Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland 4072, Australia
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ABSTRACT |
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Proteins of the
regulators of G protein signaling (RGS) family bind to G subunits to
downregulate their signaling in a variety of systems. G
-interacting
protein (GAIP) is a mammalian RGS protein that shows high affinity for
the activated state of G
i-3, a
protein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP in
epithelial cells and to investigate its potential role in the regulation of membrane trafficking.
LLC-PK1 cells were stably transfected with a c-myc-tagged GAIP
cDNA. In the transfected and untransfected cells, GAIP was found in the
cytosol and on cell membranes. Immunogold labeling showed that
membrane-bound GAIP was localized on budding vesicles around Golgi
stacks. When an in vitro assay was used to generate vesicles from
isolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgi
vesicles. Finally, the constitutive trafficking and secretion of
sulfated proteoglycans was measured in cell lines overexpressing GAIP.
We show evidence for GAIP regulation of secretory trafficking before
the level of the trans-Golgi network
but not in post-Golgi secretion. The location and functional effects of
GAIP overlap only partially with those of
G
i-3 and suggest multiple roles for GAIP in epithelial cells.
regulators of G protein signaling family of proteins; vesicle trafficking; heterotrimeric G protein regulation
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INTRODUCTION |
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HETEROTRIMERIC G PROTEINS regulate a variety of signal transduction pathways in eukaryotic cells, such as the photoreception mechanism, hormone receptor signaling, ion channel regulation, and membrane trafficking (4, 21, 48). In recent years, G protein subunits have been localized on a variety of organelle membranes, including the endoplasmic reticulum (1), the Golgi complex (20), and endosomal structures (8). In addition, it has been demonstrated that vesicle trafficking in the secretory pathway is regulated at multiple steps by heterotrimeric G proteins (33, 42, 44, 49, 51). The signal transduction pathways regulating vesicular transport are not well defined. Effectors, receptors, and other regulators of heterotrimeric G proteins in these pathways remain to be characterized.
G-interacting protein (GAIP) is a member of the recently described
regulators of G protein signaling (RGS) family (17, 30, 46). These
proteins are able to negatively regulate heterotrimeric G protein
activity through specific binding to G
subunits. The RGS functional
prototype is Sst2 in yeast, a pheromone-sensitive agent that negatively
regulates the activity of Gpa1 (6, 16, 18). There are at least 21 RGS
proteins already cloned and, with the exception of the
cholera-sensitive subunit Gs
,
all other G
subunits bind one or more of the RGS proteins with
varying affinities (38). GAIP was identified in a yeast two-hybrid
screen using G
i-3 as the bait
(14). In vitro, GAIP binds activated forms of
G
i-3 with high affinity and
other members of the
G
i subfamily
with varying but lower affinities. GAIP and other RGS proteins have
been shown to act as GTPase-activating proteins (GAPs) for
heterotrimeric G proteins; the kinetics of this activity differ
according to the binding affinity for the interacting G
subunit (3,
7, 52). RGS proteins are characterized by a stretch of 125 amino acids
(19, 46) (the RGS domain) that interacts with the G
subunit (14) and
mediates the GTPase activity of the RGS protein (43). In addition to in
vitro experiments, this GTPase activity has been demonstrated in
defined G protein pathways in intact cells or microsomal fractions (19,
26, 39). Recently, RGS9 was identified as a highly efficient GAP for
G
t (transducin) in the retina,
where, in conjunction with the cGMP phosphodiesterase effector, it
regulates recovery of visual transduction (24).
GAIP thus has the potential to act as a powerful regulator of
Gi-3 signaling pathways. Due to
the localization of heterotrimeric G proteins on Golgi membranes and
the demonstrated involvement of
G
i-3 in vesicular transport,
the RGS protein GAIP was investigated as a possible player in the
regulation of protein trafficking. Data in two recent studies indicate
that GAIP is involved in vesicular trafficking. In colonic epithelial
cells, GAIP was demonstrated to regulate trafficking in the autophagic
sequestration pathway, and the effects of overexpressing GAIP were
consistent with its function as a GAP for
G
i-3 in this pathway (41).
Another recent report shows that, in pituitary and liver cells,
membrane-bound GAIP is localized on clathrin-coated vesicles (13). The
full inventory of trafficking pathways involving GAIP now stands to be
elucidated. The locations and roles of GAIP may vary between cell
types, as do those of G
i-3. The
specific membrane locations of GAIP and other RGS proteins might
therefore serve as a preliminary indication of their links with
specific G
subunits and their potential roles in different steps in
vesicle trafficking. In this paper, we have investigated the
distribution of GAIP on intracellular membranes in kidney epithelial
cells, its interactions with membrane-bound G
i-3, and its involvement in
G
i-3-regulated trafficking in
the secretory pathway.
