Abstract
A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.
Highlights
Addition, together with thrombin-converted insoluble fibrin, fibrinogen functions as a component of the extracellular matrix in non-hemostatic normal or pathological processes promoting placenta development, angiogenesis, atherosclerosis, metastasis, as well as a variety of vascular and renal diseases [1]
Using intact fibrinogen and well characterized proteolytic fragments containing either the ␥400–411 site or the A␣572–575 RGDS site as ligands and platelets as well as Chinese hamster ovary (CHO) cells expressing resting wild type (␣IIb3wt), constitutively active (␣IIb3T562N), or non-functional (␣IIb3D119Y) receptors, we report a new functional role of the RGD motif as a molecular switch that triggers an ␣IIb3-dependent signaling cascade leading to Src-dependent RhoA activation and cell spreading
A Two-step Mechanism Underlies ␣IIb3-dependent CHO Cell Spreading on Fibrinogen—Integrins ␣v3 and ␣IIb3 mediate cell adhesion to immobilized fibrinogen through two distinct molecular mechanisms: while ␣v3-dependent cell adhesion to fibrinogen relies essentially on the C-terminal A␣572–575 RGDS recognition site in the fibrinogen ␣ chain [15], ␣IIb3-mediated cell adhesion is mediated primarily through the ␥ chain C-terminal ␥400–411 sequence [11, 15]
Summary
Addition, together with thrombin-converted insoluble fibrin, fibrinogen functions as a component of the extracellular matrix in non-hemostatic normal or pathological processes promoting placenta development, angiogenesis, atherosclerosis, metastasis, as well as a variety of vascular and renal diseases [1].
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