IDENTIFICATION OF 2 HYALURONAN-BINDING DOMAINS IN THE HYALURONAN RECEPTOR RHAMM

We have identified two discrete hyaluronan- (HA) binding domains in th e HA receptor RHAMM (Receptor for HA-Mediated Motility) that mediates the locomotion of H-ras transformed fibroblasts. A complete RHAMM cDNA (1.43 kilobases (kb)) was expressed as a fusion protein with pGEX-2T in Escherichia coli HB101 and was shown to bind specifically to both b iotin-labeled HA in a transblot assay and to HA-Sepharose. The complet e cDNA was truncated with restriction endonucleases from the 3' end re sulting in 1.30-, 1.02-, 0.71-, and 0.41-kb cDNAs which were then expr essed in HB101. Only the fusion peptide expressed from the complete cD NA and the 1.30-kb cDNA bound to HA indicating that the region located between 1.02-1.30 kb of RHAMM cDNA was critical for recognition of th is glycosaminoglycan. Deletion of 114 bases in this region virtually e liminated HA binding activity thus defining the major glycosaminoglyca n binding region to amino acids 400-434 located near the carboxyl term inus of RHAMM. Two domains containing clusters of basic amino acids we re identified within this region. Synthetic peptides mimicking these t wo domains both inhibited HA binding to the complete 1.43-kb expressed glutathione s-transferase-RHAMM fusion protein, and also directly bou nd to HA-Sepharose. Random peptides and peptides mimicking other regio ns in RHAMM did not inhibit HA-RHAMM interactions and bound weakly to HA-Sepharose. Oligonucleotides encoding either of these two peptides w ere linked to the NH2-terminal 0.71 kb of RHAMM which encoded a peptid e that did not contain HA binding activity. Fusion proteins containing either of these recombinant peptides acquired HA binding activity as assessed with a transblot assay. Thus, we have identified two domains within RHAMM that are responsible for its HA binding activity.