Abstract
Gα-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Gα subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Gαi proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5×) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Gαi3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Gαi3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Gαi3 and that membrane-associated GAIP is capable of interacting with Gαi3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.
Keywords: heterotrimeric G proteins, RGS proteins, GAP
G proteins mediate a variety of cellular processes from integration of extracellular signals to cell growth and vesicular transport (1–6). G proteins are molecular switches that oscillate between an inactive form bound to GDP and an active form bound to GTP (2). Members of the recently discovered RGS (regulators of G protein signaling) family serve as GTPase-activating proteins (GAPs) (7) that increase the rate of hydrolysis of GTP bound to the heterotrimeric Gα subunit (for review, see refs. 8–10) thereby returning Gα to its inactive, GDP-bound form (11–15). All of the RGS proteins tested thus far interact with members of the Gα family [with the possible exception of Gαs (16)] through a ≈120-aa domain called the RGS domain (10).
RGS–GAIP (Gα-interacting protein), a member of the RGS family, was identified in a yeast two-hybrid screen using Gαi3 as a bait. That GAIP is a physiologic regulator of G protein-coupled signaling has been demonstrated by the finding that GAIP (as well as RGS4) is able to reverse μ-opioid receptor-induced inhibition of adenylyl cyclase and can block Gαq-mediated signaling after reconstitution into phospholipid vesicles (15).
GAIP contains three major domains: (i) the highly conserved RGS domain (amino acids 86–205) required for GAP activity (17); (ii) the N terminus (amino acids 1–85) with a cysteine string motif, most likely responsible for membrane anchoring by palmitoylation (18); and (iii) the 12-aa C terminus (amino acids 206–217). Sequence homologies have been found between the N terminus of GAIP and two other RGS proteins, RGS-Z1 (54%) and RET-RGS (49%) (19, 20), which together constitute a distinct RGS subfamily. The C terminus of GAIP is unique and is of particular interest because it interacts with a recently identified PDZ domain protein called GIPC (21).
Previously, we localized GAIP by immunocytochemistry and found it to be associated with clathrin-coated pits and vesicles (CCVs) (22). In mouse AtT-20 pituitary cells, GAIP is located on CCVs and buds near the Golgi, whereas in rat liver it is located on CCVs found close to the cell membrane. CCVs are transport vesicles involved in endocytosis and in transport of lysosomal enzymes from the trans-Golgi network to endosomes (23–28).
Based on these findings, we proposed a model whereby GAIP, located on CCVs, is spatially separated from its target Gαi3. Here we test this model by using a biochemical approach. Because of the difficulties reported by some authors in working with highly concentrated GAIP solutions in vitro (15), we performed our experiments with vesicle fractions enriched in GAIP. Hence, this study, using endogenous GAIP in a physiologically relevant environment, provides a vital link between data obtained by other investigators with pure recombinant systems and our own localization data.
MATERIALS AND METHODS
Animals, Reagents, and Antibodies.
Male rats (150–400 g) were from Harlan–Sprague–Dawley, [γ-32P]GTP and [125I]protein A were from DuPont/NEN, the chemiluminescence detection kit was from Pierce, and the 5- and 10-nm gold, goat anti-rabbit IgG conjugates were from Amersham Pharmacia. Anti-GAIP (C) raised against a C-terminal peptide (KGGPSQSSSEA) of human GAIP209–217 (22) was affinity-purified on the same peptide and used for immunocytochemistry. Anti-GAIP (N) raised against the N terminus of GAIP, His6GAIP1–79, was used for immunoblotting (diluted 1:4,000). Antibody X22 against clathrin heavy chain was purchased from Affinity BioReagents (Golden, CO) and was used at 1:250 dilution for immunoblotting.
Subcellular Fractionation.
Procedures for subcellular fractionation and for preparation of cytosol and total microsomes from rat liver were as described (22, 29) (see Fig. 1). Briefly, total microsomes were adjusted to 1.24 M sucrose and loaded at the bottom of a discontinuous sucrose gradient composed of 8 ml each of 1.18, 1.15, 0.86, and 0.25 M sucrose. The gradient was centrifuged at 82,000 × g in an SW28 rotor for 180 min at 4°C. Aliquots of each fraction were kept frozen at −80°C; protein concentrations were determined by BCA assay (Pierce) with BSA as standard. Fractions were dialyzed for 3 h in dialysis buffer [10 mM Hepes, pH 7.5/0.05% polyoxyethylene10 lauryl ether (C12E10)/1 mM DTT] before assaying for GAP activity.
