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

Abstract

The CNS contains high levels of the glycosaminoglycan hyaluronan, and neural cells express a variety of proteins that are members of the hyaladherin family of hyaluronan-binding proteins. We have previously shown that the hyaladherin RHAMM (receptor for hyaluronan-mediated motility; CD168) is expressed by neural cells in culture; plays a role in astrocyte motility, neurite migration, and axonal growth; and is widely distributed in neurons and oligodendrocytes of developing and adult rat CNS. Here we demonstrate differential localization of various forms of RHAMM in subcellular fractions of adult rat brain. Western blotting indicated the presence of 66, 75, and 85-90 kDa molecular weight RHAMM forms in whole-brain homogenates. Subfractionation revealed enrichment of the 66 and 85-90 kDa forms in soluble fractions, whereas the 75 kDa form was enriched in mitochondrial fractions. This latter form was retained in osmotically shocked mitochondria, but was liberated by alkali carbonate, suggesting a nonintrinsic mitochondrial membrane association. By double immunohistochemical labeling for RHAMM and the mitochondrial marker cytochrome oxidase, RHAMM was localized to isolated mitochondria in vitro and to neuronal mitochondria in vivo. Hyaluronan-sepharose chromatography and cetylpiridinium chloride precipitation confirmed the hyaluronan-binding capacity of RHAMM forms. By calmodulin-affinity chromatography, endogenously expressed brain RHAMM was demonstrated to bind calmodulin in a Ca2+-dependent manner. These results, together with reports of RHAMM association with actin and microtubules in other systems, suggest a role of RHAMM in calmodulin-mediated cell signaling to cytoskeletal elements and/or mitochondria in the CNS and invoke novel functions of its interactions with hyaluronan.

RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: A different interpretation of redundancy

“…For example, RHAMM is required for activation of signaling pathways through IL-1␤ and TNF-␣, such as erk, and AP-1 (14,19). RHAMM also binds to calmodulin (16), regulates connexin expression (43), and promotes formation of diacylgycerol in response to hyaluronan, linking this hyaladherin to phospholipase C␥ (44).…”

“…RHAMM is expressed on the cell surface (where it is designated CD168) and in the cytoplasm, as well as in the cytoskeleton and nucleus. Like CD44, RHAMM is subject to alternative splicing (14)(15)(16)(17), particularly during tissue repair or after neoplastic conversion (17). After its interaction with HA, this receptor delivers signals for cell migration and proliferation in normal and malignant cells (15,(17)(18)(19).…”

Upregulation of hyaluronan-mediated motility receptor in hepatocellular carcinoma predicts poor survival

“…It has been demonstrated that RHAMM is associated with tubulin and actin cytoskeletal elements (10,23), indicating that RHAMM may be important to the movement and reorganization of blastomeres during early embryonic development. RHAMM has also been reported to be associated with cytoskeletal interaction of mitochondria, thus, it may regulate mitochondrial motility and positioning (24), which is important for the physiological process of cytoplasmic maturation and the further development of mammalian oocytes (25). In addition, RHAMM regulates Ras signaling and is involved in breast cancer progression (26).The upregulation of RHAMM is therefore closely involved in regulating tumorigenesis, proliferation, tumor vascular invasion and node metastasis of HCC cells.…”

Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

“…The mitoplasts were recovered by centrifugation at 4000 g and then resuspended in 10 mM HEPES-KOH, pH 7.4, containing 220 mM mannnitol and 70 mM sucrose. Alkaline extraction with carbonate buffer was performed as described previously (Lynn et al, 2001). Briefly, mitochondria and mitoplasts were resuspended in 100 mM sodium carbonate pH 11 for 30 minutes at 4°C, then centrifuged at 100,000 OGT localizes to mitochondria g for 30 minutes.…”