Quantification of Integrin Activation and Ligation in Adherent Cells.

Integrin activation is a crucial event for multiple biological functions. Therefore, methods to detect integrin activation are vital. Since the main cellular function of integrins is adhesion, we and others utilize this feature to measure integrin activation. Here, we describe how to detect the activity of the fibronectin-binding integrin α5ß1 using a fusion of glutathione S-transferase (GST) to the 9th, 10th, and 11th type III repeats on fibronectin (GST-FNIII9-11). Moreover, we detail how to measure αvß3 integrin activity using the ligand-mimetic WOW-1 antibody that selectively binds unoccupied αvß3 integrins. Finally, we describe methods of testing ligation of fibronectin-binding integrins utilizing monoclonal antibodies against ligand-induced binding sites (LIBS).

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5ß1 Integrin at the Macrophage-Amebae Intercellular Junction.

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5ß1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5ß1 integrin and NLRP3 into the intercellular junction, where α5ß1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5ß1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5ß1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5ß1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5ß1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.

Targeting integrins αvß3 and α5ß1 with new ß-lactam derivatives.

The αvß3 and α5ß1 integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. We report here the design, synthesis and biological activity of some new ß-lactam derivatives specifically designed to target integrins. The new molecules contain the azetidinone as the only cyclic framework armed with carboxylic acid and amine terminals spaced from 9 to 14 atoms to switch on recognition by integrins. All tested molecules showed a concentration-dependent enhancement in fibronectin-mediated adhesion of K562 and SK-MEL-24 cells; in particular 1, expressed a higher affinity towards α5ß1 integrin (EC50 of 12 nM) and 2 was more selective for integrin αvß3 (EC50 of 11 nM).

Contribution of distinct platelet integrins to binding, unfolding, and assembly of fibronectin.

Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.

alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.