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MATERIALS AND METHODS |
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Antibodies.
Polyclonal antibodies were raised in both chickens and rabbits against
human GAIP expressed as a glutathione
S-transferase (GST)
fusion protein. Animals were injected with glutathione-purified GST-GAIP protein. IgY fractions were purified from chicken egg yolks
(Eggstract, Promega, Madison, WI), and rabbit sera were affinity
purified over cyanogen bromide-activated Sepharose conjugated to the
GST-GAIP fusion protein, and then antibodies were additionally affinity
purified on thrombin-cleaved GAIP peptide columns. Antisera were tested
for reactivity and specificity by immunoblotting on GST-GAIP and on
cell extracts (see Fig. 1). An affinity-purified goat polyclonal
antibody (C-20) raised against a 20-residue GAIP peptide (197-216)
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). EC
polyclonal antibody, raised against a COOH-terminal peptide of the
Gi-3 subunit, was purchased
from DuPont (Boston, MA). A monoclonal antibody specific for the
c-myc epitope tag was purchased from
Promega. Indocarbocyanine (Cy3)-conjugated rabbit- and mouse-specific
antibodies were supplied by Jackson Immunoresearch (West Grove, PA).
AD7 monoclonal antibody, which recognizes p200/myosin II, was raised
and characterized as previously described (34). A polyclonal antibody
against the
-subunit of coatomer (
-COP) was purchased from
Sigma-Aldrich.
Cell culture and transfection. LLC-PK1 cells (pig kidney epithelial cells) were grown, passaged as confluent monolayers in DMEM containing 2% L-glutamine and 10% FCS, and transfected as previously described (5, 49). Cells were transfected with cDNA encoding human GAIP, either ligated to a c-myc expression tag (MEQKLISEEDLN) at the COOH-terminal end and cloned into a pCB7 vector (Pharmacia Biotechnology, Uppsala, Sweden) or cloned directly into a pcDNA3.1 expression vector (Invitrogen, San Diego, CA). Transfections were carried out using 10 µg cDNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate liposomal reagent (Boehringer Mannheim, Mannheim, Germany). LLC-PK1 cells transiently transfected with c-myc-GAIP, untagged GAIP, or the pcDNA3.1 vector alone were used for some experiments. Dishes of cells at 50% confluence were transfected, and duplicate dishes were screened by immunofluorescence to select dishes with 20% transfection efficiency, which is routine for this cell line. Cells were used at 24 h posttransfection for analyses. For stable expression of GAIP, transfected cells were selected using 200-500 µg/ml hygromycin B (Boehringer Mannheim) and cloned. Clones stably expressing c-myc-GAIP at levels of 2- to 20-fold were screened and selected; these were denoted GMLLC-PK1 cells. GMLLC-PK1 clones 7 and 1 were used for all experiments reported here; these clones were determined by immunoblotting with titrated antibodies and by immunofluorescence to be expressing GAIP at 5- and 20-fold over endogenous levels of GAIP, respectively.
SDS-PAGE and immunoblotting. Protein mixtures to be analyzed were separated by SDS-PAGE on 12% Laemmli denaturing gels under reducing conditions. For immunodetection, the proteins were electrotransferred onto polyvinylidene difluoride Immobilon P membrane (Millipore, Bedford, MA) and stained in 0.1% Coomassie blue (in 50% methanol and 10% acetic acid) to check protein loading. Membranes were blocked and washed in Blotto solution (20 mM Tris · HCl buffer, pH 7.5, containing 0.15 M NaCl, 0.1% Triton X-100, and 5% nonfat milk ) before an overnight incubation at 4°C in primary antibody. Bound specific antibody was detected by incubation using an alkaline phosphatase-conjugated secondary antibody (1:1,000) and the substrate 5-bromo-4-chloro-3-indolylphosphate (Sigma-Aldrich).
Immunofluorescence.
Cells grown as preconfluent monolayers on glass coverslips were washed
once in PBS (pH 7.4) before fixing in either 4% paraformaldehyde for 1 h at room temperature or in cold methanol-acetic acid (3:1) for 10 min
at 20°C. The cells were permeabilized in 0.1% Triton X-100
in PBS and then blocked in 0.5% BSA diluted in PBS. Primary antibody
incubations were carried out for 1-2 h at room temperature and
detected using a Cy3-conjugated secondary antibody. The coverslips were
mounted in 1% N-propyl gallate in
50% glycerol and viewed on an Olympus Provis AX70 microscope.
