Activation of Gαi at the Golgi by GIV/Girdin imposes finiteness in Arf1 signaling

Abstract

A long-held tenet of heterotrimeric G protein signal transduction is that it is triggered by G protein-coupled receptors (GPCRs) at the PM. Here, we demonstrate that Gi is activated in the Golgi by GIV/Girdin, a non-receptor guanine-nucleotide exchange factor (GEF). GIV-dependent activation of Gi at the Golgi maintains the finiteness of the cyclical activation of ADP-ribosylation factor 1 (Arf1), a fundamental step in vesicle traffic in all eukaryotes. Several interactions with other major components of Golgi trafficking-e.g., active Arf1, its regulator, ArfGAP2/3, and the adaptor protein β-COP-enable GIV to coordinately regulate Arf1 signaling. When the GIV-Gαi pathway is selectively inhibited, levels of GTP-bound Arf1 are elevated and protein transport along the secretory pathway is delayed. These findings define a paradigm in non-canonical G protein signaling at the Golgi, which places GIV-GEF at the crossroads between signals gated by the trimeric G proteins and the Arf family of monomeric GTPases.

Figure 1. GIV activates Gαi at the Golgi via its GEF motif

(A) COS7 cells transfected with YFP tagged Gαi3-WT or the Gαi3-WF mutant were analyzed for interaction between endogenous GIV and Gαi3-YFP by in situ PLA using mouse anti-GFP and rabbit anti-GIV antibodies. Red spots indicate the presence of interaction. Insets show the Golgi region at higher magnification (white dashed box). Bar = 10 μm. (B) Schematic for the Gαi1-YFP and Gβ1-CFP constructs used as paired FRET probes in D, E. FRET indicates the presence of inactive trimer, whereas activation of Gi is associated with loss of FRET. (C) COS7 cells expressing Gαi1-YFP (pseudo-colored green) and Man II-CFP (pseudo-colored red) were fixed and analyzed by confocal microscopy. A high degree of colocalization (yellow pixels) indicates that the Gαi1-YFP predominantly localizes on the Golgi. (D, E) Control (Scr shRNA) and GIV-depleted (GIV shRNA) COS7 cells (See also Fig. S1C) were cotransfected with Gαi1-YFP, Gβ1-CFP and Gγ2 (untagged) and live cells were analyzed by FRET imaging at steady-state, in the presence of 10% serum. Representative freeze-frame YFP, CFP and FRET images are shown. FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel. (F) Bar graphs display FRET efficiency (Y axis). FRET at the Golgi is strong in GIV-depleted cells (E; FRET efficiency = 0.37 ± 0.06), but minimal in controls (D; FRET efficiency = 0.061 ± 0.01). Results are expressed as mean ± S.D. Data represent 5 regions of interest (ROIs) analyzed over the pixels corresponding to the Golgi of 3–5 cells from 5 independent experiments. (G) Control (Scr siRNA) and GIV-depleted (GIV siRNA) COS7 cells (see also Fig. S1D) maintained in 10% serum were fixed and stained for active Gαi (green; anti-Gαi:GTP mAb) and Man II (red) and analyzed by confocal microscopy. Activation of Gαi was detected frequently in control but not in GIV-depleted cells. When detected, active Gαi colocalizes with Man II (yellow pixels in upper panel). (H) Bar graph displays % cells that stain positive for active Gαi (Y axis) in control and GIV-depleted cells analyzed in G. (I) Active Gαi is detected frequently in cells expressing GIV-WT (upper) but not in those expressing GIV-FA (lower). When detected, active Gαi colocalizes with GIV-HA (yellow pixels in merged panel). COS7 cells expressing HA tagged GIV-WT or GIV-FA were fixed and stained for GIV (red; anti-HA mAb) and active Gαi (green; anti-Gαi:GTP mAb) and analyzed by confocal microscopy. (J) Bar graph displays % cells expressing GIV-HA (WT or FA) that stain positive for active Gαi (Y axis) in I.

