The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments.
We reported recently on the intracellular localisation of the hyaluronan receptor RHAMM/IHABP in human cancer cells. Here we describe the colocalisation of RHAMM/IHABP proteins with microtubules, both in interphase and dividing cells, suggesting that RHAMM/IHABP represents a novel member of the family of microtubule-associated proteins (MAPs). We have identified four different splice variants of RHAMM/IHABP, all of which colocalise, at least transiently, with microtubules when expressed as GFP fusion proteins in HeLa cells. Using microtubule-binding assays and transient transfection experiments of deletion-bearing RHAMM/IHABP mutants, we localised the microtubule-binding region to the extreme N terminus of RHAMM/IHABP. This interaction domain is composed of two distinct subdomains, one of which is sufficient to mediate binding to the mitotic spindle while both domains are required for binding of RHAMM/IHABP proteins to interphase microtubules. Sequence analysis revealed that the projection domain of RHAMM/IHABP is predicted to form coiled-coils, implying that RHAMM/IHABP represents a filamentous protein capable of interacting with other proteins and we found that RHAMM/IHABP interacts with actin filaments in vivo and in vitro. Moreover, in vitro translated RHAMM/IHABP isoforms efficiently bind to immobilised calmodulin in a Ca(2+)-dependent manner via a calmodulin-binding site within the projection domain of RHAMM/IHABP (residues 574-602). Taken together, our results strongly suggest that RHAMM/IHABP is a ubiquitously expressed, filamentous protein capable of interacting with microtubules and microfilaments and not, as numerous previous reports suggest, a cell surface receptor for the extracellular matrix component hyaluronan.
Assmann
V
V
Richard Dimbleby Department of Cancer Research/ICRF Laboratory, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK. V.Assmann@icrf.icnet.uk
Jenkinson
D
D
Marshall
J F
JF
Hart
I R
IR
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D017398:Alternative Splicing; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000199:Actins; D002147:Calmodulin; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C098946:hyaluronan-mediated motility receptor; D049452:Green Fluorescent Proteins; D002118:Calcium