Cell fractionation. Confluent cell monolayers were scraped into homogenization buffer (10 mM Tris · HCl, pH 7.2, containing 1 mM EDTA) and left for 5 min at room temperature. The cells were homogenized in 0.5-ml lots by 20 passages through a 27.5-gauge needle and then centrifuged at 1,000 g for 10 min at 4°C. The postnuclear supernatant was centrifuged at 100,000 g for 90 min. The pellet was resuspended in homogenization buffer; this constituted the total microsomal membrane fraction. The supernatant was recentrifuged at 100,000 g for 45 min to remove membranes, and this supernatant was collected as the cytosol fraction. Protein estimations (Pierce, Rockford, IL) and SDS-PAGE analysis were carried out on all fractions.
Golgi vesicle budding assay.
A Golgi membrane fraction was prepared from homogenates of rat liver or
Madin-Darby canine kidney (MDCK) cells by density gradient
centrifugation, based on the method of Leelavathi et al. (31). Golgi
membranes were incubated in vitro with cytosol (1 mg/ml protein), 100 µM guanosine
5'-O-(3-thiotriphosphate) (GTPS), and a modified HKM buffer [in mM: 25 HEPES (pH 7.2), 20 KCl, and 2.5 Mg(CH3COO)2
(45)] containing 0.5 mM dithiothreitol and an ATP-regenerating
system (4.6 IU/ml creatine phosphokinase, 81 mM creatine phosphate, and
28.6 mM ATP) to generate budded vesicles (10). Budded vesicles and
remaining Golgi cisternae were separated by centrifugation at 17,500 g for 10 min. The vesicles in the
supernatant were then separated from the remaining cytosol by
ultracentrifugation at 100,000 g for
90 min. Fractions were analyzed by SDS-PAGE and immunoblotting using
antibodies to vesicle-associated proteins (10) and to GAIP.
Immunogold labeling.
Cells for cryosectioning were fixed in 4% paraformaldehyde-0.1%
glutaraldehyde (pH 7.4) for 1 h, scraped off the culture dish, and
pelleted in 2% gelatin. Sections (80 µm) collected on
Formvar/carbon-coated copper grids were blocked in 1% BSA in PBS and
then incubated in primary antibody for 1 h. Bound antibody was
visualized using protein A conjugated to 10-nm colloidal gold particles
(Dr. J. W. Slot, Dept. of Cell Biology, Utrecht, The Netherlands).
Sections were contrasted and embedded in 1% uranyl acetate-2%
methylcellulose on ice for 10 min and viewed in a JEOL 1010 microscope
at 80 kV. Perforated MDCK cells were prepared from monolayers plated at confluence on 24-mm Transwell filters (Corning Costar, Cambridge, MA)
(28). Briefly, the filters were incubated at 20°C for 20 min,
washed, and partially dried, and then the apical membranes were
perforated by application and removal of a nitrocellulose filter. The filters were incubated at 37°C for 15 min
in wash buffer [in mM: 25 HEPES-KOH (pH 7.2), 2.5 Mg(CH3COO)2,
50 KCH3COO, 5 EGTA, and 1.8 CaCl2] containing cytosol to
1 mg/ml, aluminum fluoride (AlF4:
50 µM AlCl3, 30 mM NaF), and an
ATP-regenerating system (1 mM creatine phosphate, 8 U/ml creatine phosphokinase, and 50 µM ATP-Na). The filters were then fixed in
0.1% glutaraldehyde-4% paraformaldehyde in 0.1 M phosphate buffer (pH
7.4) for 1 h at room temperature; strips were excised from the filter
and immunogold labeled as described above. The filter strips were then
postfixed for 1 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate
containing 0.1 g/ml sucrose, 12.5 mM CaCl2, 70 mM KCl, and 12.5 mM
MgCl2, followed by 1% osmium
tetroxide in potassium ferrocyanide for 30 min. The samples were then
stained en bloc in 0.5% uranyl acetate in 50% ethanol, dehydrated
through a graded series of ethanols, and embedded in Epon 812 resin.
Ultrathin sections were cut (Reichardt Ultracut S), contrasted in lead
acetate and uranyl acetate, and viewed as described above.
Secretion of sulfated proteoglycan.
