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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02486

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The Arf GAPs AGAP1 and AGAP2 distinguish between the adaptor protein complexes AP-1 and AP-3

¹ Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
² Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA

View larger version (68K): [in a new window]   Fig. 1. Distribution and biochemical characterization of AGAP2. (A) Similarities between different domains of AGAP1 and AGAP2. Alignment was performed using MacVector. Identical residues were marked in red and similar residues were marked in green. The different domains were boxed with different colors, GLD (GTP-binding protein-like domain) in red; PH (pleckstrin homology domain) in green; ArfGAP (Arf directed-GTPase activating protein domain) in blue; Ank (ankyrin repeat domain) in black. (B) Distribution of AGAP1 and AGAP2 message by RT-PCR. The top panel shows the message of AGAP1, the middle panel shows the message of AGAP2, and the bottom panel shows the message of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) in different human tissues. AGAP1, AGAP2 and G3PDH cDNAs were amplified for 34 cycles using a human cDNA panel from Clontech. AGAP1 cDNA was detected using primers to amplify the region between nucleotide 648 and 1369 of the open reading frame. AGAP2 cDNA was detected using primers to amplify the region between nucleotide 1303 to 1770 of the open reading frame. G3PDH cDNA was detected using primers provided by Clontech. (C) Differential distribution of AGAP1 and AGAP2 in different cell lines. Lysates from different cell lines were subjected to SDS-PAGE and western blot for AGAP1 (detected using the antibody 1158) and AGAP2 (detected using 4572). Jurkat, T lymphocyte; HeLa, cervical adenocarcinoma; H4, neuroglioma; U87, U118, U138 and U1620, glioblastoma; Mel 14 and 526, melanoma; A549, squamous carcinoma; AGS, gastric adenocarcinoma; HepG2, hepatocellular carcinoma; SKOV3, ovary cancer; SUM149, breast cancer; VW373, astrocytoma. (D) Arf specificity of AGAP2. Arf GAP activity of AGAP2 was measured as described in Materials and Methods, using 360 µM PA together with either 45 µM PIP2 or 1 µM PIP3 presented in micelles with 0.1% Triton X-100. (E) Phospholipid specificity of AGAP2. AGAP2 activity was measured in the presence of the lipids as indicated at concentrations as follows, PA, 360 µM; PI3P, 45 µM; PI3,4P2, 45 µM; PI3,5P2, 45 µM; PI4,5P2, 45 µM presented in micelles with 0.1% Triton X-100.

View larger version (44K): [in a new window]   Fig. 2. Interaction of AGAP2 with adaptor protein complex AP-1. (A) Effect of AGAP1 and AGAP2 on the membrane association of AP-3. NIH3T3 cells were transfected with AGAP1 and AGAP2 for 24 hours. Cells were fixed and stained for AP-3 (using anti- antibody) and the FLAG tag (using a polyclonal anti-FLAG antibody) to visualize the transfected cells (arrows). (B) Co-immunoprecipitation of AP-1 with AGAP2. FLAG tagged AGAP1, AGAP2 or empty vector were transfected into HEK293 cells at 10 µg DNA/10cm dish, using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 hours after transfection and lysed into a buffer containing 20 mM Tris, pH 8.0, 100 mM NaCl, 1% Triton X-100 and 10% glycerol. Protease inhibitors (Complete®, Roche) were included in the lysis buffer. AGAP1 and AGAP2 were immunoprecipitated through the FLAG tag using anti-FLAG M2 gel. Different coat protein complexes in the precipitates were detected by antibodies against the subunit for AP-1, subunit for AP-2, subunit for AP-3 and subunit for AP-4. (C) Pulldown assay of AP-1 with different GST fusion proteins of AGAP2. Different domains of AGAP2, including the GLD2, PH2 and ZA2, were fused with the glutathione S-transferase (GST) and expressed in E. coli. GST was included as a control. The purified proteins were incubated with the soluble extracts of bovine brain at 4°C overnight. The beads were washed and the proteins precipitated were resolved by SDS-PAGE and transferred to nitrocellulose. Immunoblots were performed using antibody to AP-1 (top panel) and AP-2 (middle panel). Only AP-1 was detected. The GST or the GST fusion proteins used for precipitation were shown in the bottom panel by Coomassie Blue staining. (D) Inhibition of AGAP2 activity by AP-1. His-tagged PZA2 domain of AGAP2 (25 nM) was preincubated with different concentrations of AP-1 or AP-2 for 30 minutes at room temperature before addition of [32P]GTP-labeled myristoylated Arf1. The phospholipids were presented in vesicles as described in Materials and Methods. The reaction was stopped after 2 minutes and the amount of GTP hydrolyzed quantified by a PhosphorImager (Molecular Dynamics). (E) Time course of GAP activity of AGAP2. The activity of AGAP2 was measured at the time intervals as indicated. For inhibition by AP-1 and clathrin, 100 nM of AP-1 and 100 nM of clathrin were incubated with AGAP2 at room temperature for 30 minutes before addition of [32P]GTP labeled myristoylated Arf1. (F) Effect of AP-1 and clathrin on AGAP2 activity. His-tagged PZA2 of AGAP2 was incubated with 88 nM AP-1, and/or 80 nM clathrin at room temperature for 30 minutes before addition of [32P]GTP labeled myristoylated Arf1. *P<0.001 compared with AGAP2 as analyzed by one-way ANOVA with Tukey post-test. (G) Differential effect of AP-1 on AGAP2 and ASAP1. AP-1 (75 nM) was incubated with AGAP2 or ASAP1 at room temperature for 30 minutes before the addition of [32P]GTP labeled myristoylated Arf1. GAP assay was performed as described in panel D. *P<0.05 compared with AGAP2 alone as analyzed by one-way ANOVA with Tukey post-test.

