatment would shift
its location along with other surface proteins. As shown in
Fig. 7 A, the distribution does change drastically. Fig. 7 A,
1 and 2, shows an elongated cell involved in random movement, with a number of pseudopods. Con A (final volume
20-50 µg/ml) was added 10 s before Fig. 7 A, 2 was taken.
A detectable enrichment of signal on the bottom right corner of the cell quickly occurred (Fig. 7 A, 2, arrowhead).
Massive patching and/or capping of the receptor then took
place at three regions (Fig. 7 A, 3, arrowheads). After this,
the cell gradually rounded up, and one major capping event proceeded to completion at the bottom edge of the
cell. The patch on the opposite end persisted but did not
culminate into an equally impressive cap. During this
stage, certain regions of the membrane lost receptors, as
was evident from the depletion of fluorescence signals
(Fig. 7 A, 5 and 6, arrowheads). The rounded cells were
not able to respond to micropipettes containing cAMP. Higher Con A concentration (>0.5 mg/ml) resulted in
very inefficient patching and no subsequent capping (data
not shown), as might be expected for a multivalent ligand.
Fig. 7.
(A) Con A treatment of cAR1-GFP cells to induce receptor capping. All images are fluorescence ones. Frame 1: cell
before treatment. Frames 2-6: after application of 50 µg/ml Con
A. Con A was applied 15 s before frame 2 was taken. Time interval is 45 s. In frames 2 and 3, arrowheads indicate patching and
capping of signals. In frames 5 and 6, they show the regions devoid of signals. (B) Desensitized cells can still carry out efficient
Con A-induced receptor capping. Cells were either pretreated
with buffer (1) or 105 M cAMP (2) for 10 min to induce desensitization. Con A was then added to induce receptor capping. Images were taken after 5 min of Con A addition.
[View Larger Version of this Image (76K GIF file)]
; Espinosa-Cantellano et al., 1994), the above
result may indicate a close association between chemoattractant receptors and the cytoskeleton. We were interested in knowing whether receptor phosphorylation and
cell desensitization would affect the capacity of the receptor to be capped by Con A treatment, hence reflecting a
corresponding change of this presumptive association. Towards this end, we treated two identical sets of cells with
either buffer (Fig. 7 B, 1) or 10
5 M cAMP (Fig. 7 B, 2) for
10 min to induce receptor desensitization. Con A was then
added to both sets of cells and changes in receptor distribution were monitored. The photographs were taken after 5 min of Con A treatment. According to the previous test,
this time should be sufficient for capping to take place.
This experiment clearly demonstrated that the receptors
on completely desensitized cells were still able to be efficiently capped. Further study also indicated that the time
frames of the capping process were not significantly different between the two sets of cells (data not shown).
Discussion
; Devreotes and Zigmond, 1988
). In this model, cells
constantly extend pseudopods in various directions: those in the correct direction are more likely to persist and become part of the migrating front; those in the wrong direction are more easily suppressed. Since the receptors coat
these projections, we can speculate that receptor-mediated
sensing mechanisms are continuously involved in these decisions. As the cell thrusts a portion of its periphery into a
chemoattractant gradient to form a pseudopod, receptors
on this protrusion experience an increase in agonist occupancy leading to recruitment of further cytoskeletal and
cytoplasmic components into this region. Conversely, receptors on filopods or pseudopods extending down the
gradient or in other directions will experience a decrease
or no increase in occupancy, so no further recruitment of
cellular material into this domain would occur. These
events could bias orientations and overall directions of cell
migration.
). We do not know the cause of this expression level
polymorphism, but it was useful in analyzing the results.
We carefully observed both bright and less intense cells.
These should have been receptor populations both greater
than and less than wild-type cells. Nevertheless, the behaviors of cells with high or low receptor levels were indistinguishable (Fig. 4). This indicates that our observations are
not complicated by overexpression-caused artifacts.