Figure 2. GIV associates with COPI vesicles and is required for uncoating of COPI vesicles

(A) COPI membranes were immunoisolated from a crude membrane (100,000 g pellet; see Fig. S2A) fraction prepared from COS7 cells using mAb CM1A10 (which recognizes coatomer), and bound immune complexes were analyzed for GIV, β-COP, Gαi3, and transferrin recepter (TfR, a negative control) by immunoblotting (IB). (B) HeLa cells expressing GalT-YFP (Golgi marker, pseudocolored green) were fixed and analyzed for interactions between GIV and β-COP by in situ PLA (red). Nuclei = DAPI (blue). Bar = 10 μm. Negative control (S2B) showed no signal. (C) HeLa cells expressing GIV-FLAG were treated or not with 5 μg/mL BFA for 30 min prior to lysis. Equal aliquots of lysates were immunoprecipitated with anti-FLAG mAb and immune complexes were analyzed for β-COP by immunoblotting. (D) Membrane (P, 100,000 g pellet) and cytosolic (S, 100,000 g supernatant) fractions (left panel) were prepared from post-nuclear supernatants (PNS, right panel) of control (Ctrl siRNA) or GIV-depleted (GIV siRNA) COS7 cells and analyzed for the indicated proteins by immunoblotting. (E) Bar graphs display the relative abundance of β-COP in membrane fractions in D, derived from the equation [(P)/(S + P)] × 100. Data was normalized to control and expressed as % changes. Results are expressed as mean ± S.E.M. (F) GIV-depleted cells were treated with adenovirus containing RNAi-resistant GIV-WT or GIV-FA. Homogenates of these cells were used to prepare membrane and cytosolic fractions as in D and analyzed by immunoblotting. (G) Bar graphs display the relative abundance of β-COP in membrane fractions in F. (H) Control (Ctrl siRNA) or GIV-depleted (GIV siRNA) HeLa cells were prepermeabilized with 0.1% Saponin (to release cytosolic β-COP), stained for β-COP (green) and the nucleus (DAPI, blue), and analyzed by confocal microscopy. Bar = 10 μm. (I) Bar graphs display the number of COPI vesicles per cell in H, as determined by 3D-reconstruction using Imaris software (n=3; mean ± SEM; >20 cells/experiment). (J) Bar graphs display the number of COPI vesicles in adeno-GIV-WT-HA or -GIV-FA-HA infected cells (see Fig. S2F) that were analyzed exactly as in H, I (n=3; mean ± SEM; >10 cells/experiment/condition). (K) Whole cell lysates (WCL) of control (Scr shRNA) or GIV-depleted (GIV shRNA) cells were subjected to differential centrifugation to obtain a 15,000 g (P2) fraction enriched in Golgi membranes and a 100,000 g (P3) fraction containing COPI vesicles, and a cytosolic fraction (S, 100,000 g supernatant). Fractions were analyzed for GIV, β-COP and GM130 (Golgi marker) by immunoblotting. (L) Bar graphs display the quantification of β-COP in P3 fractions normalized to WCL. Increased membrane-association of β-COP as determined by membrane-cytosol fractionation or increased peripheral vesicular staining of β-COP we observe in GIV-depleted cells (2D, E, H, I) or in cells expressing GIV-FA (2F, G, J) is not due to changes in absolute cellular levels of β-COP (see S2G, H).

Figure 3. Activation of Gαi by GIV terminates Arf1 signaling

(A) Depletion of GIV promotes accumulation of COPI vesicles in the ER-Golgi intermediate compartment (ERGIC). Left: Control (Ctrl siRNA; upper panel) or GIV-depleted (GIV siRNA; lower panel) HeLa cells were fixed and stained for β-COP (red) and ERGIC-53 (green). Merged panels display the overlay of β-COP (red) and ERGIC-53 (green) panels. Insets on merge panels viewed at higher magnification shows limited colocalization of ERGIC-53 with β-COP in the cis-Golgi in control cells and that ERGIC-53 is also found in vesicles scattered throughout the cytoplasm which do not stain for β-COP. In GIV-depleted cells there is increased colocalization between β-COP and ERGIC-53 and that these vesicular structures that co-stain for both β-COP and ERGIC-53 are more dispersed. The white line in the insets indicates the pixels used for the RGB profile plots shown (see Experimental Procedures). Bar = 10 μm. Right: RGB profiles of ERGIC-53 (green) and β-COP (red) corresponding to lines in merge of (A) as shown in insets. (B) COS7 cells were treated with control (−) or GIV siRNA (+), and adenoviral vectors expressing siRNA-resistant GIV-WT or GIV-FA as indicated. Equal aliquots of lysates (bottom panels, 5% input) were analylzed for Arf1•GTP by carrying out GST pulldown assays with GST-GGA3. Bound proteins (top panel) were analyzed for active Arf1 by immunoblotting. (C) Bar graphs display the band densitometry quantification of Arf1•GTP (bound)/total Arf1 (inputs) in B. Data was normalized to control and expressed as % changes. Results are expressed as mean ± S.E.M. (D) COS7 cells expressing Gαi3-WT, Gαi3-WF mutant, or control vector were analyzed for levels of Arf1•GTP as in B. (E) Bar graphs display the band densitometry quantification of Arf1•GTP (bound)/total Arf1 (inputs) in D. Results are expressed as mean ± S.E.M.

