In the present study, we showed that SPA-1, a Rap1 GTPase-activating protein (GAP), was bound to a cytoskeleton-anchoring protein AF-6. SPA-1 and AF-6 were co-immunoprecipitated in the 293T cells transfected with both cDNAs as well as in normal thymocytes. In vitro binding studies using truncated fragments and their mutants suggested that SPA-1 was bound to the PDZ domain of AF-6 via probable internal PDZ ligand motif within the GAP-related domain. The motif was conserved among Rap1 GAPs, and it was shown that rapGAP I was bound to AF-6 comparably with SPA-1. RapV12 was also bound to AF-6 via the N-terminal domain, and SPA-1 and RapV12 were co-immunoprecipitated only in the presence of AF-6, indicating that they could be brought into close proximity via AF-6 in cells. Immunostaining analysis revealed that SPA-1 and RapV12 were co-localized with AF-6 at the cell attachment sites. In HeLa cells expressing SPA-1 in a tetracycline-regulatory manner, expression of AF-6 inhibited endogenous Rap1GTP and ␤ 1 integrin-mediated cell adhesion to fibronectin in SPA-1-induced conditions, whereas it affected neither of them in SPA-1-repressed conditions. These results suggested that AF-6 could control integrin-mediated cell adhesion by regulating Rap1 activation through the recruitment of both SPA-1 and Rap1GTP via distinct domains.

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CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral overexpression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process. ß

S U M M A R YThe high-affinity IgE receptor (Fc ε RI) on mast cells and basophils consists of a ligand-binding ␣ -chain and two kinds of signaling chains, a ␤ -chain and disulfide-linked homodimeric ␥ -chains. Crosslinking by multivalent antigen results in the aggregation of the bound IgE/ ␣ -chain complexes at the cell surface, triggering cell activation, and subsequent internalization through coated pits. However, the precise topographical alterations of the signaling ␤ -and ␥ -chains during stimulation remain unclarified despite their importance in ligand binding/signaling coupling. Here we describe the dynamics of Fc ε RI subunit distribution in rat basophilic leukemia cells during stimulation as revealed by immunofluorescence and immunogold electron microscopy. Immunolocalization of ␤ -and ␥ -chains was homogeneously distributed on the cell surfaces before stimulation, while crosslinking with multivalent antigen, which elicited optimal degranulation, caused a distinct aggregation of these signaling chains on the cell membrane. Moreover, only ␥ -but not ␤ -chains were aggregated during the stimulation that evoked suboptimal secretion. These findings suggest that high-affinity IgE receptor ␤ -and ␥ -chains do not co-aggregate but for the most part form homogenous aggregates of ␤ -chains or ␥ -chains after crosslinking.

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