BINDING-SITE IN HUMAN PLASMA FIBRONECTIN TO HL-60 CELLS LOCALIZES IN THE C-TERMINAL HEPARIN-BINDING REGION INDEPENDENTLY OF RGD AND CS1

A 29-kDa monomeric dispase-digestive fragment of human plasma fibronec tin has been purified by heparin affinity chromatography. The NH2-term inal sequence was determined as Ala(1687)-Val-Thr-Thr-Ile-Pro-Ala-Pro. By mass spectrometry the molecular weight was determined to be 30,241 .9 with standard deviation of 3.9 amu. Therefore, we defined the C-ter minal sequence of the 29-kDa fragment as Arg(l957)-Lys-Lys-Thr-Gly-Gln -Glu, This indicates that the fragment is composed of 277 amino acids. I-125-fibronectin and the I-125-labeled 29-kDa fragment bound to HL-6 0 (human acute promyelocytic leukemia) cells in a time-dependent, satu rable, and reversible manner. Approximately 120 min was required to re ach maximal binding. There were no differences in quantity or rate of binding of labeled fibronectin and 29-kDa fragment at temperatures of 4 degrees, 22 degrees, and 37 degrees C. The number of binding sites p er HL-60 cell of fibronectin and the 29-kDa fragment were 140,000 with a K-d of 133 nM and 108,000 with a K-d of 250 nM, respectively. The b inding of fibronectin to HL-60 cells was completely inhibited by this fragment, and by the peptides of RGDS and CS1 with IC(50)s of 3.6, 840 , and 670 mu M, respectively. Native fibronectin inhibited the direct binding of the 29-kDa fragment to HL-60 cells; however, RGDS peptide, peptide CS1, or two melanoma cell adhesion-promoting domain peptides i n this 29-kDa fragment (peptide I; Tyr(1906)-Val(1924), peptide II; As p(1946)-Thr(1960)) did not block this binding. Neither heparitinase no r chondroitinase treatment of cells had any effect on these bindings. These results indicate that the C-terminal cell- and heparin-binding d omain of fibronectin mediates HL-60 cell binding by direct interaction independently of RGD, CS1, and melanoma cell adhesion domains in this fragment. (C) 1995 Academic Press, Inc.