CCVs were obtained from rat liver by differential centrifugation as described (30). Briefly, four livers were excised and homogenized in Mes buffer (0.1 M Mes, pH 6.5/1 mM EGTA/0.5 mM MgCl2/0.02% sodium azide) in the presence of protease inhibitors. Homogenates were centrifuged at 19,000 × g for 40 min, and the resulting postmitochondrial supernatants were centrifuged at 43,000 × g for 70 min. Pellets were resuspended in a 10 ml of Mes buffer, homogenized, diluted with an equal volume of 12.5% sucrose and 12.5% Ficoll 400, and centrifuged at 43,000 × g for 40 min at 4°C. The supernatant containing the CCVs was saved and diluted with 4–5 volumes Mes buffer, and CCVs were pelleted for 70 min at 33,000 × g at 4°C. Pellets were resuspended in Mes buffer and stored frozen in aliquots at −80°C until used.
Purification of Recombinant Gαi3 and GAIP.
Gαi3 (31) and GAIP1–217 were subcloned into pET28a (Novagen). GAIP80–206 and GAIP1–79 were cloned by PCR in pET28a. For protein expression, Escherichia coli strain BL21(DE3) was used as host. Bacteria were induced with 0.4 mM IPTG at 20°C. Bacterial pellets were resuspended in lysis buffer A (25 mM Tris, pH 8/500 mM NaCl/5 mM imidazole/1% Tween 20) and 200 μg/ml lyzozyme for GAIP constructs or in lysis buffer B (20 mM Hepes, pH 7.4/500 mM NaCl/5 mM imidazole/2 mM MgCl2/30 μM AlCl3/20 mM NaF/100 μM GDP/1% Tween 20) and 200 μg/ml lyzozyme for Gαi3. Histidine-tagged proteins were purified by affinity chromatography on Ni2+ beads (Qiagen, Chatsworth, CA). GAIP proteins were eluted with buffer A containing 250 mM imidazole and Gαi3 with buffer B containing 50 mM imidazole. Concentrations of recombinant proteins were estimated by SDS/PAGE after Coomassie staining with known amounts of BSA as standards. Proteins were dialyzed overnight in dialysis buffer, and aliquots were kept frozen at −80°C until used for GTPase assays.
Hydrolysis of Gαi3-bound [γ-32P]GTP.
Gαi3 (250 nM) was loaded with 1 μM [γ-32P]GTP (30 Ci/mmol, DuPont/NEN; 1 Ci = 37 GBq) for 40 min in 50 mM Hepes, pH 8.0/5 mM EDTA/1 mM DTT/0.05% C12E10 at 30°C, and the temperature was reduced to 4°C. Initially, Gαi3-dependent hydrolysis was determined after removing free nucleotides by size exclusion chromatography on Sephadex G50 (Amersham Pharmacia). Enrichment in GAP activity did not depend on the G50 chromatographic step, hence, this step was subsequently eliminated. All hydrolysis measurements were done in solution at 4°C under single-turnover conditions as described (7). For all fractions, the nonspecific hydrolysis of GTP (in the absence of Gαi3) was estimated to be 3% of the Gαi3-dependent hydrolysis. Reactions were started by addition of RGS mix, i.e., 15 mM MgSO4/375 μM GTP/5 μM of each GAIP peptide or different fractions (as indicated in the figures). Aliquots (50 μl) were removed at different times, and reactions were stopped by additon of 750 μl of 5% (wt/vol) charcoal slurry in 50 mM NaH2PO4 (pH 2). Zero time point was obtained by adding 30 μl of [γ-32P]GTP-loaded Gαi3 in charcoal slurry. After vortexing, 20 μl of RGS mix was added. Stopped reactions were centrifuged at 4°C for 15 min at 12,000 × g. Aliquots (400 μl) were counted by Cerenkov scintillation. No GAP activity could be detected in any fractions after heat inactivation by boiling.
Negative Staining and Immunolabeling.
Enriched vesicle fractions (CV2 or CCVs) were fixed for 10 min at room temperature in 2% formaldehyde in PBS, pH 7.4, and either adsorbed onto freshly ionized formvar/carbon-coated grids for 20 min or filtered onto 1-cm, 0.45-μm cellulose nitrate filter paper circles. Some grids were negatively stained in 2% uranyl acetate in water. Other grids and filters were washed in PBS containing 0.01 M glycine and subsequently processed like coverslips as described (32). Briefly, after blocking in PBS supplemented with 10% FCS, the grids/filters were incubated in anti-GAIP (C) IgG, (4 μg/ml) in 10% FCS (4 h to overnight), washed in PBS containing 0.01 M glycine and 5% FCS, and incubated in the secondary goat anti-rabbit 5 nm gold conjugate diluted 1:50 in 10% FCS (2 h). For double labeling, anti-GAIP (C) IgG and monoclonal anti-clathrin antibodies were mixed, and goat anti-rabbit 5-nm gold and goat anti-mouse 10-nm gold conjugates were also mixed.
