Molecular cloning and functional expression of the rat 175-kDa hyaluronan receptor for endocytosis.
We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and approximately 300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5'-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig kappa-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of (125)I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the approximately 300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Bin
B
Zhou
Janet A
JA
Weigel
Amit
A
Saxena
Paul H
PH
Weigel
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002469:Cell Separation; D002966:Clathrin / Q000378:metabolism; D003001:Cloning, Molecular; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
D002966:Clathrin; D018960:Hyaluronan Receptors; D011994:Recombinant Proteins; C472814:hyaluronan receptor 175-kDa, rat; D006820:Hyaluronic Acid