Pre-B cells adhere to fibronectin via interactions of integrin alpha 5/alpha V with RGDS as well as of integrin alpha 4 with two distinct V region sequences at its different binding sites.
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-pyridyldithio) propionic acid N-hydroxysuccinimide ester and subsequently with dithiothreitol (DTT) allowed the plates to adsorb DTT-treated extracellular matrix (ECM) proteins as well as synthetic peptides containing cysteine at the N-terminal. These treatments produced culture plates coated with ECM proteins or peptides 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 peptides, 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 peptide used for coating, because each binding to a given peptide-coated plate was inhibited only by addition of a homologous free peptide. This assay system further demonstrated that the pre-B cell binding to RGDS was mediated by the alpha 5 and alpha v integrins, whereas the binding to LDVP and RGDV was mediated by the alpha 4 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.2+ interaction involving complex molecular mechanisms.
Narumiya
S
S
Biomedical Research Center, Osaka University Medical School, Japan.
Abe
Y
Y
Kita
Y
Y
Miyake
K
K
Nakajima
K
K
Watanabe
T X
TX
Oka
Y
Y
Sugiyama
H
H
Yagita
H
H
Okumura
K
K
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D005353:Fibronectins / Q000378:metabolism*; D039441:Integrin alpha4; D039482:Integrin alpha5; D039564:Integrin alphaV; D016023:Integrins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*
D015703:Antigens, CD; D005353:Fibronectins; D039482:Integrin alpha5; D039564:Integrin alphaV; D016023:Integrins; D009842:Oligopeptides; D039441:Integrin alpha4; C046617:arginyl-glycyl-aspartyl-serine