GMLLC-PK1
(clones
1 and
7), untransfected
LLC-PK1 cells, and transiently
transfected LLC-PK1 cells were
plated at confluence on 24-mm Transwell filters (Corning Costar) and
used to measure the sulfation and secretion of
[35S]sulfate-labeled
basement membrane heparan sulfate proteoglycan (bmHSPG), as described
previously (11, 49). Transiently transfected cells with equivalent
levels of transfection were plated in triplicate wells for each
experiment. Cells were washed and preincubated in modified sulfate-free
Fischer's medium (GIBCO BRL, Grand Island, NY) and then metabolically
labeled for 3 h at 37°C using 500 µCi/ml [35S]sulfate (carrier
free; ICN Biomedical) added to the basal medium. Duplicate filters were
also labeled with
[35S]cysteine (500 µCi/ml; to measure total bmHSPG synthesis and secretion) or with
[35S]sulfate in the
presence of 1 mM -D-xyloside
to measure total 35S uptake into
glycosaminoglycans. After the labeling, the apical and basal media were
collected and the cells were scraped off the filters into RIPA buffer
(25 mM Tris · HCl, pH 7.4, containing 1% Triton
X-100, 1% deoxycholate, 0.1% SDS, and 0.15 M NaCl). The bmHSPG was
immunoprecipitated from each of the fractions, immunoprecipitates were
run on 5-15% gradient SDS-PAGE gels, and [35S]bmHSPG in
intracellular (cell layer) and secreted pools (apical or basal media)
were compared by fluorography and densitometry (11).
Immunoprecipitation of GAIP and
Gi-3.
Whole cell homogenates or total membrane fractions from
GMLLC-PK1 cells were extracted by
incubation for 10 min at room temperature in HKM buffer (30 mM HEPES,
pH 7.4, with 20 mM KCl and 5 mM magnesium acetate) containing 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Extracts were then centrifuged at 14,000 g for 10 min at 4°C to remove
unextracted material. Incubations and washes were carried out in HKM
buffer with 1% CHAPS. The supernatant was incubated overnight at
4°C with EC antibody, raised against G
i-3. Protein A-Trysacryl beads
(Pierce) resuspended in 10 mM Tris · HCl (pH 7.4)
were added to the supernatant and incubated for 1 h at room temperature
with constant mixing. After brief centrifugation, the supernatant was
collected as the depleted starting material and the beads were
collected as the immunoprecipitate. The beads were washed several times
in excess 10 mM Tris · HCl (pH 7.4), and finally
immunoprecipitated proteins were solubilized in SDS-PAGE sample buffer.
Samples were analyzed by SDS-PAGE and immunoblotted using both EC and
GAIP-specific antibodies.
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RESULTS |
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GAIP is found in membrane and cytosol cell fractions.
Affinity-purified chicken and rabbit antibodies raised against a
GST-GAIP fusion protein were shown to recognize the fusion protein and
a single, 29-kDa band, corresponding to GAIP, in cell extracts (Fig.
1); this same band was also recognized by an antibody (C-20) raised
against a COOH-terminal peptide of GAIP (Fig.
1A). The chicken and rabbit antibodies did not recognize bands corresponding to other proteins or other known RGS proteins in these extracts and
were determined to be specific for GAIP. The antibodies were used to
probe for endogenous GAIP in a number of cell lines. Cell extracts were
fractionated into total microsomal membranes and cytosol and
immunoblotted for GAIP using the chicken antibody; four of these are
shown in Fig. 1B. Endogenous GAIP was
expressed at roughly equivalent, relatively low levels in all cells
tested. In cultured epithelial cell lines, GAIP was detected
predominantly (>90%) in the cytosol fractions with much smaller
amounts in the membrane fractions. In macrophages (RAW 264.7), a band
of slightly faster mobility was recognized by the GAIP antibody in
membrane fractions. This shift in electrophoretic mobility could
reflect conformational differences between cytosolic and membrane-bound forms of GAIP due to posttranslational modifications such as
palmitoylation (12).
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Localization of GAIP in cells.
Immunofluorescence staining, carried out on a variety of cell lines and
transfected cells, showed distributions of GAIP similar to that found
by immunoblotting. GMLLC-PK1 cells
overexpressing c-myc-tagged GAIP
showed predominantly cytoplasmic staining (Fig. 2). Although a heavier concentration of
GAIP staining was often observed in the perinuclear region (Fig.
2A), no specific staining of any
intracellular membranes or organelles, or any staining of the cell
surface, was detected at this level. The more sensitive method of
immunogold labeling at the electron microscopy level also was used to
localize GAIP. Ultrathin cryosections of
GMLLC-PK1 cells showed
c-myc-GAIP labeling throughout the
cytoplasm (Fig. 3A).