After immunolabeling, grids were washed through several puddles of PBS containing 5% FCS and 0.01 M glycine and then washed and negatively stained as described above. Filters were washed in PBS containing 5% FCS and 0.01 M glycine, followed by 100 mM cacodylate buffer (pH 7.4) fixed in 1% OsO4 in 100 mM cacodylate buffer for 1 h at 4°C, treated with 1% tannic acid in 100 mM cacodylate buffer (pH 7.4) and subsequently stained enbloc in 2% uranyl acetate for 2 h. The filters were then dehydrated in ethanol, embedded in Epox, and processed for routine electron microscopy. Sections were picked up onto 300 mesh nickel grids and stained with lead citrate and uranyl acetate. Grids were observed in a JEOL 1200 EXII transmission electron microscope at 80 kV.
Immunodepletion of the CV2 Fraction.
To reduce DTT to 5 μM, 10 μg of GAIP1–217 was diluted in 10 μM Hepes (pH 7.5) and rocked overnight with 10 μl of either GAIP(C) antiserum or CALNUC antiserum as a control (33). The CV2 fraction, 0.84 mg/ml, was diluted to 0.4 mg/ml in 50 mM Tris, pH 8/150 mM NaCl and incubated in the presence of 20 μg of either anti-GAIP (C) IgG or an irrelevant anti-IgG antibody (Vector Laboratories). GAP assays were performed as described above except that 2 μg of protein was used per assay.
RESULTS
GAP Activity of GAIP1–217 and GAIP80–206 on Recombinant Gαi3.
Initially, we carried out GAP assays using recombinant proteins and confirmed that GAIP1–217 is able to increase the basal GTPase activity of [γ-32P]GTP-loaded Gαi3 (Fig. 2) in a concentration-dependent manner (7). We also confirmed earlier findings (17) that the RGS domain alone, GAIP80–206, also has GAP activity on Gαi3, whereas the N terminus, GAIP1–79, has no activity (data not shown). The curve obtained with recombinant GAIP1–217 showed that GAP activity was dose-dependent and could be inhibited by preincubation with anti-GAIP (C) serum (Fig. 2, Inset).
GAIP Is Enriched in Carrier Vesicle and Microsomal Fractions.
We previously established that the level of expression of GAIP is high in rat liver (31) and that GAIP is found in fractions containing transport vesicles (CV1 and CV2) (22). This suggested that such fractions might be suitable for testing the ability of isolated vesicles containing GAIP to stimulate GTPase activity. GAIP enrichment in well characterized Golgi and vesicular carrier fractions (29) was assessed by quantitative immunoblotting. Fig. 3A confirms that the membrane pool of GAIP is present mainly in carrier vesicle fractions (CV1, CV2) and in residual microsomes. The enrichment for GAIP in CV1, CV2, and RM fractions was 2.4-, 4.5-, and 3.9-fold, respectively, over homogenate (Table 1).
Table 1.
Fraction added | Hydrolyzed Pi, pmol | Ratio of activity | Enrichment of GAIP |
---|---|---|---|
— | 2.02 ± 0.10 | 1.25 | — |
H | 1.73 ± 0.28 | 1 | 1 |
PNS | 1.94 ± 0.11 | 1.21 | ND |
Cytosol | 1.89 ± 0.15 | 1.12 | 1.1 |
TM | 2.39 ± 0.22 | 1.42 | 2.6 |
GL | 1.69 ± 0.25 | .98 | ND |
GH | 2.25 ± 0.02 | 1.38 | ND |
CV1 | 2.51 ± 0.22 | 1.49 | 2.4 |
CV2 | 5.88 ± 0.48 | 3.49 | 4.5 |
RM | 1.63 ± 0.12 | 0.97 | 3.9 |
Pi values are given as average ±SEM (n = 3). Ratio of activity values are averages of activity of the fraction added divided by the homogenate activity. GAIP enrichment values were obtained after densitometry ([125I]protein A) for each fraction (see Fig. 3) divided by the value of homogenate.
Carrier Vesicle Fraction 2 (CV2) Increases the GTPase Activity of Gαi3.
