α3aβ1 integrin localizes to focal contacts in response to diverse extracellular matrix proteins

Abstract

In vitro binding assays and inhibition of cell adhesion with monoclonal antibodies have implicated the integrin alpha 3 beta 1 as a receptor for a variety of extracellular ligands. However, reports of alpha 3 beta 1-ligand interactions are inconsistent, and transfection studies have suggested that alpha 3 beta 1 is not sufficient for cell attachment to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the alpha 3A cytoplasmic domain variant in different cultured cell types. Using standard fixation and permeabilization methods, antibodies specific for alpha 3A stained most cell types in a diffuse pattern, consistent with previous reports. Surprisingly, however, chemical cross-linking of integrins to the extracellular matrix and extraction of the cytoskeleton prior to immunofluorescence revealed alpha 3A in focal contacts of most cells tested, suggesting that the cytoplasmic domain was concealed in intact focal contacts by cytoskeletal or other cytoplasmic proteins. The alpha 3A subunit localized to focal contacts in several cell types cultured on fibronectin, kalinin/laminin 5, EHS-laminin/laminin 1, type IV collagen, or vitronectin. In contrast, alpha 5 and alpha V integrins were detected in focal contacts only in cells grown on their known ligands (fibronectin, and fibronectin or vitronectin, respectively). Therefore, our results show that alpha 3A beta 1 responds to a broad spectrum of extracellular ligands. Time course comparisons of the recruitment of alpha subunits from different fibronectin receptors suggested that localization of alpha 3A beta 1 to fibronectin-induced focal contacts was independent of the recruitment of alpha 5 and alpha 4 integrins. However, other studies have shown that alpha 3A beta 1 does not mediate initial cell adhesion to many of the ligands that induced its focal contact localization, including fibronectin. Therefore, we suggest that alpha 3A beta 1 may be a secondary receptor with post-cell-adhesion functions for a broad spectrum of extracellular matrices.

Effect of Ligand Conformation on Melanoma Cell α3β1Integrin-Mediated Signal Transduction Events: Implications for a Collagen Structural Modulation Mechanism of Tumor Cell Invasion

“…We believe that binding to type IV collagen is initiated via the R 2 β 1 and R 1 β 1 integrins, and then R 3 β 1 integrin is recruited to sites on the substrate including R1(IV)531-543. This model is consistent with that of DiPersio et al (49), who demonstrated that the R 3A β 1 integrin is localized to focal contacts following cell adhesion to type IV collagen, but does not initiate cell adhesion to this ligand. The primary role of R 3 β 1 would then be in initiating postadhesion signal transduction events.…”

“…The R 3 β 1 integrin is upregulated in certain tumor cell types, such as metastatic melanoma (55,56). DiPersio et al (49) and Menter et al (57) have shown that the R 3 β 1 integrin is recruited to the site of focal contacts, and we have demonstrated direct binding of melanoma and ovarian carcinoma cells to the R1(IV)531-543 site in type IV collagen (24,26). (b) Once the R 3 β 1 integrin binds R1(IV)531-543, the cytoplasmic tail (C-terminus) of the β 1 integrin subunit may interact directly with the N-terminal region of p125 FAK (58) or with talin, which binds to the C-terminus of p125 FAK (59).…”

Integrin α6β4 requires plectin and vimentin for adhesion complex distribution and invasive growth

“…S2). This observation is in line with previous studies on integrin α6β4 (Elaimy et al, 2019;Lotz et al, 1997) and a prevailing concept that laminin integrins tend to form smaller FCs (DiPersio et al, 1995). Previous work in keratinocytes suggests that hemidesmosomes and FAs associate but treadmill as separate entities, particularly at the leading edge of a migrating cell (Pora et al, 2019).…”

The effects of inhibition and siRNA knockdown of collagen-binding integrins on human umbilical vein endothelial cell migration and tube formation

“…Finally, integrins form focal adhesions, which contain many other signalling molecules and scaffolding proteins, in the absence of integrin expression these focal adhesions will have a different composition that could affect the downstream signalling processes. For example, α3β1 has been found in focal adhesions even when its ligand is not present 51 . In our case, inhibition of α2β1 disrupts tube formation because HUVECs rely on this integrin to facilitate appropriate adhesion to the ECM, when we knockdown this integrin the cells have time to upregulate expression of other ECM receptors to compensate for the loss of α2β1 and there is therefore no disruption of tube formation.…”