Again, no concentrated immunogold labeling of plasma membrane or of
other organelles was obvious in the cryosectioned cells, perhaps due to
the abundant cytoplasmic staining. Immunogold labeling was therefore
also carried out on perforated MDCK cells from which the bulk of the
cytosolic proteins had been removed, leaving only intracellular
organelles and the proteins associated specifically with them (Fig. 3,
B-D).
Specific gold labeling of endogenous GAIP was found predominantly
adjacent to membranes, showing the effective removal of cytosolic GAIP.
Membrane labeling of GAIP was seen on a proportion of vesicles in the
vicinity of Golgi stacks and not on any other membranes or organelles
in MDCK cells. GAIP labeling was commonly on newly budded vesicles and
on the budding ends of Golgi cisternae, with only occasional labeling on Golgi cisternal membranes. The specific population of GAIP-labeled vesicles could not be identified in these samples. Some, but not all,
of the vesicles had electron-dense coats, and yet other vesicles, with
identifiable electron-dense, clathrin coats, were not labeled (Fig. 3).
In double-labeling experiments, GAIP-labeled vesicles were not
colabeled for p200/myosin II, which is found on a population of
trans-Golgi network (TGN)-derived
vesicles (27) (data not shown). The low level of GAIP labeling in these
sections was consistent with the relatively small amount of
membrane-bound endogenous GAIP detected by immunoblotting in MDCK cell
fractions. In all, immunostaining showed a diffuse distribution of GAIP
throughout the cytoplasm and specific labeling of membrane-bound GAIP
on a population of Golgi-derived vesicles.
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Fractionation of Golgi vesicles from rat liver and MDCK cells.
Density gradient centrifugation was used to further study the
association of GAIP with Golgi-derived vesicles. Rat liver Golgi membranes and cytosol from rat liver homogenates and MDCK cells were
used to reconstitute in vitro vesicle budding in the presence of
GTPS (10). The distribution of GAIP was compared with other peripheral, vesicle-associated proteins, p200/myosin II and
-COP, which are known to bind to distinct populations of Golgi-derived vesicles under these conditions (10, 35). In this assay,
-COP and
p200/myosin II derived from the cytosol bind to vesicles in a
GTP-dependent fashion (Fig. 4,
lane
3). Most of the GAIP was also
initially found in the cytosol; it was not detected in the freshly
isolated stacked-Golgi membrane fraction (Fig. 4,
lane 2). After incubation, GAIP was
enriched in budded vesicle fractions (lanes
3 and
6) but was not present in
significant amounts on remnant Golgi cisternae
(lane
4). This pattern suggests that GAIP binds to vesicles during budding and is not retained on cisternal membranes. These results are consistent with the immunogold labeling in
showing that GAIP is found primarily on Golgi-derived vesicles and that
little, if any, GAIP appears to reside on Golgi cisternae. These data
also highlight the temporal segregation of GAIP and G
i-3. As can be seen in Fig. 4,
all of the G
i-3 is membrane bound, and it resides on the Golgi cisternal membranes
(lanes 2 and
4). After budding, some of the
G
i-3 fractionates with the vesicles (lane
6), and this is the only site where
GAIP and G
i-3 may be colocated.
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Membrane-bound GAIP associates with activated
Gi-3.
The partial overlap of G
i-3 (on
Golgi membranes and vesicles) and GAIP (in the cytosol and on vesicles)
raises the question of whether or not these two proteins do interact in
cells. In assays using purified components, RGS proteins have been
shown to bind specifically to G
subunits and with highest affinity to a transitionally activated state of G
, induced by the addition of
AlF
4 and GDP in the presence of
Mg2+ (2). We tested the ability of
c-myc-GAIP expressed in
GMLLC-PK1 cells to bind to
G
i-3, which is found on the
Golgi membranes in these cells (49).
G
i-3 was immunoprecipitated
from both whole cell extracts and membrane fractions of
GMLLC-PK1 cells in buffers
containing combinations of Mg2+,
AlF
4, and GDP (Fig.
5). The 41-kDa
G
i-3 was immunoprecipitated
under all conditions from these samples. Immunoprecipitates were
analyzed by immunoblotting to detect coprecipitated GAIP. Only small
amounts of c-myc-GAIP were
coprecipitated with G
i-3 from
untreated cell extracts or in the presence of added
Mg2+ and
AlF
4 alone. The coprecipitation of
GAIP with G
i-3 was
substantially enhanced in the presence of both AlF
4 and GDP, suggesting a
specific and conformation-dependent interaction between these two
proteins (Fig. 5A). To decipher whether this bound GAIP was derived primarily from the cytosol or from
the smaller membrane-associated GAIP pool,
G
i-3 was immunoprecipitated from separate extracts of membranes or whole cell lysates (Fig. 5B). Quantitation of coprecipitated
proteins revealed that G
i-3 bound a higher proportion of GAIP from its membrane-bound pool (~30%) than of GAIP from the whole cell extract (~5%), which is mostly cytosolic. These results suggest that, in cells, membrane-bound GAIP can bind to the transitional activation state of
G
i-3 and that GAIP and
G
i-3 are unlikely to
sustain this interaction throughout the whole GTP-GDP
cycle.