Figure 4. GIV and its GEF function are required for ER-Golgi vesicle transport and integrity of the Golgi structure

(A) Control (Ctrl siRNA) or GIV-depleted (GIV siRNA) COS7 cells were transfected with VSVG-tsO45-GFP and incubated at 40°C overnight before shifting to 32°C for the indicated times. Cell surface proteins were labeled with membrane-impermeable Sulfo-NHS-SS-Biotin as described in Methods. Surface biotinylated and total VSVG-GFP were analyzed by immunoblotting with anti-GFP. (B) Graphs display the quantification of VSVG trafficking in control or GIV-depleted cells expressed as surface biotinylated to total VSVG-GFP, as determined by band densitometry. Data was normalized to control and expressed as % changes. (n=3; error bars = S.E.M). (C) Graphs display the quantification of VSVG trafficking (see Figure S4A) in cells expressing GIV-WT or GIV-FA calculated as in B (n=4; error bars = S.E.M). (D) Control (Ctrl siRNA) and GIV-depleted (GIV siRNA) HeLa cells transfected with myc-cochlin were pulsed with [³⁵S]Met-Cys (100 mCi/mL) for 30 min, washed, and chased for the indicated times prior to lysis. Equal aliquots of media and cell lysates were immunoprecipitated with anti-myc mAb and analyzed for [³⁵S]-labeled-cochlin by autoradiography. (E) Graphs display the quantification of results in D, as determined by band densitometry and expressed as % myc-cochlin secreted in media/total myc-cochlin in lysates (n=5). (F) Graphs display the quantification of myc-cochlin secretion from HeLa cells stably expressing GIV-WT or GIV-FA (see Figure S4B) expressed as % myc-cochlin secreted in media/total myc-cochlin in lysates (n=5). (G) Control (Ctrl siRNA) or GIV-depleted (GIV siRNA) COS7 cells were infected with VSVG-tsO45 retrovirus for 1 h at 32°C and shifted to 40°C for the next 16 h. Cells were then shifted to 32°C for the indicated period of time prior to lysis. Equal aliquots of lysates were incubated with Endo-H (+) and subsequently analyzed for Endo-H-resistance (upward shift, upper panel) and GIV depletion (lower panel) by immunoblotting using anti-VSV and anti-GIV, respectively. (H) Graphs display the quantification of the data presented in G, expressed as the ratio between Endo-H-resistant (VSVG^r; upper band) vs Endo-H-sensitive VGV-G (VSVG^s; lower band) at various time points (n=4). (I) Graphs display the ratio between Endo-H-resistant vs sensitive VGV-G in cells expressing GIV-WT or GIV-FA (n=3) (see Figure S4E). (J) Control (Ctrl siRNA) or GIV-depleted (GIV siRNA) COS7 cells transfected with chimeric tsO45-VSVG-KDELr-Myc were first incubated at 32°C and then shifted to 40°C (to allow transport of the chimeric receptor to the ER) for the indicated times, fixed and stained for myc (white pixels). Bar = 10 μm. (K) Bar graphs display the % of the total tsO45-VSVG-KDELr-Myc within the Golgi region, quantified from confocal images of cells at 30 min (see Methods for details) (n=3; 5 cells/experiment). (L) Control (Ctrl siRNA) or GIV-depleted (GIV siRNA) COS7 cells were fixed and stained for Man II (red) and nuclei (DAPI; blue), and analyzed by confocal microscopy. Representative images of control or GIV-depleted cells are shown with a magnified view of the boxed area enlarged to the right. Bar = 10 μm. (M) Quantitative analysis of the Golgi phenotype in control and GIV-depleted cells by Image J (NIH) is shown (left panels). A fixed threshold was applied to all images, and objects were measured using the Analyze Particles function. Bar graphs (right) display the average number of Golgi elements per cell (n=3; >14 cells/experiment).

