IDENTIFICATION OF A RECEPTOR CANDIDATE FOR THE CARBOXYL-TERMINAL CELL-BINDING DOMAIN OF THROMBOSPONDINS

The carboxyl-terminal cell binding domain (CBD) of thrombospondin-1 (T S1) contains two cell attachment peptides, 4N1s (RFYVVMWK) and 7N3 (FI RVVMYGKK), which share the sequence VVM. These peptides, and more solu ble derivatives have been radiolabeled with I-125 and used in conjunct ion with a variety of membrane impermeant cross-linking reagents to id entify and characterize receptor candidates on several cell types. All of the VVM containing peptides tested with five different cross-linki ng reagents specifically labeled a 52-kDa protein, which was also affi nity labeled by the recombinant TS1 CBD. After cross-linking peptide t o K562 cells to block the 52-kDa protein, both cell adhesion to and af finity labeling by VVM peptides were inhibited in a concentration-depe ndent manner. Peptide labeling, like cell adhesion, was partially inhi bited by heparin and stimulated by EDTA The 52-kDa protein did not app ear to contain sulfated glycan chains and was trypsin sensitive. It wa s recovered in a membrane fraction and was readily solubilized with Tr iton X-100 and X-114. Upon phase separation of the Triton X-114, the 5 2-kDa protein partitioned into the hydrophobic detergent phase. The de tergent-solubilized receptor candidate bound selectively to wheat germ agglutinin-Sepharose, and after cell surface labeling with a membrane impermeant biotinylating reagent, bound 60 streptavidin-Sepharose. Fu rthermore, fluorescent beads covalently derivatized with peptide speci fically decorated intact K562 cells. Thus the properties of the 52-kDa protein are consistent with those of a receptor for the CBD of TS1 an d other TS isoforms.