Recombinant GST/CD36 fusion proteins define a thrombospondin binding domain. Evidence for a single calcium-dependent binding site on CD36.
CD36 is a multifunctional cell surface glycoprotein that acts as a surface receptor for thrombospondin (TSP), and thereby may mediate adhesive interactions between cells and substrata, platelets and other cells, and macrophages and apoptotic neutrophils. The identity of the TSP binding site on CD36 is controversial and may involve more than one structural domain. We have constructed a series of recombinant bacterial GST/CD36 fusion proteins that span nearly all of the CD36 molecule and have demonstrated that fusion proteins containing the region extending from amino acid 93 to 120 formed specific, saturable, and reversible complexes with TSP. As with intact CD36, binding was calcium-dependent, was independent of which ligand was immobilized, and was blocked by monoclonal antibodies to both CD36 and TSP. Stoichiometry and affinity of the fusion proteins for TSP were consistent with that of the intact protein. We also demonstrated that these fusion proteins competitively inhibited binding of TSP to purified platelet CD36 and to cell surface CD36 on peripheral blood monocytes and CD36 cDNA-transfected melanoma cells. These data demonstrate that the region between amino acids 93 and 120 has all of the characteristics required of the TSP binding domain.
Frieda
S
S
Department of Medicine (Hematology/Oncology), Cornell University Medical College, New York, New York 10021.
Pearce
A
A
Wu
J
J
Silverstein
R L
RL
D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D018955:CD36 Antigens; D002118:Calcium / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008545:Melanoma; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D019699:Thrombospondins; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018955:CD36 Antigens; D015815:Cell Adhesion Molecules; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins; D005982:Glutathione Transferase; D002118:Calcium