). Studies have shown that the sterol content of the
plasma membrane can have a dramatic impact on sorting
and trafficking of membrane proteins. Stieger et al. (1994)
have been able to disrupt the asymmetric distributions of
various apical and basolateral membrane markers in rat
hepatocytes by altering the cholesterol content of cellular
membranes. If the high sterol content or low phospholipids level inhibits the recruitment of these domains into endocytic vesicles, the persistent presence of cAR1 on plasma membranes might be expected.
; Freedman and Lefkowitz, 1996
; Lohse et al., 1996
).
The most frequently discussed phenomena include internalization through endosomal-like structures or "sequestration" into coated or noncoated vesicles near the cell
surface. Typically, only a small fraction (15-25%) of receptors is "sequestered." It is not clear whether receptor phosphorylation is required for this process (von Zastrow
and Kobilika, 1992; Barak et al., 1994
; Roettger et al.,
1995a
). Our study indicates that cAR1 does not undergo a
significant rearrangement during desensitization, although
we cannot rule out slight changes. Most of the receptor remains at the cell surface but with dramatically reduced affinity. It has been shown definitively that phosphorylation of two serine residues on cAR1 is absolutely required for
this affinity decrease (Caterina et al., 1995
).
). Studies on the
N-formyl chemotactic peptide receptors in human neutrophils also revealed that while basal state receptors reside
on the regular plasma membrane fraction, desensitized receptors are segregated into different lateral microdomains of the plasma membrane enriched in cytoskeletal components and devoid of G proteins (Jesaitis et al., 1988
, 1989
).
In both cases, the proposed major functions for these domains are to prevent the coupling between the desensitized receptor with G proteins and facilitate the resensitization processes of receptors. However, in neither case were
these presumptive domains morphologically observable.
Received for publication 9 April 1997 and in revised form 11 July 1997.
Address all correspondence to Peter N. Devreotes, Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205. Tel.: (410) 955-3225. Fax: (410) 955-5757.We wish to thank Stephen M. Mattessich for technical help during the micropipette assay and Dr. Roger Tsien for providing the GFP S65T construct.
This work is supported by the National Institutes of Health Grant GM34933 to P.N. Devreotes.
F-actin, filamentous actin; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; LLB, loss-of-ligand binding.
1. |
Baiocchi, M.,
E. Olivetta,
C. Chelucci,
A.C. Santarcangelo,
R. Bona,
P. d'Aloja,
U. Testa,
N. Komatsu,
P. Verani, and
M. Federico.
1997.
Human immunodeficiency virus (HIV)-resistant CD4+ UT-7 megakaryocytic human cell line
becomes highly HIV-1 and HIV-2 susceptible upon CXCR4 transfection: induction of cell differentiation by HIV-1 infection.
Blood.
89:
2670-2678
|
2. |
Barak, L.S.,
M. Tiberi,
N.J. Freedman,
M.M. Kwatra,
R.J. Lefkowitz, and
M.G. Caron.
1994.
A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated ![]() |
3. | Behar, T.N., A.E. Schaffner, H.T. Tran, and J.L. Barker. 1994. Correlation of gp140trk expression and NGF-induced neuroblast chemotaxis in the embryonic rat spinal cord. Brain Res. 664: 155-166 |
4. |
Caterina, M.J.,
P.N. Devreotes,
J. Borleis, and
D. Hereld.
1995.
Agonist-
induced loss of ligand binding is correlated with phosphorylation of cAR1, a
G protein-coupled chemoattractant receptor from Dictyostelium.
J. Biol.
Chem.
