Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N‐terminal domain of the ost proto‐oncogene and was highly tumorigenic in nude mouse assays. The proto‐ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP‐binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP‐bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha‐tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.

The Eph family of receptors is the largest family of RTKs. Eph receptors are stimulated by a family of membrane-linked ligands designated ephrins (6, 7). Both biochemical and genetic studies have established the central role that ephrins have in the control of cell contact repulsion, boundary formation, cell migration, and repulsive axon guidance (6). Repulsive axon guidance appears to be caused by modulation of cytoskeletal organization leading to regulation of neural growth-cone development (8). Eph-receptors also regulate cell-matrix interaction and cell proliferation by affecting signaling by integrins (9-11) and by modulation of MAPK response (12)(13)(14).In this article, we demonstrate that EphA4 binds directly and specifically via the N-terminal portion of its protein tyrosine kinase core to the juxtamembrane (JM) region of FGFRs. In cells that express EphA4 and FGFRs, the interactions between the cytoplasmic domains of EphA4 and FGFRs can lead to transreceptor activation, resulting in tyrosine phosphorylation of FRS2␣ and MAPK activation. The synergistic effect of ephrin-A1 stimulation on FGF2-induced cellular responses may influence the biological outcome of the activation of these two families of RTKs. Materials and MethodsYeast Two-Hybrid Experiments. The yeast two-hybrid system was used as described (15). The bait used for screening was 81 aa (amino acids 398-478), derived from the JM domain of human FGFR3. A human brain cDNA library in a pJG4-5 vector consisting of 3.5 ϫ 10 6 primary transformants (Clontech) was used for screening for proteins that interact with the JM domain of FGFR3. Fig. 1A shows the constructs used to detect interactions between the cytoplasmic domain of EphA4 and the JM domain of FGFR3.Cells. HEK293 cells were maintained in DMEM supplemented with 10% calf serum. For neural differentiation, P19 cells were maintained in ␣-MEM supplemented with 10% FBS containing 0.5 M retinoic acid for 3 days. Rat L6 myoblasts were maintained in DMEM supplemented with 10% FBS.Preparation of Ephrin-A1. Ephrin-A1 fused to human IgG-Fc was purchased from Sigma-Aldrich. Before application to the cells, 5 g of ephrin-A1-Fc was oligomerized by mixing with 12 g of rabbit anti-human IgG-Fc (Jackson ImmunoResearch) in 1 ml of PBS at 4°C for at least 1 h. As a control, a human IgG-Fc fragment (Jackson ImmunoResearch) was also applied after oligomerization.Expression Plasmids. Full-length cDNA of human EphA4 was prepared by RT-PCR using total RNA from a human brain extract (Clontech) as the template. The cDNA of human FGFR4 was prepared by RT-PCR using K562 cell-derived RNA as the template. The cDNAs for FGFR1 and FGFR2 were provided by W. McKeehan (Texas A&M University, College Station, TX). The cDNA for FGFR3 was provided by D. E. Johnson (University of Pittsburgh, Pittsburgh). Receptor mutants were prepared by applyConflict of interest statement: No conflicts declared.

Familial multiple endocrine neoplasia type 1 (MEN-1) is characterized by tumors of the parathyroids, endocrine pancreas, and anterior pituitary. Since the gene associated with MEN-1, located on chromosome 11 (11q13), may normally inhibit tumor proliferation, tumors could arise from inactivation of one or both of the alleles. However, parathyroid tumors in patients with MEN-1 have been considered to result from polyclonal hyperplasia. Using genetic probes, we tested parathyroid tumors for a monoclonal component, represented by a loss of alleles at any of eight loci along chromosome 11. Ten of 16 tumors from 14 patients with familial MEN-1 had losses of alleles from chromosome 11. Tumors with losses were larger than those without (1.6 vs. 0.2 g; P less than 0.002), suggesting that a monoclonal adenoma may develop after a phase of polyclonal hyperplasia. In 7 of 10 tumors, the subregion of loss was less than the full length of chromosome 11 but always included one copy of the MEN-1 locus. Of 34 sporadic adenomas from patients without MEN-1, 9 showed similar allelic losses in chromosome 11; in 7 the losses included the apparent MEN-1 locus. We conclude that many "hyperplastic" parathyroid tumors in familial MEN-1 are in fact monoclonal and may progress or even begin to develop by inactivation of the MEN-1 gene (at 11q13) in a precursor cell. Some sporadic adenomas have allelic losses on chromosome 11, which may also involve the MEN-1 gene.