Abstract. A eDNA encoding a unique hyaluronan receptor has been molecularly cloned from a ~GTll 3T3 eDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the eDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the eDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58-and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58-and 52-kD proteins, which block ms-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronanmediated motility. THE transforming oncogene H-ras has been reported to promote cell locomotion (17), although the regulatory mechanisms remain unknown. Several observations suggest that when this oncogene promotes locomotion, the mechanisms are complex and involve, at least, the release of autocrine motility factor(s) (14,20), growth factors (14), and the glycosaminoglycan hyaluronan (HA) ~ (20, 34). In particular, HA appears to function as an autocrine mechanism for stimulating maximal locomotion in ms-transformed cells (34). Further, it is also required for the ability of an autocrine motility-stimulating factor to promote breast carcinoma cell locomotion (20). We have shown that HA-promoted, rastransformed cell locomotion requires the presence of a novel hyaluronan receptor complex termed I/ARC (34). This complex of proteins occurs at the cell surface or is released as soluble proteins of 72, 68, 58, and 52 kD (32). The complex is tightly regulated in vitro (30) and expressed on the leading lamellae and perinuclear region only on rapidly locomoting cells (29, 31). Both polyclonal and monoclonal antibodies (pAbs and mAbs, respectively) prepared against this complex block cell locomotion regulated by mutant ms (34). In Address all correspondence and requests for reprints to E. A. Turley, Manitoba Institute of Cell Biology, 100 Olivia St., Winnipeg, Manitoba R3E OV9 Canada.1. Abbreviations used in this paper: HA, hyaluronan; HARC, hyaluronan receptor complex; pAb, polyclonal antibody; RHAMM, receptor for HAmediated motility, a recent study, we have shown that these blocking mAbs are specific to the 58-and 52-kD proteins, that these proteins are isoforms of each other, and, further, that these proteins are the HA-binding component of HARC (Turley, E. A., K. Home, and V. Cripps, manuscript submitted for publication).We have used the blocking antibodies specific to the 58-and 52-kD HARC proteins to screen...

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Tnk1/Kos1 is a non-receptor protein tyrosine kinase implicated in negatively regulating cell growth in a mechanism requiring its intrinsic catalytic activity. Tnk1/Kos1 null mice were created by homologous recombination by deleting the catalytic domain. Both Tnk1 +/À and Tnk1 À/À mice develop spontaneous tumors, including lymphomas and carcinomas, at high rates [27% (14 of 52) and 43% (12 of 28), respectively]. Tnk1/Kos1 expression is silenced in tumors that develop in Tnk1 +/À mice but not in adjacent uninvolved tissue, and silencing occurs in association with Tnk1 promoter hypermethylation. Tissues and murine embryonic fibroblasts derived from Tnk1/Kos1-null mice exhibit proportionally higher levels of basal and epidermal growth factor-stimulated Ras activation that results from increased Ras-guanine exchange factor (GEF) activity. Mechanistically, Tnk1/Kos1 can directly tyrosine phosphorylate growth factor receptor binding protein 2 (Grb2), which promotes disruption of the Grb2-Sos1 complex that mediates growth factor-induced Ras activation, providing dynamic regulation of Ras GEF activity with suppression of Ras. Thus, Tnk1/Kos1 is a tumor suppressor that functions to down-regulate Ras activity.

A method for detecting hyaluronan (HA)-binding proteins in transblot assays using biotinylated HA (BHA) is described. Some of the binding characteristics of a novel HA receptor termed RHAMM (Receptor for HA-Mediated Motility) are characterized using this assay. The method is also used to detect other HA-binding proteins in tissue homogenates. This method is semiquantitative, rapid, reproducible, sensitive and therefore of potential use in identifying the levels of HA-binding proteins in different cells and tissues.