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...

Abstract. The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 rain of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 #g/ml or herbimycin A, 0.5 /zg/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125 F~, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is critical for cell locomotion.

An expression vector was constructed in which TGF‐beta 1 was placed under the control of the metallothionein promoter. Cys223 and Cys225 in the TGF‐beta 1 propeptide were converted to serines, mutations which result in dissociation of the pro‐peptide and secretion of bioactive TGF‐beta 1 [Brunner, A.M., Marquardt, H., Malacko, A.R., Lioubin, M.N. and Purchio, A.F. (1989) J. Biol. Chem., 264, 13660–13664]. A fibrosarcoma was transfected with this plasmid and a clone (17.18) was selected in which TGF‐beta 1 mRNA was able to be induced six‐fold following zinc sulphate treatment. These cells increased the secretion of bioactive TGF‐beta 1 14‐fold and exhibited a coincidental increase in jun‐B mRNA expression, suggesting that secreted TGF‐beta 1 was acting to induce this early response gene by autocrine activation. Following zinc sulphate induction, the tumor cells became progressively more motile and able to invade collagen gels. In contrast to parental tumor not bearing the TGF‐beta 1 expression vector, zinc sulphate stimulation of clone 17.18 enhanced collagenase IV and procathepsin L mRNA levels and enhanced the secretion of many collagenolytic proteases into the medium. Since the action of TGF‐beta generally decreases proteolysis by suppression of protease transcription, we compared the response of normal parental fibroblasts to ras‐transformed fibrosarcomas and confirmed that TGF‐beta could greatly enhance collagenase IV and procathepsin L mRNA levels while having little effect on non‐transformed fibroblasts. These experiments indicate that induction of TGF‐beta secretion can enhance motility and protease production through autocrine activation, thus increasing the invasion potential of fibrosarcomas.

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (Mr,e) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with [35S]methionine label. The three protein species isolated by hyaluronate affinity chromatography (Mr,e 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of 3H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The apparent dissociation constant of HABP for hyaluronate was approximately 10(-8) M, and kinetic analyses showed these binding interactions were complex and of a positive cooperative nature.(ABSTRACT TRUNCATED AT 250 WORDS)

The recently cloned and characterized hyaluronan (HA) receptor RHAMM (receptor for HA-mediated motility) has been shown to play a critical role in mechanisms underlying the motile capacity of a variety of peripheral cell types. Similarities in molecular processes that govern cell locomotion and growth cone migration prompted us to investigate whether RHAMM also contributes to neurite migration in vitro. In immunohistochemical studies of PC12 cells, NG108–15 cells and a neuroblastoma/spinal cord neuronal hybrid cell line (NSC-34 cells) as well as rat and human primary neurons, a punctiform RHAMM labeling pattern was detected in cell bodies, along processes, and at growth cones. By Western blot analysis, the cells lines expressed major RHAMM forms with apparent MW of 60, 75, and 116 kDa. Treatment of NG108–15 cells with dibutyryl-cAMP led to a clear increase in immunolabeling for RHAMM and enhanced expression of the 60 and 75 kDa forms. A polyclonal anti-RHAMM antibody that interferes with HA/RHAMM interaction significantly reduced neurite migration of each cell type examined, while another directed against a RHAMM repeat sequence thought to promote RHAMM receptor aggregation significantly stimulated neurite migration of NSC-34 and rat primary neurons. Different monoclonal anti- RHAMM antibodies had differential inhibitory actions on neurite movement. Low concentrations (ng/ml) of a peptide corresponding to an HA binding domain within RHAMM inhibited neurite migration. These results are the first to implicate RHAMM in the mediation of neurite motility and migration and to point to the potential importance of HA in this process.