title: Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.
abstract: Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for ERK2. These measures of FAK and ERK2 activity were found to correlate with short term cell-substratum adhesivity, indicating that signaling via FAK and ERK2 is proportional to the number of integrin-fibronectin bonds.
authors.affiliation: Department of Chemical Engineering, Division of Bioengineering and Environmental Health, and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Asthagiri
authors.forename: C M
authors.initials: CM
authors.lastname: Nelson
authors.forename: A F
authors.initials: AF
authors.lastname: Horwitz
authors.forename: D A
authors.initials: DA
authors.lastname: Lauffenburger
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006224:Cricetinae; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007700:Kinetics; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017469:Receptors, Fibronectin / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
chemical_list: D015815:Cell Adhesion Molecules; D008024:Ligands; D017469:Receptors, Fibronectin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019950:Mitogen-Activated Protein Kinase 1