270:
8667-8672
|
5. | Chen, X., J. Berrou, G. Nguyen, J.D. Sraer, and E. Rondeau. 1995. Endothelin-1 induces rapid and long lasting internalization of the thrombin receptor in human glomerular epithelial cells. Biochem. Biophys. Res. Commun. 217: 445-451 |
6. | Chen, M.Y., R.H. Insall, and P.N. Devreotes. 1996. Signaling through chemoattractant receptors in Dictyostelium. Trends Genet. 12: 52-57 |
7. | Chuang, T.T., L. Iacovelli, M. Sallese, and A. De Blasi. 1996. G protein-coupled receptors: heterologous regulation of homologous desensitization and its implications. Trends Pharmacol. Sci. 17: 416-421 |
8. | Cubitt, A.B., R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20: 448-455 |
9. | de Priester, W., A. Bakker, and G. Lamers. 1990. Capping of Con A receptors and actin distribution are influenced by disruption of microtubules in Dictyostelium discoideum. Eur. J. Cell Biol. 51: 23-32 |
10. | Devreotes, P.N.. 1994. G protein-linked signaling pathways control the developmental program of Dictyostelium. Neuron. 12: 235-241 |
11. | Devreotes, P.N., and S.H. Zigmond. 1988. Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium. Annu. Rev. Cell Biol. 4: 649-686 . |
12. | Devreotes, P.N., D. Fontana, P. Klein, J. Sherring, and A. Theibert. 1987. Transmembrane signaling in Dictyostelium. In Methods in Cell Biology. Vol. 28. J. Spudich, editor. Academic Press, Inc., New York. 299-329. |
13. | Espinosa-Cantellano, M., and A. Martinez-Palomo. 1994. Entamoeba histolytica: mechanism of surface receptor capping. Exp. Parasitol. 79: 424-435 |
14. | Fisher, P.R., R. Merkl, and G. Gerisch. 1989. Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients. J. Cell Biol. 108: 973-984 [Abstract]. |
15. | Freedman, N.J., and R.J. Lefkowitz. 1996. Desensitization of G protein-coupled receptors. Recent Prog. Horm. Res. 51: 319-351 |
16. | Gerisch, G., and H.U. Keller. 1981. Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J. Cell Sci. 52: 1-10 [Abstract]. |
17. | Gerisch, G., D. Hulser, D. Malchow, and U. Wick. 1975. Cell communication by periodic cyclic AMP pulses. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 272: 181-192 |
18. | Gerisch, G., R. Albrecht, C. Heizer, S. Hodgkinson, and M. Maniak. 1995. Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein-coronin fusion protein. Curr. Biol. 5: 1280-1285 |
19. |
Hampton, R.Y.,
A. Koning,
R. Wright, and
J. Rine.
1996.
In vivo examination
of membrane protein localization and degradation with green fluorescent
protein.
Proc. Natl. Acad. Sci. USA.
93:
828-833
|
20. | Hogan, S.P., and P.S. Foster. 1996. Cellular and molecular mechanisms involved in the regulation of eosinophil trafficking in vivo. Med. Res. Rev. 16: 407-432 |
21. |
Hunyady, L.,
M. Bor,
T. Balla, and
K.J. Catt.
1994.
Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of
the AT1 angiotensin receptor.
J. Biol. Chem.
269:
31378-31382
|
22. | Jesaitis, A.J., G.M. Bokoch, J.O. Tolley, and R.A. Allen. 1988. Lateral segregation of neutrophil chemotactic receptors into actin- and fodrin-rich plasma membrane microdomains depleted in guanyl nucleotide regulatory proteins. J. Cell Biol. 107: 921-928 [Abstract]. |
23. | Jesaitis, A.J., J.O. Tolley, G.M. Bokoch, and R.A. Allen. 1989. Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils. J. Cell Biol. 109: 2783-2790 [Abstract]. |
24. | Johnson, R.L., R.A. Vaughan, M.J. Caterina, P.J. Van Haastert, and P.N. Devreotes. 1991. Overexpression of the cAMP receptor 1 in growing Dictyostelium cells. Biochemistry. 30: 6982-6986 |
25. |
Johnson, R.L.,
P.J. Van Haastert,
A.R. Kimmel,
C.L. Saxe III,
B. Jastorff, and
P.N. Devreotes.
1992.
The cyclic nucleotide specificity of three cAMP receptors in Dictyostelium.
J. Biol. Chem.
267:
4600-4607
|
26. |
Kim, J.Y., and
P.N. Devreotes.
1994.
Random chimeragenesis of G protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium.
