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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.

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