Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Abstract

Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60csrc, ppl25FAKphosphatidylinositol-3-kinase, phospholipase C-'y, and Na+/H+ antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp6Ocsrc, pp125FAK, phospholipase G-y, and the Na+ /H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60csrc and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.

BAX inhibitor-1 enhances cancer metastasis by altering glucose metabolism and activating the sodium-hydrogen exchanger: the alteration of mitochondrial function

“…NHE inhibition reduces the migration rate of MDCK-F cells (Klein et al, 2000), whereas NHE activation augments the motility and the invasiveness of human breast carcinoma cells (Reshkin et al 2000). Interestingly, the NHE and integrin receptor molecules are often colocalized at the focal adhesion sites of the leading edges of lamellipodia (Grinstein et al, 1993;Plopper et al, 1995). Thus, NHE could create a proton-enriched microenvironment in the immediate vicinity of the focal adhesion complexes, that is, close to integrin molecules.…”

“…NHE could be strongly inhibited by 5-(Nethyl-N-isopropyl) amiloride (EIPA, an inhibitor of NHE1) in BI-1 cells ( Figure 5c). NHE extrudes H þ through intrusion of Na þ to regulate intracellular pH (Plopper et al, 1995;Klein et al, 2000). To understand the physiological meaning of NHE1 in BI-1 cells, intraand extracellular pH were measured in Neo, BI-1 and CDBI-1 cells with or without EIPA.…”

“…The acidic environment is produced by the activity of ion channels and transporters such as Na þ /H þ or Cl/HCO 3 exchangers (Klein et al, 2000;Saadoun et al, 2005). The ubiquitously expressed Na þ /H þ exchanger isoform, sodium hydrogen exchanger (NHE1), also contributes to this acidic environment (Plopper et al, 1995). NHE1 activity is important for many tumor cells (Lagarde et al, 1988;Rotin et al, 1989;McLean et al, 2000).…”

Bax Inhibitor 1 in apoptosis and disease

“…Tumour development is promoted by the formation of a patho-physiological microenvironment characterised by hypoxic conditions, nutrient deprivation and acidification. This acidic environment is regulated by ion channels and transporters, for example, Na þ /H þ (NHE) or Cl/HCO 3 exchangers (Plopper et al, 1995;Lee et al, 2010b). Elevated NHE levels enhance the acidification of extracellular space by extruding excess protons from tumour cells (Tannock and Rotin, 1989;Wahl et al, 2000), which by activating proteases for example, can promote the invasiveness of carcinoma cells (Reshkin et al, 2000;Lee et al, 2010b).…”

Microenvironmental regulation of biomacromolecular therapies

“…This approach may potentially increase the effect of therapeutic protein expression 129 . Because growth-factor receptors are spatially associated with integrins at the focal-adhesion complex 130 , the binding of bFGF might have multiple effects. The inclusion of a pDNA encoding dendritic-cell growth factor and fmsrelated tyrosine kinase 3 (FLT3) ligand in a DNA vaccine similarly enhanced the level of the resultant immune response 131 .…”