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Overexpression of GAIP retards constitutive secretory trafficking.
We and others have previously measured the constitutive trafficking and
secretion of
[35S]sulfate-labeled
bmHSPG to assay regulatory effects of G subunits on vesicular
trafficking (5, 33, 49). Proteoglycans, such as bmHSPG, are heavily
sulfated at the level of the TGN. In
LLC-PK1 cells, we have shown that
the intracellular trafficking of bmHSPG can be retarded at pre- and
post-TGN steps by overexpression of G
i-3 (5, 49). A
similar assay was used in this study to investigate a functional role
for GAIP by measuring the effects of GAIP overexpression on
constitutive trafficking in
GMLLC-PK1 cells. Untransfected
LLC-PK1 cells,
GMLLC-PK1 cells, and
LLC-PK1 cells transiently
transfected with vector only were labeled with [35S]sulfate;
radiolabeled bmHSPG precursors in cell extracts and secreted bmHSPG,
collected from the apical and basal media, were analyzed by SDS-PAGE,
fluorography, and densitometry (Fig. 6). Compared with control LLC-PK1
cells and those transfected with vector alone (pcDNA3.1), all
GMLLC-PK1 cells produced
significantly lower amounts of sulfated bmHSPG (Fig. 6), although
equivalent levels of early bmHSPG precursors were produced in all
cell lines, as determined by
[35S]cysteine
labeling of bmHSPG (data not shown). Overexpression of GAIP
in stably transfected GMLLC-PK1
cells reduced
[35S]sulfate bmHSPG in
a dose-dependent manner; two- to nine-fold less
[35S]sulfate bmHSPG
was present in transfected cells than in the controls. Because
[35S]sulfate is added
to the bmHSPG at the level of the TGN, these results imply
that overexpression of GAIP significantly retards delivery of bmHSPG
precursors to the TGN, i.e., that trafficking in earlier steps of the
secretory pathway is retarded. Sulfation function in
GMLLC-PK1 cells was tested in the
presence of
-D-xyloside acceptor and was found not to be compromised by GAIP overexpression (data not shown). GAIP had little or no detectable effect on post-TGN secretion of
[35S]sulfate-labeled
bmHSPG into the basal medium of
GMLLC-PK1 cells. The proportion of
[35S]bmHSPG in cell
layers and in medium was the same in
GMLLC-PK1 cells and control cells.
Also, the polarity of bmHSPG secretion (90% basal; 10% apical) was
unchanged in GMLLC-PK1 cells.
Together these data suggest that overexpression of GAIP does not affect G
i-3-regulated post-TGN
constitutive secretion of bmHSPG. However, the data are consistent with
overexpressed GAIP playing a significant role in Golgi trafficking
before the level of the TGN.
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DISCUSSION |
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The intracellular distribution of GAIP and its potential participation
in protein trafficking were investigated in kidney epithelial cells.
Our current studies on endogenous GAIP in a variety of cells and on
recombinant GAIP in transfected
LLC-PK1 cells indicate that GAIP
exists in both membrane-bound and cytosolic pools. The majority of
endogenous GAIP was found as a soluble pool in the cytoplasm, and
relatively small amounts were found on membranes. In our hands,
transfected cells expressed proportionately more (10-30% more)
membrane-bound GAIP than untransfected cells, suggesting that membrane
binding sites for GAIP are not saturated at steady state. This may lead
to, and be reflected in, differences in the relative levels of
membrane-bound and cytosolic GAIP measured at different times. De Vries
et al. (12) showed variable amounts of membrane-bound GAIP in different
cell lines; only 30% of overexpressed GAIP was in membrane fractions
in transfected COS cells, whereas >80% of GAIP in AtT20 cells was in
the membrane fraction, which coincided with heavy and predominant
labeling of GAIP on membranes in pituitary cells (13). The differences
observed, between this and previous studies (12, 13), in the relative
distribution of GAIP between membrane and cytosol might reflect
differences in the cell types used or be due to cycling of GAIP between
the two pools. The relationship between pools of membrane-bound and cytosolic GAIP remains unclear at this stage. GAIP has been shown to be
modified for membrane attachment through palmitoylation at a number of
cysteine residues near the NH2
terminus (12), and palmitoylation in other proteins, such as
Gs subunits, provides a basis
for their transient and regulated attachment to membranes (32). GAIP
may move dynamically between the two sites, as is suggested by the
GTP-dependent attachment of GAIP to Golgi membranes in the vesicle
budding assay.