View larger version (69K): [in a new window]   Fig. 3. Specific interaction between AGAP2 and AP-1. (A) Redistribution of AP-1 by AGAP2. HeLa cells were transfected with FLAG-tagged AGAP1 or AGAP2 for 24 hours. The cells were fixed and stained with antibodies against the subunit of AP-1. The transfected cells were detected using polyclonal anti-FLAG antibody. (B) Membrane dissociation of AP-3 by AGAP1. HeLa cells were transfected with FLAG-AGAP1 or AGAP2 for 24 hours. Cells were stained for the epitope tag and AP-3 using anti- antibody. (C) No effect of AGAP2 on the staining pattern of COPI or TGN46. HeLa cells were transfected with FLAG-AGAP2. The COPI coat was visualized by staining with antibody against ßCOP (panels a, b). The TGN was visualized by staining with an antibody against TGN46 (panels c, d). Transfected cells were indicated by arrows.

View larger version (52K): [in a new window]   Fig. 4. Dependence of GAP activity of AGAP2 for interaction with AP-1. (A-D) Effect of [Q71L]Arf1 on AGAP2 induced AP-1 redistribution. HeLa cells were transfected with FLAG-AGAP2 (B), [Q71L]Arf1-HA (C) or both (A,D) for 24 hours. Cells were stained for the AP-1 and FLAG tag (B,D), or HA-tag (C), or stained for both FLAG and HA tag (A). (E) Requirement of the conserved arginine for GAP activity. The catalytic core of AGAP2, PZA2, or its point mutant with the conserved arginine mutated to lysine, [R618K]PZA2, were expressed as GST-fusion proteins in E. coli and purified. The proteins were eluted from the beads with glutathione and dialyzed overnight against PBS with 1 mM dithiothreitol. Increasing concentrations of PZA2 or [R618K]PZA2 were titrated into the GAP assay as described in Materials and Methods. (F) Effect of GAP dead AGAP2 on AP-1 association with TGN. FLAG-tagged [R618K]AGAP2 was transfected into HeLa cells. Cells were fixed 24 hours after transfection and stained for AP-1 and the FLAG tag. Transfected cells were indicated by arrows.

View larger version (69K): [in a new window]   Fig. 5. Effect of AGAP2 on endosomal markers. (A) Colocalization of AGAP2 with endosomal markers. (A, a-d) AGAP2 colocalized with AP-1 and transferrin receptor. FLAG-AGAP2 under the control of SV40 promoter (in pSI vector) was transfected into HeLa cells for 24 hours. The cells were fixed and stained for AP-1 and transferrin receptors (TfnR). The overexpressed AGAP2 was detected by staining for the FLAG tag. Colocalization of AGAP2 with AP-1 and TfnR was indicated by arrows. (A, e-j) Colocalization of AGAP2 with Rab4. FLAG-AGAP2 in pSI vector was transfected with GFP-Rab4, GFP-Rab5 and GFP-Rab11 into HeLa cells for 24 hours. The overexpressed AGAP2 was detected by staining for the FLAG tag. AGAP2 colocalized with Rab4 (arrows in e, f), but not with Rab5 (g,h) or Rab11 (i,j). (B) Effect of AGAP2 on endogenous Rab4 and TfnR. HeLa cells were transfected with FLAG-AGAP2 driven by the CMV promoter (in pCI vector) for 24 hours. Endogenous Rab4 (B, a,b), Rab11 (B, c,d) and transferrin receptors (B, e,f) were visualized by staining with specific antibodies. Transfected cells were visualized by staining for the FLAG tag and indicated by arrows. (C) Relative expression level of [FLAG]AGAP2. HeLa cells were transfected with empty vector, pSI-[FLAG]AGAP2 or pCI-[FLAG]AGAP2 for 24 hours. Cells were harvested and lysed. The lysates were subjected to SDS-PAGE and western blot using polyclonal anti-FLAG antibody.

View larger version (56K): [in a new window]   Fig. 6. Effect of AGAP2 on transferrin recycling. (A) AGAP2 promoted transferrin recycling. HeLa cells were transfected with FLAG-AGAP2 for 24 hours. The cells were starved for 30 minutes and then incubated with Rhodamine-conjugated transferrin for 10 minutes (a,b) or 30 minutes (c,d). (B) Effect of [S22N]Rab4 and [S25N]Rab11 on transferrin uptake. Cells were transfected with GFP-[S22N]Rab4 (a,b) and GFP-[S25N]Rab11 (c,d) for 24 hours. Cells were incubated with transferrin for 10 minutes before washing and fixing. (C,D) Interaction of [S22N]Rab4 and [S25N]Rab11 with AGAP2 on transferrin recycling. AGAP2 was transfected into HeLa cells with either GFP-tagged [S22N]Rab4 (C, a-c) or [S25N]Rab11 (D, a-c) for 24 hours. The cells were then incubated with transferrin for 10 minutes. Cells overexpressing AGAP2 were identified by staining for the FLAG tag (arrows). (E) Quantification of intracellular transferrin fluorescence intensity. Intracellular fluorescence intensity of transferrin from single cells was quantified using LSM510 software. The fluorescence intensity of the transfected cells was compared to that of the non-transfected cells on the same coverslip. *P<0.05; **P<0.01 compared with control as analyzed by one-way ANOVA with Tukey post-test.

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