DEFINING THE TOPOLOGY OF INTEGRIN ALPHA-5-BETA-1-FIBRONECTIN INTERACTIONS USING INHIBITORY ANTI-ALPHA-5 AND ANTI-BETA-1 MONOCLONAL-ANTIBODIES - EVIDENCE THAT THE SYNERGY SEQUENCE OF FIBRONECTIN IS RECOGNIZED BY THE AMINO-TERMINAL REPEATS OF THE ALPHA-5 SUBUNIT

The high affinity interaction of integrin alpha 5 beta 1 with the cent ral cell binding domain (CCBD) of fibronectin requires both the Arg-Gl y-Asp (RGD) sequence (in the 10th type III repeat) and a second site ( in the adjacent 9th type III repeat) which synergizes with RGD, We hav e attempted to map the fibronectin binding interface on alpha 5 beta 1 using monoclonal antibodies (mAbs) that inhibit ligand recognition, T he binding of two anti-alpha 5 mAbs (P1D6 and JBS5) to alpha 5 beta 1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (cont aining both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha 5 beta 1. In contrast, binding o f the anti-beta 1 mAb P4C10 to alpha 5 beta 1 was inhibited to a simil ar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding, P1D6 inhibited the interaction of a wild type CCBD fragment with alpha 5 beta 1 but had no effect on the binding of a mutant fragment that lacked the synergy region, The e pitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alp ha 5 subunit, Our results indicate that the synergy region is recogniz ed primarily by the alpha 5 subunit (in particular by its NH2-terminal repeats) but that the beta 1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mech anisms, specificity, and topology of integrin-ligand interactions.