In kidney epithelial cell lines, immunolocalization and cell
fractionation studies revealed that GAIP is attached to vesicles associated with the Golgi complex. Labeling of the relatively small
pool of membrane-bound GAIP cannot be distinguished at the immunofluorescence level or by immunogold labeling on transfected cells, where staining of the abundant cytosolic GAIP dominated. Membrane-bound GAIP was localized by immunogold labeling in perforated MDCK cells. The use of perforated MDCK cells has proven useful, and
sometimes uniquely successful, for immunogold labeling of peripheral
proteins bound to the cytoplasmic face of Golgi membranes or vesicle
membranes (22, 27, 28). Labeling of membrane-bound GAIP was restricted
to a subpopulation of vesicles or budding membrane structures around
the Golgi stack. This distribution indicates that GAIP associates
selectively with only some of the budding vesicles; similar
distributions (on selected subpopulations of vesicles) are seen for
other vesicle-associated proteins, such as p200/myosin II, -COP, and
-adaptin, in these preparations. Further characterization of these
vesicle populations is needed to identify the particular vesicles
binding GAIP in MDCK cells. De Vries et al. (13) have shown
localization of GAIP on clathrin-coated vesicles, both those budding
from the Golgi and from the plasma membrane, in pituitary cells and
liver cells. The vesicles labeled here in MDCK cells could include
Golgi-derived clathrin-coated vesicles, although not all labeled
vesicles had recognizable coats and no plasma membrane-derived vesicles
were labeled in perforated cells. The paucity of labeling on Golgi
cisternae, together with the very small amount of GAIP detected in rat
liver Golgi cisternae fractions (Fig. 4), suggests that GAIP associates
with the vesicle membranes only during or after budding and that it
does not reside on Golgi membranes.
Just how the localization of GAIP on these Golgi vesicles relates to
its interaction with Gi-3 is
not clear. The presence of both GAIP and
G
i-3 on Golgi-associated
membranes hints at a Golgi-specific functional link between the two
proteins. G
i-3 is found on the
Golgi membranes of many cell types by immunofluorescence labeling
(reviewed in Ref. 50); in LLC-PK1
cells, G
i-3 is exclusively located on Golgi membranes (20, 49). At an ultrastructural level,
G
i-3 appears across the Golgi
stack in LLC-PK1 cells (49), and
in exocrine pancreatic cells
G
i-3 is also found from
cis- to
trans-Golgi and on vesicles at both
sides of the Golgi stack by immunogold labeling (15). Studies in
several cell types show that
G
i-3 regulates multiple pre-
and post-Golgi trafficking steps, suggesting that it may function at
multiple and variable sites across the Golgi stacks of different cells
(5, 33, 49, 53). Current and previous data (13) showing that
membrane-bound GAIP is restricted to budded vesicles seemingly limit
the overlapping locations of
G
i-3 and GAIP, and their
potential to interact, within the Golgi milieu. Coimmunoprecipitation
of GAIP with G
i-3 confirmed
that indeed the two proteins do, at some time, interact. The binding of
GAIP to Golgi vesicle membranes does not apparently depend on
G
i-3 interaction, since
vesicles are produced in the presence of GTP
S and GAIP does not bind
with high affinity to G
i-3-GTP
S (Fig. 5) (2). Any
interaction of G
i-3 on Golgi membranes with GAIP on vesicle membranes would be most likely to occur
during the process of vesicle budding.
Studies using fusion proteins (3, 14, 52) have previously demonstrated
high-affinity binding of GAIP to the
Gi-3 subunit, and one very
recent study has now confirmed that recombinant GAIP binds to
G
i-3 in intestinal cells (41).
We also showed that c-myc-GAIP in
extracts of overexpressing
GMLLC-PK1 cells is coprecipitated with G
i-3. Interaction occurred
with highest affinity in the presence of
AlF
4 and GDP, which emulates the transitional state on the G
subunit. The GAP function of RGS proteins has been attributed to the ability of these proteins to
interact with, and stabilize, this transitional form (2). Our
experiments additionally show that the membrane-bound form of GAIP, and
not that in the cytosol, accounts for the majority of binding to
G
i-3. Soluble forms of GAIP and
G
i-3 must be able to interact,
since their binding in solution has been demonstrated and GAIP was
originally identified as a soluble binding partner for
G
i-3 in the yeast two-hybrid
system (14). It is likely that in intact cells
GAIP-G
i-3 interactions do occur
at the membrane, since G
i-3 in
cells is always tightly bound to membranes (5). In the context of
intact cells, it is also possible that intervening protein interactions
might either enhance or prevent
G
i-3 interactions with
membrane-bound or cytosolic pools of GAIP.