Figure 5. GIV terminates Arf1 by targeting ArfGAP3 to COPI vesicles and releasing free Gβγ dimers

(A) Lysates of COS7 cells coexpressing pCEFL-GST-GIV or GST alone (pCEFL-GST) and either ArfGAP1-myc (lanes 1 and 4) or ArfGAP3-myc (lanes 2 and 5) were incubated with glutathione-Sepharose beads and bound proteins were analyzed by immmunoblotting. (B) COS7 cells co-transfected with β-GalT-YFP (Golgi marker) and either ArfGAP1-Myc or ArfGAP3-Myc were analyzed for interaction between GIV and ArfGAPs by in situ PLA using rabbit anti-GIV and mouse-anti-myc antibodies. Similar distribution and expression of ArfGAPs 1 and 3 was verified by immunofluorescence (see Fig. S5C). Incubation with primary antibodies was excluded for negative controls. Red dots = sites of interaction; Bar =10 μm. (C) Immunoprecipitation was carried out on lysates from control (−) or GIV-depleted (GIV siRNA +) cells expressing ArfGAP3-myc using the CM1A10 (anti-coatomer) antibody. Inputs and immunoprecipitates were analyzed by immunoblotting. Band densitometry confirmed a ~60% reduction in ArfGAP3 in immune complexes from GIV-depleted cells. (D) Control (Ctrl siRNA) or GIV-depleted (GIV siRNA) COS7 cells cotransfected with β-GalT-YFP (Golgi marker) and ArfGAP3-Myc were analyzed for interaction between β-COP and ArfGAP3-Myc by in situ PLA. Incubation with primary antibodies was excluded for negative controls. Red dot = sites of interaction; Bar = 10 μm. Depletion of GIV and equal transfection of ArfGAP3-Myc were confirmed by immunoblotting (IB; inset). (E) Bar graphs display the quantification of interactions (n=3; >20 cells/experiment) in D. (F) Lysates of COS7 cells expressing GIV-WT, βARK-CT and control vector (−) as indicated were analyzed for Arf1•GTP as in 3B. (G) Bar graphs display the quantification of the data in F, expressed as the ratio of Arf1•GTP to total Arf1. Results were presented as mean ± S.E.M (n = 5).

