Subcellular distribution, calmodulin interaction, and mitochondrial association of the hyaluronan-binding protein RHAMM in rat brain

The CNS contains high levels of the glycosaminoglycan hyaluronan, and neura l cells express a variety of proteins that are members of the hyaladherin f amily of hyaluronan-binding proteins. We have previously shown that the hya ladherin RHAMM (receptor for hyaluronan-mediated motility; CD168) is expres sed by neural cells in culture; plays a role in astrocyte motility, neurite migration, and axonal growth; and is widely distributed in neurons and oli godendrocytes of developing and adult rat CNS. Here we demonstrate differen tial localization of various forms of RHAMM in subcellular fractions of adu lt rat brain. Western blotting indicated the presence of 66, 75, and 85-90 kDa molecular weight RHAMM forms in whole-brain homogenates. Subfractionati on revealed enrichment of the 66 and 85-90 kDa forms in soluble fractions, whereas the 75 kDa form was enriched in mitochondrial fractions. This latte r form was retained in osmotically shocked mitochondria, but was liberated by alkali carbonate, suggesting a nonintrinsic mitochondrial membrane assoc iation. By double immunohistochemical labeling for RHAMM and the mitochondr ial marker cytochrome oxidase, RHAMM was localized to isolated mitochondria in vitro and to neuronal mitochondria in vivo. Hyaluronan-sepharose chroma tography and cetylpiridinium chloride precipitation confirmed the hyalurona n-binding capacity of RHAMM forms. By calmodulin-affinity chromatography, e ndogenously expressed brain RHAMM was demonstrated to bind calmodulin in a Ca2+-dependent manner. These results, together with reports of RHAMM associ ation with actin and microtubules in other systems, suggest a role of RHAMM in calmodulin-mediated cell signaling to cytoskeletal elements and/or mito chondria in the CNS and invoke novel functions of its interactions with hya luronan. J. Neurosci. Res. 65.6-16, 2001. (C) 2001 Wiley-Liss, Inc.