Gi-3 has been demonstrated to
regulate protein trafficking in a variety of pathways (5, 33, 40, 41,
49, 53). We have previously shown that in
LLC-PK1 cells overexpression of
G
i-3 subunits downregulates
polarized (basolateral) constitutive secretion (49). The assay used for
these studies measures the trafficking of precursors and the secretion
of terminally sulfated bmHSPG from polarized
LLC-PK1 cells. This same assay was
applied here to cells overexpressing GAIP. GAIP overexpression reduced the amount of
[35S]sulfate-labeled
bmHSPG in cells, although levels of
[35S]cysteine-labeled
bmHSPG precursors were unchanged, suggesting that trafficking of bmHSPG
before the level of the TGN, where sulfation occurs, was retarded. GAIP
overexpression, even at high levels, did not alter the kinetics of
post-TGN secretion of bmHSPG. GAIP is thus implicated in regulating
constitutive trafficking early in the secretory pathway. This
correlates with one of the stages of trafficking affected by
overexpression of G
i-3, which retards trafficking of bmHSPG precursors in the pre-TGN Golgi (by
~3-fold). Overexpression of
G
i-3 also significantly reduces post-TGN secretion of
[35S]sulfate-labeled
bmHSPG (5, 49), a step that is not affected by GAIP. Thus GAIP appears
to function in only one of the steps of constitutive trafficking
similarly regulated by G
i-3.
The known G protein-coupled functions for GAIP, to date, are as a GAP
and as a potential effector antagonist (25, 54). In the current assay,
GAIP did not downregulate the
G
i-3 response, as would be
expected for GAP activity. The retardation of secretory trafficking
caused by GAIP in LLC-PK1 cells
either masks a G
i-3-GAP activity of GAIP or reflects an additional or alternative function for
GAIP in this trafficking pathway. Recently, GAIP functioning as a GAP
was demonstrated in another trafficking pathway. GAIP overexpression
downregulated the G
i-3 response
in an autophagic sequestration pathway in colonic epithelial cells
(41). This study also found that GAIP mRNA expression levels were
altered during differentiation and were regulated by
G
i-3 activity, suggesting that,
in this pathway, GAIP is a highly reactive modulator of G protein
signaling (41). The precise requirements for GAPs and other modulating
proteins to downregulate G
i
signaling in the constitutive secretory pathway has not yet been well
established, since the roles of receptor activation and G protein
cycling in this system are not known.
It is possible that GAIP has more than one function in vesicular
trafficking. This is supported by our finding that GAIP binds only to
the transitionally active state of
Gi-3 and that only a small
proportion of the total GAIP (membrane-bound pool only) interacts with
G
i-3 in these cells. This
leaves open the possibility that the vast majority of overexpressed,
cytosolic GAIP performs additional functions. RGS proteins are known to
have diverse structural features. The presence of a COOH-terminal
cysteine string motif (12) potentially links GAIP to the family of
cysteine string proteins that are found on secretory vesicles and
granules, although the function of this protein family is not yet known
(9, 23, 29). RGS3 has an
NH2-terminal coiled-coil domain
(19), whereas RGS12 has a COOH-terminal coiled-coil domain that has
been proposed as a potential site for cytoskeletal interactions (47).
Binding of RGS proteins to Switch I/II regions of G
supports their
function as effector antagonists for some, but not all, effectors of
G
subunits (36, 37) The data reported here demonstrate the
localization of GAIP on Golgi-derived vesicles in epithelial cells.
Functional data are consistent with the regulatory participation of
GAIP in early stages of the constitutive secretory pathway, but GAIP does not appear to modulate
G
i-3-regulated post-Golgi
secretion. Future mutational analyses of functional domains in GAIP
will ultimately reveal the extent and diversity of its regulatory roles in protein trafficking pathways.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Brandon Sullivan and Dennis Ausiello (Massachusetts General Hospital, Harvard Medical School) for providing the GAIP cDNA constructs and for helpful discussions and comments on the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by a grant from the National Health and Medical Research Council of Australia (to J. L. Stow).
J. L. Stow is a Wellcome Trust Senior Research Fellow.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. L. Stow, Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, QLD 4072, Australia.
Received 16 July 1998; accepted in final form 2 November 1998.
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