Abstract
4N1K is a peptide fragment derived from the C-terminal, globular domain of thrombospondin which has been shown to mediate integrin-dependent cell adhesion and promote integrin activation acting via the cell-surface receptor, CD47. However, some studies found that 4N1K could act independently of CD47, putting in question the specificity of 4N1K for CD47. This led us to characterize the cellular and non-cellular effects of 4N1K. We found that 4N1K stimulated a potent increase in binding of a variety of non-specific IgG antibodies to cells in suspension. We also found that these same antibodies, as well as CD47-deficient cells, could bind substrate-immobilized 4N1K significantly better than a control peptide, 4NGG. Furthermore, we found that cells treated with 4N1K at higher concentrations inhibited, while lower concentrations promoted cell adhesion to immobilized fibronectin as an integrin substrate. Importantly, both the stimulatory and the inhibitory activity of 4N1K occurred as efficiently in the CD47-deficient JinB8 cells, as it did in the CD47-expressing parental or in JinB8 cells reconstituted with CD47 expression. Given these results, we suggest that 4N1K interacts non-specifically with epitopes commonly found on the cell surface, and conclude that it is not a suitable peptide for use to study the consequences of CD47 receptor ligation.
Highlights
Integrins are a family of cell adhesion receptors that can be regulated by conformational changes in their extracellular domains which modulate their affinity state for binding to ligands [1]
Compared to untreated or 4NGG-treated controls, MnCl2 treatment did not alter binding of 6A or A1 cells to either: an activation-independent b1-integrin antibody TS2/16 (Fig. 2B), a non-specific mouse IgG control antibody (Fig. 2C), or to the goat anti-mouse (GAM) secondary antibody used for all assays (Fig. 2D)
Since binding of the fluorophore-conjugated secondary antibody alone is sufficient to explain the ‘pseudo’ integrin activation data, we titrated the concentration of 4N1K incubated with the cells to show that GAM secondary antibody binds to A1 cells in a dose-dependent manner (Fig. 1E)
Summary
Integrins are a family of cell adhesion receptors that can be regulated by conformational changes in their extracellular domains which modulate their affinity state for binding to ligands [1]. CD47, known as ‘‘Integrin Associated Protein’’ (IAP) is a penta-spanning receptor protein with a highly glycosylated, IgV-containing extracellular domain that mediates binding to signal regulatory protein (SIRP) and to thrombospondins (TSP) [4,5,6]. All five members of the TSP family share a common C-terminal domain containing a VVM motif that mediates cell binding to CD47 [4,9]. The 10mer KRFYVVMWKK peptide, commonly known as 4N1K, is derived from the C-terminal globular domain that was characterized as the main site responsible for TSP-mediated cell adhesion [10,11]. CD47 was subsequently determined to be the receptor responsible for 4N1K-mediated cell adhesion since adhesion to this peptide was enhanced in CD47-transfected OV10 cells compared to non-transfected cells [12]
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