Figure 6. Active Arf1 binds GIV and triggers its membrane association

(A) Membrane-cytosol fractionations were carried out on COS7 cells expressing HA-tagged wild-type (WT), constitutively active (Q71L) or dominant-negative (T31N) Arf1, and fractions were analyzed for GIV and β-COP as in 2D. (B) Bar graphs display the % GIV in membrane (P) fractions derived from (P)/(S + P) × 100. Results are expressed as mean ± S.E.M. (C) COS7 cells transfected with HA-tagged WT Arf1 or Arf1[Q71L] mutant were fixed and stained for Arf1 (HA; green) and GIV (red) and nuclei (DAPI; blue). Panels on the left show overlay of all 3 stains (see S6A for individual stains), whereas panels on the right display the red pixels in grayscale which represent GIV. (D) Immunoprecipitation was carried out from lysates of HEK293T cells expressing Arf1-HA alone or Arf1-HA and GIV-FLAG using anti-FLAG mAb, and bound immune complexes were analyzed for Arf (HA) and GIV by immunoblotting. (E) Purified myristoylated Arf1 pre-loaded with GDP or GMP-PNP was incubated with GST or GST-GIV (expressed and purified from HEK293 cells) immobilized on glutathione beads. Bound proteins were analyzed for Arf1 and GIV by immunoblotting. (F) Lysates of HEK293 cells coexpressing HA-tagged Arf1[Q71L] and GST or GST-tagged GIV constructs (GIV-NT, aa 1–220; GIV-CT, aa 1660–1870; GIV-fl, aa 1–1870) were incubated with glutathione beads. Bound proteins were analyzed for Arf1 and GST proteins by immunoblotting. (G) Sequence alignment of GIV with GGA proteins reveals a putative GGA-GAT like region within the Hook domain of GIV. The sequence corresponding to the N-terminal Hook domain of human GIV (BAE44387, aa 90–150) was aligned with the sequence of human GGA1/2 proteins using CLUSTAL W. Conserved residues are shaded in black; similar residues in gray. This alignment reveals that the key amino acids that are essential for Arf1•GGA-GAT interaction are conserved in GIV. The residues marked with red star were mutated in this work to confirm that they are essential for the Arf1:GIV interaction. (H) 3D model of aa 90–150 within GIV’s Hook domain (purple) superimposed on the solved structure of the Hook-like patch on GAT domains of human GGA1 protein (green), generated based on the alignment in G. Key residues in GGA that are important for coupling to Arf1•GTP and the corresponding residues in GIV are labeled. (I) 3D model of a complex between Arf1•GTP (colored by blue/green/yellow/red rainbow from N- to C-terminus) and the N-terminal helix-loop-helix motif of GIV (dark red). GTP and a Mg ion are shown as colored sticks and a blue sphere, respectively. Left panel shows the overall complex with the most important interface residues in GIV. The right panel shows the most important interface residues in Arf1. The binding occurs at the loosely helical Arf1 loop “switch 1” (blue, containing I49 and G50) and also involves β2/3-strands (blue, containing F51 and W66) and the second alpha helix (green, containing H80 and Y81). Residues L94, I98, I106, and F113 form the core of the hydrophobic patch on GIV’s helix-loop-helix motif that interacts with residues F51, W66, and Y81 in Arf1. Multiple polar contacts surrounding this patch may additionally strengthen the Arf1•GTP:GIV interaction and provide specificity. (J) Lysates of HEK cells coexpressing HA-tagged Arf1[Q71L] and WT or mutant GST-tagged GIV-NT aa 1–220 constructs (L94D, I98D and L106D) were incubated with glutathione beads. Bound proteins were analyzed for Arf1 and GST proteins by immunoblotting. As expected for a Hook domain, WT and all mutant GIV-NT proteins bound tubulin.

Figure 7. Summary and working model

(Upper): Schematic of the multimodular GIV protein is shown. A patch of residues within the N-terminal Hook-domain of GIV (purple) resembles the Hook-like patch within the GAT domain of GGA1/2/3 proteins and directly binds GTP-bound Arf1. The coiled-coil domain (blue) assists in homooligomerization, whereas the PI(4)P-binding domain mediates binding to phosphoinositides at the Golgi and the PM (Enomoto et al., 2005). The C-terminally located GEF motif (red) of GIV binds and activates Gαi and releases ‘free’ Gβγ. Homology models shown for Arf1:GIV-Hook (left) is validated in current work (Fig 6) and that for Gαi3(green):GIV-GEF(red) was validated previously (right; Garcia-Marcos et al., 2009). (Middle): Schematic of perinuclear Golgi compartment is shown. GIV is a GEF for Gαi that activates trimeric G proteins on Golgi membranes and releases free Gβγ dimers. (Lower): Schematic of hierarchical steps that enable GIV to terminate Arf1 signaling and trigger COPI coatomer dissociation during anterograte trafficking. From Left to Right: Membrane-associated Arf1•GTP recruits COPI coatomer (represented here as β-COP) onto membranes and promotes vesicle budding from the ER/ERGIC compartment. GIV is recruited to COPI vesicles, presumably as an effector of active Arf1, and further stabilized by its ability to interact with the coat protein, β-COP. Once on vesicles, GIV binds and recruits ArfGAP3 which is required for terminating Arf1 activity and initiating the process of uncoating (STEP 1). Termination of Arf1 activity is further perpetuated by a second step (STEP 2) which involves activation of Gi and release of ‘free’ Gβγ by GIV on Golgi membranes. This second step presumably takes place only when COPI vesicles dock on Golgi membranes (i.e., acceptor membrane), and serves to terminate any remaining active Arf1 on those vesicles via pathway(s) that remain unclear (“?”) and ensure completion of the uncoating process just prior to vesicle fusion.