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. Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metaUothionein (MT-1) hybrid gene. Promotion of the rashybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two-to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1/zg/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the-experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.

No abstract