Ser 752 mutation to Pro or Ala in the beta 3 integrin subunit differentially affects the kinetics of cell spreading to von Willebrand factor and fibrinogen

The beta 3 cytoplasmic domain of the alpha v beta 3 integrin is essential f or intracellular signals required for cytoskeletal rearrangements. Expressi on of beta 3Ser752Pro mutation in heterologous cells profoundly affects cel l spreading and beta 3 localization into focal contacts. However, the beta 3Ser752Ala substitution mostly restores normal integrin functions, suggesti ng that the presence of Pro is responsible for the receptor's loss of funct ion. To further assess the role of the Ser752 of the beta 3 cytoplasmic dom ain in the cytoskeletal organization of adherent cells, we developed a comp uter-assisted method of image analysis allowing the automatic classificatio n of spread cells according to the quantitative analysis of their cell morp hology. We compared adhesion and spreading to von Willebrand factor (VWF) o r fibrinogen (Fg) of cells expressing beta 3 wild type, beta 3Ser752Pro or beta 3Ser752Ala mutated integrin subunit as a chimeric alpha v beta 3 recep tor. The beta 3Ser752Ala substitution did not impair the general ability of cells to spread, but resulted in a delayed and reduced spreading on both V WF and Fg. Moreover, the beta 3Ser752Ala mutation produced modifications of the morphology of spread cells, suggesting a disorganization of their cyto skeleton. Attachment studies showed that the beta 3Ser752Ala mutation did n ot modify the capacity of cells to attach to the substrate, indicating no c hange in the ligand binding affinity of the alpha v beta 3 integrin. Furthe rmore, we identified a slight defect of beta 3Ser752Pro cell attachment to vWF and Fg, beside their impairment of spreading. Taken together, these res ults suggest a role of Ser752 of the beta 3 cytoplasmic domain in the optim al cytoskeletal organization of adherent cells.