We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPaseactivating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding proteinlike domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the transGolgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.

We sought to determine whether osteoblasts (OBs) can serve as accessory cells (ACs) for T-cell activation and whether T cells directly activate OB production of IL-6, using primary human OBs (NHOst), the transformed fetal osteoblast line hFOB1.19, and an osteosarcoma line SaOS-2. Robust, bidirectional activating interactions were shown using each of these three human ostoblast lines.Introduction: Osteoblasts (OBs) could come into contact with lymphocytes during inflammatory joint destruction and fracture repair. Materials and Methods: We used several in vitro assays to assess the ability of T cells and OBs to interact in the generation of immune and inflammatory responses. Results: By flow cytometry, three OB cell lines all were found to express ligands for T-cell co-stimulation. The integrin ligand CD54/ICAM-1 was constitutively expressed by hFOB1.19 and NHOst and was upregulated on SaOS-2 by IFN-␥. MHC Class II was upregulated on all three lines by IFN-␥. CD166/ALCAM, a ligand of the T-cell molecule CD6, was constitutively expressed on all three lines. A second putative CD6 ligand designated 3A11 was expressed on hFOB1.19 and NHOst, but not consistently on SaOS-2. The ectoenzyme CD26 (dipeptidyl peptidase IV) was expressed on hFOB1.19 and NHOst, but not on SaOS-2. All three cell lines presented superantigen to T cells, especially after treatment with IFN-␥. Superantigen presentation was inhibited by antibodies to the leukocyte integrin CD11a/CD18 (LFA-1), MHC Class II, and CD54/ICAM-1. T cells, particularly when cytokine activated for 7 days before co-culture, stimulated all three osteoblast lines to produce interleukin (IL)-6, and this effect was boosted when IL-17 was added to the co-cultures with either resting T cells or cytokine-activated T cells. Conclusions: Bidirectional activating interactions are readily shown between human T cells and several types of human OBs. The expression by OBs of ligands for the T cell-specific molecule CD6, as well as other molecules involved in immune interactions, strongly suggests that such in vitro interactions are representative of physiologic or pathologic events that occur in vivo.

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The role of ADP‐ribosylation factor (Arf) in Golgi associated, γ‐adaptin homologous, Arf‐interacting protein (GGA)‐mediated membrane traffic was examined. GGA is a clathrin adaptor protein that binds Arf through its GAT domain and the mannose‐6‐phosphate receptor through its VHS domain. The GAT and VHS domains interacted such that Arf and mannose‐6‐phosphate receptor binding to GGA were mutually exclusive. In vivo, GGA bound membranes through either Arf or mannose‐6‐phosphate receptor. However, mannose‐6‐phosphate receptor excluded Arf from GGA‐containing structures outside of the Golgi. These data are inconsistent with predictions based on the model for Arf's role in COPI veside coat function. We propose that Arf recruits GGA to a membrane and then, different from the current model, ‘hands‐off’ GGA to mannose‐6‐phosphate receptor. GGA and mannose‐6‐phosphate receptor are then incorporated into a transport intermediate that excludes Arf.