Abstract
We investigated the influence of the activation state of integrin alpha 5 beta 1 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked alpha v beta 3 expression and adhered to fibronectin through alpha 5 beta 1. Mel57 cells adhered through alpha v beta 3 and alpha 5 beta 1. 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 alpha v beta 3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory beta 1-integrin antibodies or Mn2+ induced alpha 5 beta 1-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn2+, alpha 5 beta 1-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn2+ or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn2+, and TS2/16, alpha 5 beta 1-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for alpha 5 beta 1-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin.
Highlights
The CCBD consists of type III repeats, each containing approximately 90 amino acids [6]
We show that requirement for the PHSRN synergy site for cell adhesion to the CCBD depends on the integrins expressed and on the activity of the integrins involved
K562, MV3, and Mel57 Differentially Adhere to the CCBD—We investigated adhesion of K562 human erythroleukemic cells and MV3 and Mel57 human melanoma cells to the CCBD
Summary
The CCBD consists of type III repeats, each containing approximately 90 amino acids [6]. For ␣51 binding to Fn, the synergy region in 3Fn9 is the most important of these two regions [21], and recently, a short amino acid sequence ProHis-Ser-Arg-Asn (PHSRN) was identified in this repeat that synergistically enhances the cell adhesion promoting activity of the RGD sequence [24]. This sequence is present in an 11-amino acid integrin binding site from 3Fn9 that is recognized by ␣IIb3 [25]. We show that requirement for the PHSRN synergy site for cell adhesion to the CCBD depends on the integrins expressed and on the activity of the integrins involved
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