We investigated the influence of the activation state of integrin 51 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked v3 expression and adhered to fibronectin through 51. Mel57 cells adhered through v3 and 51. A recombinant fibronectin polypeptide, containing five type III repeats from the central cell binding domain 3Fn6-10, and a mutated polypeptide lacking the synergy site were equally effective in promoting Mel57 adhesion. For K562 and MV3, the mutated polypeptide was not or poorly active compared to the control polypeptide. Expression of v3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory 1-integrin antibodies or Mn induced 51-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn, 51-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn, and TS2/16, 51-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for 51-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin.

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