We next tested the fractions for GAP activity. When 1 μg of each fraction as a source of RGS was added to recombinant Gαi3, we found that only the CV2 fraction increased total GTPase activity over 15 min (Fig. 3B). The average activity (average ±SEM; n = 4) expressed as pmol of phosphate hydrolyzed after 15 min was 3-fold greater for CV2 (6.01 ± 0.44) than for the homogenate (2.16 ± 0.28). The enrichment of GTPase activity in CV2 is similar to the enrichment for GAIP as assessed by Western blotting (Table 1), suggesting that GAIP may be responsible for the increased GTPase activity. No significant increase in GTPase activity over basal turnover could be detected when homogenate was added or after boiling the CV2 fraction (Fig. 3B). When CV2 was added, the time course was slower than with recombinant GAIP (Fig. 2), probably because of the low amount of GAIP contained in the fraction. Using quantitative immunoblotting with known amounts of recombinant GST-GAIP1–217 as standard, we determined that there is 800 ng of GAIP in 1 mg of CV2. By comparison, we added 1 μg of GAIP per assay when carrying out the assay with recombinant proteins, which is 1,000 times the amount of GAIP present in 1 μg of CV2 (0.8 ng). To determine the relationship between GAIP on vesicles and GAP activity, we added increasing amounts of vesicle protein to the assay. Fig. 3C shows that GAP activity measured after 2 and 5 min of hydrolysis increased in a concentration-dependent fashion in the presence of the CV2 fraction. When the amounts of GAIP in CV2 and the cytosolic fraction were equal, similar hydrolysis rates were obtained (data not shown). This result suggests that GAIP is responsible for the GAP activity and that membrane association is not required for its activity.
GAIP Is Detected on a Subpopulation of Vesicles in the CV2 Fraction by Immunogold Labeling.
To determine the distribution of GAIP within the CV2 fraction, we carried out immunogold labeling for GAIP on the isolated fraction. Fig. 4 shows that the CV2 fraction contains a mixed population of 70- to 90-nm vesicles with no obvious coats. Previous data indicate that this fraction contains a mixture of vesicles involved in transport along different intracellular pathways (i.e., transcytosis, endoplasmic reticulum to Golgi, and Golgi to cell membrane) (29). After immunogold labeling with affinity-purified anti-GAIP (C), relatively few of the vesicles (≈7–10%) were labeled.
GAIP Is Responsible for the Increased GTPase Activity of CV2.
To confirm that GAIP is responsible for the GAP activity of the CV2 fraction, we incubated CV2 in the presence of an antibody raised against the C terminus of GAIP (amino acids 209–217) or an irrelevant rabbit antibody. Fig. 5 shows that the GTP hydrolysis stimulated by CV2 is decreased after immunodepletion with anti-GAIP IgG compared with the control antibody. After 2 min of hydrolysis, GAP activity was inhibited 30% (1.15 vs. 1.7 pmol of Pi hydrolyzed). These results demonstrate that GAIP present on vesicles in CV2 fractions is at least partly responsible for the increased activity of Gαi3 measured in our assay.
GAIP Is Found on CCV by Immunoelectron Microscopy.
We reasoned that the subpopulation of vesicles in the CV2 fraction containing GAIP were most likely CCVs lacking coats because GAIP was previously localized by immunofluorescence and immunogold labeling on CCVs in hepatocytes. To extend our findings, we carried out immunolocalization and GAP assays on CCVs isolated using an established protocol (30) that maintains clathrin coats. The typical polypeptide pattern expected of CCV could be visualized after electrophoresis of these fractions and Coomassie blue staining (Fig. 6A). Clathrin heavy chain (Fig. 6B) and GAIP (Fig. 6C) were enriched in CCV fractions by immunoblotting.
By negative staining (Fig. 7A), the CCV fraction was seen to be composed of a heterogeneous population of vesicles, many with typical clathrin coats. To confirm the presence of GAIP double labeling for GAIP (5 nm gold) and clathrin heavy chain (10 nm gold) was performed on the CCV fraction followed by negative staining to simultaneously visualize clathrin coats and gold particles. Both sizes of gold particles were found in a halo surrounding these vesicles (Fig. 7 B and C), indicating the presence of clathrin and GAIP on the same vesicles. In specimens labeled for GAIP and processed for routine electron microscopy, gold particles were found on vesicles with morphologically recognizable clathrin coats (Fig. 7 D–F).
CCVs Exhibit GAP Activity for Gαi3.
We next assessed the GAP activity of the CCV fraction on Gαi3 loaded with [γ-32P]GTP. Again, a significant increase in GAP activity could be detected after addition of CCV as compared with homogenate (Fig. 8). The GAP activity was dose-dependent from 0.2 to 8 μg. Addition of 2 μg of CCVs resulted in release of 1.5 pmol of Pi after 2 min of hydrolysis. Thus, the CCV fraction had the same activity as the same amount of CV2 (1.61 pmol of Pi, Fig. 3B). These data confirm that GAP activity correlates with GAIP enrichment.
