Pre-B cells adhere to fibronectin via interactions of integrin α5/αv with RGDS as well as of integrin α4 with two distinct V region sequences at its different binding sites

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Abstract
The present study aimed to develop an assay system capable of directly examining the adhesion of cells to each cell-binding site on fibronectin (FN) and to investigate molecular mechanisms underlying the pre-B cell – FN interaction. Treatment of culture plates with 3-(2-pyrtdyldlthlo) proplonlc acid N-hydroxysuccinlmlde ester and subsequently with dtthlothreltol (DTT) allowed the plates to adsorb DTT-treated extracellular matrix (ECM) proteins as well as synthetic peptldes containing cystelne at the N-termlnal. These treatments produced culture plates coated with ECM proteins or peptldes corresponding to Its cell-binding sequences, I.e. three sites on FN termed RGDS, LDVP, and RGDV. Pre-B cells exhibited potent adhesiveness to FN-coated plates. Its FN binding was most efficiently inhibited by adding a combination of free forms of RGDS, LDVP, and RGDV peptldes, Indicating the involvement of these three cell-binding sites in the pre-B cell – FN interaction. In accordance with this, pre-B cells exhibited considerable and potent binding to the respective RGDS-, LDVP-, or RGDV-coated plates. Such binding was specific for the peptlde used for coating, because each binding to a given peptlde-coated plate was inhibited only by addition of a homologous free peptlde. This assay system further demonstrated that the pre-B cell binding to RGDS was mediated by the α5 and αv integrins, whereas the binding to LDVP and RGDV was mediated by the α4 Integrin. It was also shown that LDVP binding was inhibited by LDVP but not by RGDV and, likewise, RGDV binding was inhibited by RGDV but not by LDVP. These results indicate that pre-B cells adhere to FN molecules at three different cell-binding sites via two integrins, α5 and αv for RGDS and α4 for LDVP/RGDV sequences, and that the α4 Integrin interacts with LDVP and RGDV at its mutually different binding sites. The results are discussed in the context of the biological significance for the existence of the cell – ECM (FN) interaction involving complex molecular mechanisms.

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