Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins.
The carboxyl-terminal cell binding domain (CBD) of thrombospondin-1 (TS1) contains two cell attachment peptides, 4N1s (RFYVVMWK) and 7N3 (FIRVVMY-EGKK), which share the sequence VVM. These peptides, and more soluble derivatives have been radiolabeled with 125I and used in conjunction with a variety of membrane impermeant cross-linking reagents to identify and characterize receptor candidates on several cell types. All of the VVM containing peptides tested with five different cross-linking reagents specifically labeled a 52-kDa protein, which was also affinity labeled by the recombinant TS1 CBD. After cross-linking peptide to K562 cells to block the 52-kDa protein, both cell adhesion to and affinity labeling by VVM peptides were inhibited in a concentration-dependent manner. Peptide labeling, like cell adhesion, was partially inhibited by heparin and stimulated by EDTA. The 52-kDa protein did not appear to contain sulfated glycan chains and was trypsin sensitive. It was recovered in a membrane fraction and was readily solubilized with Triton X-100 and X-114. Upon phase separation of the Triton X-114, the 52-kDa protein partitioned into the hydrophobic detergent phase. The detergent-solubilized receptor candidate bound selectively to wheat germ agglutinin-Sepharose, and after cell surface labeling with a membrane impermeant biotinylating reagent, bound to streptavidin-Sepharose. Furthermore, fluorescent beads covalently derivatized with peptide specifically decorated intact K562 cells. Thus the properties of the 52-kDa protein are consistent with those of a receptor for the CBD of TS1 and other TS isoforms.
Gao
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AG
Department of Biochemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Frazier
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WA
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D018955:CD36 Antigens; D002478:Cells, Cultured; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D016024:Receptors, Cytoadhesin / Q000378:metabolism*; D019699:Thrombospondins
D015703:Antigens, CD; D018955:CD36 Antigens; D008562:Membrane Glycoproteins; D016024:Receptors, Cytoadhesin; D019699:Thrombospondins