J. Biol. Chem.
269:
28724-28731
|
27. | Klein, P., A. Theibert, D. Fontana, and P.N. Devreotes. 1985. Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum. J. Biol. Chem. 260: 1757-1764 [Abstract]. |
28. | Lohse, M.J., S. Engelhardt, S. Danner, and M. Bohm. 1996. Mechanisms of
![]() |
29. | Maloteaux, J.M., and E. Hermans. 1994. Agonist-induced muscarinic cholinergic receptor internalization, recycling and degradation in cultured neuronal cells. Cellular mechanisms and role in desensitization. Biochem. Pharmacol. 47: 77-88 |
30. | Maniak, M., R. Rauchenberger, R. Albrecht, J. Murphy, and G. Gerisch. 1995. Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell. 83: 915-924 |
31. | Marshall, J., R. Molloy, G.W. Moss, J.R. Howe, and T.E. Hughes. 1995. The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron. 14: 211-215 |
32. | Patton, W.F., M.R. Dhanak, and B.S. Jacobson. 1990. Analysis of plasma membrane protein changes in Dictyostelium discoideum during concanavalin A induced receptor redistribution using two-dimensional gel electrophoresis. Electrophoresis. 11: 79-85 |
33. | Petty, H.R., and J.W. Francis. 1986. Polymorphonuclear leukocyte histamine receptors: occurrence in cell surface clusters and their redistribution during locomotion. Proc. Natl. Acad. Sci. USA. 83: 4332-4335 [Abstract]. |
34. | Perkins, J.P., W.P. Hausdorff, and R.J. Lefkowitz. 1991. The ![]() |
35. |
Prado, G.N.,
H. Suzuki,
N. Wilkinson,
B. Cousins, and
J. Navarro.
1996.
Role of
the C terminus of the interleukin 8 receptor in signal transduction and internalization.
J. Biol. Chem.
271:
19186-19190
|
36. | Raposo, G., I. Dunia, S. Marullo, C. Andre, J.G. Guillet, A.D. Strosberg, E.L. Benedetti, and J. Hoebeke. 1987. Redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands. Biol. Cell. 60: 117-123 |
37. | Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller. 1995a. Dual pathways of internalization of the cholecystokinin receptor. J. Cell. Biol. 128: 1029-1041 [Abstract]. |
38. | Roettger, B.F., R.U. Rentsch, E.M. Hadac, E.H. Hellen, T.P. Burghardt, and L.J. Miller. 1995b. Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell. J. Cell Biol. 130: 579-590 [Abstract]. |
39. | Sengupta, P., J.H. Chou, and C.I. Bargmann. 1996. ord-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell. 84: 899-909 |
40. | Stieger, B., P.J. Meier, and L. Landmann. 1994. Effect of obstructive cholestasis on membrane traffic and domain-specific expression of plasma membrane proteins in rat liver parenchymal cells. Hepatology. 20: 201-212 |
41. |
Strader, C.,
T. Fong,
M. Graziano, and
M. Tota.
1995.
The family of G-protein-coupled receptors.
FASEB J.
9:
745-754
|
42. | Swanson, J.A., and D.L. Taylor. 1982. Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis. Cell. 28: 225-232 |
43. |
Szekanecz, Z.,
M.R. Shah,
L.A. Harlow,
W.H. Pearce, and
A.E. Koch.
1994.
Interleukin-8 and tumor necrosis factor-![]() |
44. |
von Zastrow, M., and
B.K. Kobilka.
1992.
Ligand-regulated internalization and
recycling of human ![]() |
45. | Wang, M., P.J. Van Haastert, P.N. Devreotes, and P. Schaap. 1988. Localization of chemoattractant receptors on Dictyostelium discoideum cells during aggregation and down-regulation. Dev. Biol. 128: 72-77 |
46. | Wells, T.N., A.E. Proudfoot, C.A. Power, and M. Marsh. 1996. Chemokine receptors: the new frontier for AIDS research. Chem. Biol. 3: 603-609 . |
47. | Xiao, Z., and P.N. Devreotes. 1997. Identification of detergent-resistant plasma membrane microdomains in Dictyostelium: enrichment of signal transduction proteins. Mol. Biol. Cell. 8: 855-869 [Abstract]. |