THROMBOSPONDIN MODULATES ALPHA(V)BETA(3) FUNCTION THROUGH INTEGRIN-ASSOCIATED PROTEIN

Integrin-associated protein (IAP) is a receptor for the carboxyl-termi nal ''cell-binding domain'' (CBD) of thrombospondin 1 (TS1). IAP assoc iates with alpha(v) beta(3) integrin and mAbs against IAP inhibit cert ain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adh esion and spreading on vitronectin (VN) of C32 human melanoma cells wh ich express IAP, alpha(v) beta(3), and alpha(v) beta(5). Cells adhere to VN at low surface densities via alpha(v) beta(5) and spread very sl owly while adhesion to higher density VN involves both alpha(v) beta(5 ) and alpha(v) beta(3) and results in rapid spreading. Spreading of th e cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the ''mutant'' peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against alpha(v ) beta(3) and the anti-IAP mAb B6H12. Correlated with this enhanced sp reading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading i nduced by TS1 and 4N1K and the constitutive spreading on higher densit y VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), an d tyrosine kinase inhibitors. In contrast, pertussis toxin specificall y blocks only the TS1 stimulated spreading on low density VN, indicati ng that IAP exerts its effects on signal transduction via a heterotrim eric Gi protein acting upstream of a common cell spreading pathway whi ch includes PI-3 kinase, PKC, and tyrosine kinases.