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 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.
Lynn
B D
BD
Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
Turley
E A
EA
Nagy
J I
JI
D000468:Alkalies; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D001923:Brain Chemistry / Q000502:physiology; D002147:Calmodulin / Q000032:analysis / Q000378:metabolism*; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018960:Hyaluronan Receptors / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D009836:Oligodendroglia / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions
D000468:Alkalies; D000906:Antibodies; D002147:Calmodulin; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; C098946:hyaluronan-mediated motility receptor; D006820:Hyaluronic Acid