A New Functional Role of the Fibrinogen RGD Motif as the Molecular Switch that Selectively Triggers Integrin aIIbb3-Dependent Cell Spreading, but not Cell Attachment

Abstract number: OR105

Salsmann A, Schaffner-Reckinger E, Kabile F, Margue C, Plançon S, Kieffer N

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 aIIbb3 occurs exclusively through the synergistic g^400–411 sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen g^400–411 sequence and the RGD motif in the molecular events leading to ligand-induced aIIbb3-dependent cell spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the g^400–411 sequence (fragment D), and CHO cells expressing resting wild type (aIIbb3wt), constitutively active (aIIbb3T562N) or non-functional (aIIbb3D119Y) receptors. Our data provide evidence that the g^400–411 site by itself is able to initiate aIIbb3 clustering and recrutement 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 b3 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 b3 subunit leading to maximal RhoA activation and stress fiber organization. Finally, aIIbb3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and PI3 kinase-independent.