Experimental Investigation of the Relationship Between Integrin Clustering and Focal Adhesion Kinase Phosphorylation

Erik S. Welf, Chemical Engineering, University of Delaware, 150 Academy Street, Colburn Lab, Newark, DE 19716, Babatunde A. Ogunnaike, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, and Ulhas P. Naik, Biological Science, University of Delaware, 150 Academy St., Newark, DE 19716.

Integrins are transmembrane proteins that bind extra-cellular matrix (ECM) proteins to facilitate cell adhesion and migration. In this role, the two main functions of integrins are to sense the extra-cellular environment and to regulate cell attachment. Accomplishing these tasks requires that integrins interact dynamically with numerous multi-functional cytoplasmic proteins that alter integrins' ability to bind ECM and transduce signals as a result of integrin binding to ECM. As a consequence of these multi-functional protein-protein interactions that occur where integrins cluster, it is still unknown how integrins and integrin clustering regulate cell adhesion and migration as a function of ECM properties, and how integrins themselves are regulated by the molecules that bind integrins.

To determine how cells use integrins to sense and respond to their environment, we investigate the relationship between integrin clustering and the intracellular protein focal adhesion kinase (FAK) experimentally. FAK senses integrin clustering by phosphorylating itself at locations where integrins cluster [1], and it regulates integrin cluster size by associating with a host of proteins that affect integrin binding and clustering [2]. Because FAK acts as both a sensor and a regulator of integrin clustering, it has been identified as a key protein that facilitates integrin function in both their adhesive role and their signaling role. In this work, we use an in vitro experimental system to investigate how cells use integrin clustering and FAK phosphorylation to sense and respond to ECM protein concentration.

Cells stably expressing the IIb3 integrin were allowed to spread on varying concentrations of the ECM molecule fibrinogen (Fg), a specific ligand for IIb3. By measuring integrin cluster sizes on different concentrations of Fg, we show that lower concentrations of Fg result in larger integrin clusters. To investigate how cells sense and respond to changes in Fg concentration, we examined the role of FAK in integrin cluster regulation by measuring FAK phosphorylation as a function of Fg concentration. Differences in the magnitude and dynamics of FAK phosphorylation resulting from cell adhesion to different ECM ligand densities suggest bi-directional relationships between FAK activation and integrin clustering. The quantitative relationships between integrin clustering and FAK phosphorylation that we elucidate provide new insight into how integrin clustering and intracellular signaling are coupled to relay important information about the ECM into the cell and simultaneously move the cell across the ECM.

[1] Miyamoto S et al. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. (1995) J Cell Biol 131, 791-805.

[2] Sieg DA, Hauck CR and Schlaepfer DD (1999) Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci 112, 2677-2691.