Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin- 1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Ga o, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1 996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melan oma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spr eading of A2058 melanoma cells on several gp, integrin ligands, including v itronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independen t of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repea ts expressed as a glutathione S-transferase fusion protein also enhance spr eading on vitronectin and TSP1, Activation of alpha(v)beta(3) integrin by t he soluble peptide 246 stimulates organization of F-actin and increases tyr osine phosphorylation of focal adhesion kinase, In contrast, direct adhesio n of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphoryl ation of focal adhesion kinase, Stimulation of alpha(v)beta(3) integrin fun ction by the type 1 repeat peptide differs from that induced by CD47-bindin g TSP1 peptides in that heparan sulfate proteoglycans are required and pert ussis toxin does not inhibit the former activity. Thus, the type I repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronect in and TSP1 by the alpha(v)beta(3) integrin.