The objective of this proposal is to analyze the mechanism of Fibronectin (Fn) recognition by alpha-5-beta-1 and other integrins important for the biological properties of normal and transformed cells. Fn, a prototype adhesion molecule, is composed of 3 types of repeating modules and the solution structures for representatives of each type have been elucidated. The cell binding domain of Fn, which is made up of type III modules (3Fn), is ideal for structure-function studies using prokaryotic expression systems. It is hypothesized that the recognition of Fn, and possibly other ligands recognized by integrins, involves extended complementary surfaces between the two molecules. Part of the Fn surface recognized by alpha-5- beta-1 involves the Arg- Gly-Asp sequence located in a turn between F and G beta strands in the 10th 3Fn module (3Fn10) and other residues located in the 9th 3Fn module (3Fn9). The hypothesis will be tested using a random mutagenesis approach combined with the power of in vitro selection. A filamentous phage display system expressing 3Fn9l0 module pairs with random substitutions of the 20 possible residues will be selected for specific properties. First, pseudorevertants of a D1495E mutation that improve the alpha-5-beta-1 binding affinity will be isolated. This positive selection will provide insight into which residues in the 3Fn9I0 module pair are in close proximity to alpha-5-beta-1. A second selection will enrich for substitutions that decrease the affinity for alpha-5-beta-1, but have a lesser affect on other integrins that recognize the cell binding domain of Fn. An alternative strategy employing alanine scanning mutagenesis will also be used. Loops between the ~ strands of the 3Fn910 module pair and Fn residues spanning D137&T1383 will be specifically targeted and analyzed for affects on alpha-5-beta-1 interactions. Finally, residues involved in 3Fn910 binding will be substituted with corresponding regions from immunoglobulin domains of VCAM-1 recognized by alpha-5-beta-1 to test whether specific residues presented in the same location of a different beta-barrel structure can alter the integrin specificity. These studies will provide fundamental information on the mechanisms of integrin-ligand recognition and may provide a starting point for the identification of novel integrin recognition structures. Specific control of integrin function may permit the inhibition or enhancement of cell adhesion, migration. and proliferation.