DISCUSSION
Understanding the molecular mechanisms that regulate GAIP in vivo requires an in vitro system based on components isolated by fractionation. We created such a system to study the interaction between GAIP on vesicles and recombinant Gαi3. Based on previous data obtained with the yeast two-hybrid system by one-to-one interaction (18), Gαi3 is the GAIP-interacting partner of choice for our system. Recent reports show that, in vitro, the RGS domain of GAIP serves as a GAP for Gαi3 as well as for both Gαz (17) and Gαq (15).
To validate the system, we first demonstrated that the recombinant Gαi3 we purified is functional in an in vitro GAP assay using recombinant GAIP. We further confirmed, using recombinant proteins, that the RGS domain of GAIP is responsible for its GAP activity and that the N terminus of GAIP has no activity.
We next showed that vesicles prepared using two different methods contain GAIP by immunoblotting and verified GAIP’s presence on the vesicles by immunoelectron microscopy. Using an enzymatic assay, we also provided data demonstrating that these vesicles are functional as GAPs for recombinant Gαi3. Initially, a carrier vesicle-enriched fraction, CV2, from rat liver was used because we knew that this fraction was enriched in GAIP. Subsequently, we prepared a CCV fraction. Both CV2 and CCVs were able to stimulate the GTPase activity of Gαi3.
Our results clearly indicate that GAP activity can be observed only after sufficient enrichment of GAIP-bearing vesicles. This is consistent with the finding of Wang et al. (34), who did not find any significant GAP activity in liver homogenate.
Potential problems in the interpretation of our data might come from contaminating and/or nonspecific enzymatic activity measured in our assay. CV2 fractions are known to contain Rab proteins (29), which could be responsible for the activity. However, given the slow rates of exchange of GDP for GTP for ras (koff Mg-GDP = 1.3 10−4 s−1 at 37°C) (35, 36) in the absence of any exchange factor, it seems unlikely that the hydrolysis of free radiolabeled GTP by monomeric GTPases occurs under our experimental conditions. In addition, because two different methods of fractionation were used to obtain the CV2 and CCV fractions, it is unlikely that the same irrelevant activity was measured in both cases.
Another possible complication is that GAP activity could be due to the presence of RGS proteins other than GAIP. Although no other RGS has been described in CV2 or CCV fractions to date, we cannot rule out the presence of other RGS proteins in these fractions. However, the fact that the GAP activity of the CV2 fraction was reduced by incubation with an antibody raised against the unique C terminus of GAIP provides direct evidence that GAIP is responsible for at least part of the observed GAP activity.
From the data obtained in this study, we propose a model whereby GAIP is anchored on the membrane of CCVs by its N terminus, allowing the RGS domain to be exposed and therefore able to interact with Gαi3 bound to a separate membrane. This model proposes that the Gα subunit and its GAP are separated in space and come into contact at the time of vesicle fusion. Hence, membrane-bound Gαi3 could recruit GAIP-containing vesicles to the G protein-bearing compartment, interaction thereby returning the GTP-Gαi3 to its GDP-bound form. The G protein-bearing compartment could be either the Golgi or the plasma membrane (6). Alternatively, Gαi3 and GAIP could be located on the same membrane and separate by lateral diffusion at the time of vesicle budding.
The example of RGS–GAIP in the hepatocyte might allow us to extrapolate to the role of other membrane-associated RGS proteins, such as RGS-Z1, that have common biochemical features. Both GAIP and RGS-Z1 are tightly membrane-associated, detergent extraction-resistant, hydrophobic, and have a propensity to aggregate in solution. We can speculate that RGS-Z1 might be the equivalent of GAIP in the brain, where GAIP was not detected by Northern blotting. Further characterization of the subclass of CCV-containing GAIP by using a combination of biochemical and morphological techniques should help us to understand the role of GAIP in vesicular trafficking and provide a useful tool to study the role of Gα subunits (Gαs excepted) on intracellular membranes.
Acknowledgments
This work was supported by National Institutes of Health Grants CA58689 and DK17780 to M.G.F.; T.F. was funded by la Fondation pour la Recherche Médicale (FRM) (1996) and the l’Association pour la Recherche sur le Cancer (ARC) (1997). E.E. is a graduate student in the Biomedical Sciences Graduate program and is supported by National Institutes of Health Training Grant CA67754.
ABBREVIATIONS
- CCV
clathrin-coated vesicle
- GAP
GTPase-activating protein
- RGS
regulators of G protein signaling
- GAIP
Gα-interacting protein
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