Identification of sequence, protein isoforms, and distribution of the hyaluronan‐binding protein RHAMM in adult and developing rat brain

Abstract

The protein RHAMM (for "receptor for hyaluronan-mediated motility"; CD168) is a member of the hyaladherin family of hyaluronan-binding proteins. RHAMM has a role in cell signaling, migration, and adhesion via interactions with hyaluronan, microtubules, actin, calmodulin, and components of the extracellular regulated kinase (erk) signaling pathway. Based on previous findings of potentially similar roles in neural cells in culture, we investigated the molecular characteristics, protein expression profile, and distribution of RHAMM in rat brain. Reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA isolated from adult rat brain yielded a single RHAMM sequence of 2.1 kilobases encoding a protein of 82.4 kDa. RHAMM is subject to alternate splicing in other systems, but no RT-PCR evidence was found for splice variants in brain, although our analysis does not rule out this possibility. The amino acid sequence displayed homology with human and murine RHAMM (74% and 80%, respectively) but contained only one copy of a 21-amino-acid sequence that is repeated five times in the murine homologue. By using anti-RHAMM antibodies, several RHAMM isoforms were identified in brain. Immunohistochemically, RHAMM was found in the vast majority of neurons and in many oligodendrocytes throughout brain, with heterogeneous levels among cell populations, and was confined to the somata and initial processes of these cells. RHAMM was detected in neurons of cerebral cortex and most subcortical and brainstem structures at postnatal day 1 and exhibited an adult distribution pattern by postnatal day 5. High levels were detected in oligodendrocytes by postnatal day 10. The widespread expression of RHAMM in adult and developing brain implies a role for this protein and its ligand hyaluronan in key events of cell signaling and cytoskeletal regulation in the CNS.

Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and cx47 association with zonula occludens-1 (zo-1) in mouse brain

“…Total RNA was isolated from adult mouse brain using the guanidine isothiocyanate method and reverse transcription using 500 ng oligo(dT)15 primer (Promega, Madison, WI, USA) was conducted as previously described (Lynn et al, 2001a;Li et al, 2002). Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).…”

Genetic deletion of receptor for hyaluronan-mediated motility (Rhamm) attenuates the formation of aggressive fibromatosis (desmoid tumor)

“…We did not detect the expression of a protein corresponding to the predicted 950 bp mRNA in RhammÀ/À tissue using Western blot assays. Since it is possible that antibodies to sequences encoded in exons 17 and 18 are not represented in the polyclonal population of antibodies used for Western analysis, we utilized polyclonal antibodies prepared against the peptide sequence encoded in exon 7, which would be expressed from the 920 bp mRNA fragment of RhammÀ/À tissue (Lynn et al, 2001). Positive immunofluorescence was detected in wt but not RhammÀ/À MEF with this antibody.…”

“…Nonspecific binding sites were blocked by incubation with 3% bovine serum albumin (BSA) in PBS for 2 h at RT. Polyclonal rabbit Rhamm antibodies prepared against sequence encoded in exons 7 and 8 (Lynn et al, 2001) were used at 1 : 2000 dilution in 1% BSA/PBS containing 0.3% Triton X-100. After 24 h incubation at 41C, cells were washed 4 Â 15 min with PBS followed by incubation with FITC-labeled goat anti-rabbit antibodies,1 : 200 dilution in 1% BSA/PBS and 30 min at room temperature.…”

“…Here we use Rhamm to distinguish this protein from other, similar HA-binding proteins. Rhamm occurs on the cell surface, as a peripheral or GPI-linked protein, and such intracellular compartments as the cell nucleus, cytoskeleton, podosomes, lamellae and mitochondria (Turley et al, 1990;Pilarski et al, 1994;Zhang et al, 1998;Assmann et al, 1999;Lynn et al, 2001). Rhamm expression is low or undetectable in most normal tissues, but is upregulated following wounding in vivo , culture at low confluence in vitro in the presence of growth factors (Turley and Auersperg, 1989;Hardwick et al, 1992;Cheung et al, 1999;Savani et al, 2001) or upon neoplastic transformation in vitro (Hall et al, 1995) and in vivo (Tammi et al, 2002;Turley et al, 2002).…”

Expression patterns of CD168 correlate with the stage and grade of squamous cell carcinoma of head and neck

“…It is involved in cell signaling, migration, and adhesion via interactions with hyaluronan, microtubules, actin, calmodulin, and components of the extracellular regulated kinase (ERK) signaling pathway (11). Hyaluronan accumulates during high tissue turnover (e.g.…”