Ser752 mutation to Pro or Ala in the beta3 integrin subunit differentially affects the kinetics of cell spreading to von Willebrand factor and fibrinogen.
The beta3 cytoplasmic domain of the alpha v beta3 integrin is essential for intracellular signals required for cytoskeletal rearrangements. Expression of beta3Ser752Pro mutation in heterologous cells profoundly affects cell spreading and beta3 localization into focal contacts. However, the beta3Ser752Ala substitution mostly restores normal integrin functions, suggesting that the presence of Pro is responsible for the receptor's loss of function. To further assess the role of the Ser752 of the beta3 cytoplasmic domain in the cytoskeletal organization of adherent cells, we developed a computer-assisted method of image analysis allowing the automatic classification of spread cells according to the quantitative analysis of their cell morphology. We compared adhesion and spreading to von Willebrand factor (vWF) or fibrinogen (Fg) of cells expressing beta3 wild type, beta3Ser752Pro or beta3Ser752Ala mutated integrin subunit as a chimeric alpha v beta3 receptor. The beta3Ser752Ala substitution did not impair the general ability of cells to spread, but resulted in a delayed and reduced spreading on both vWF and Fg. Moreover, the beta3Ser752Ala mutation produced modifications of the morphology of spread cells, suggesting a disorganization of their cytoskeleton. Attachment studies showed that the beta3Ser752Ala mutation did not modify the capacity of cells to attach to the substrate, indicating no change in the ligand binding affinity of the alpha v beta3 integrin. Furthermore, we identified a slight defect of beta3Ser752Pro cell attachment to vWF and Fg, beside their impairment of spreading. Taken together, these results suggest a role of Ser752 of the beta3 cytoplasmic domain in the optimal cytoskeletal organization of adherent cells.
Perrault
C
C
INSERM U143, Bicetre, France.
Mekrache
M
M
Schoevaert
D
D
Kieffer
N
N
Melchior
C
C
Warszawski
J
J
Baruch
D
D
D000409:Alanine / Q000502:physiology; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D002452:Cell Count / Q000379:methods; D002460:Cell Line; D006224:Cricetinae; D003599:Cytoskeleton / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D039661:Integrin beta3; D007700:Kinetics; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D011392:Proline / Q000502:physiology; D012694:Serine / Q000502:physiology*; D014841:von Willebrand Factor / Q000378:metabolism
D015703:Antigens, CD; D039661:Integrin beta3; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012694:Serine; D005340:Fibrinogen; D011392:Proline; D000409:Alanine