Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.Ral is a member of small G protein 1 superfamily and consists of RalA and RalB (1, 2). As well as other small G proteins, Ral has the GDP-bound inactive and the GTP-bound active forms. The GDP-bound form of Ral is converted to the GTP-bound form by RalGDS, and inversely the GTP-bound form is changed to the GDP-bound form by RalGAP (3, 4). We and other groups have found that RalGDS is a putative effector protein of Ras (5-7). Since RalGDS stimulates the GDP/GTP exchange of Ral (4), it is possible that there is a new signaling pathway from Ras to Ral through RalGDS. Indeed, it has been shown that RalGDS stimulates the GDP/GTP exchange of Ral in a Ras-dependent manner in COS cells and that a dominant negative form of Ral blocks a Ras-dependent transformation in NIH3T3 cells (8). It has been also demonstrated that Ral is required for Src-and Ras-dependent activation of phospholipase D and that it regulates the initiation of border cell migration induced by Ras in Drosophila oogenesis (9, 10). Furthermore, it has been shown that RalGDS and Raf synergistically stimulate cellular proliferation and gene expression (11, 12). These results indicate that RalGDS and Ral act downstream of Ras and mediate Ras functions. However, the mechanism by which Ral regulates cellular functions is not known.One possible clue to clarify the mode of action of Ral is RalBP1 (13-15). RalBP1 has a Ral-binding domain in its Cterminal region and binds to the GTP-bound form of Ral but not to the GDP-bound form. A mutation in the effector loop of Ral impairs its interaction with RalBP1 and RalBP1 inhibits the RalGAP activity for Ral (13, 16). These results suggest that RalBP1 is an effector protein of Ral. RalBP1 also has a RhoGAP homology domain in its central region and exhibits the GAP activity for Rac1 and CDC42 but not for Rh...
The articular cartilage consists of resident chondrocytes embedded within the extracellular matrix which contains several components such as collagen and hyaluronic acids (HA). CD44 is a major cell surface receptor for HA and is homologous to cartilage-link proteins. Although CD44 is present in cartilage, it is not clear if chondrocytes adhere to HA through CD44 or whether such adhesion changes the function of chondrocytes. We studied the molecular mechanisms of CD44-related chondrocyte adhesion to HA and the effects of such adhesion on chondrocyte function. Experiments were performed using the human chondrosarcoma-
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