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...
Mü ller cells, the principal glia of the retina, generate tractional forces in response to IGF-I and plateletderived growth factor and are present in diabetic fibrovascular scar tissues causing traction retinal detachment. While diabetes-associated increases in vitreous IGFs have been reported, paradoxically high concentrations of these same growth factors in normal vitreous suggest the presence of more complex mechanisms regulating growth factor bioavailability. To define diabetes-associated changes in vitreous biological activity, the stimulatory effects of 68 samples were evaluated using Mü ller cell tractional force generation as a target bioassay. Dose-response profiles were used to calculate vitreous specific activity (per unit protein) and total vitreous activity (per unit volume). Vitreous samples from patients lacking diabetes or other retinal pathology had undetectable or low activities, whereas diabetic retinopathy was associated with 6.9-and 8.7-fold increases in vitreous specific and total activities, respectively. Secondary analyses revealed no activity differences associated with patient sex, age, or the presence of vitreous hemorrhage. However, compared with diabetes alone, the presence of proliferative diabetic retinopathy was associated with additional 2.3-fold increases in vitreous specific and total activities. Vitreous dose-response assays performed with and without growth factor-neutralizing antibodies enable attribution of vitreous activity to IGFs (53.9%) and, to a lesser extent, platelet-derived growth factors (14.5%). Because the observed increases in vitreous growth factor activity grossly exceed the reported increases in growth factor concentration, these data indicate that diabetes-associated changes in vitreous biological activity involve more complex biochemical changes that ultimately yield increased growth factor bioavailability and/or Mü ller cell responsiveness. Diabetes 53: 2428 -2435, 2004
The use of fluoresceinated BSS infusion yields larger vitreous samples from which the native biochemical characteristics can be determined. Patient safety during collection is enhanced because ocular hypotony and collapse can be avoided.
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