title: L718P mutation in the membrane-proximal cytoplasmic tail of beta 3 promotes abnormal alpha IIb beta 3 clustering and lipid microdomain coalescence, and associates with a thrombasthenia-like phenotype.
abstract: BACKGROUND
  Support for the role of transmembrane and membrane-proximal domains of alpha IIb beta 3 integrin in the maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bi-directional transmembrane signaling.
  
  
  METHODS
  We report the functional characterization of a mutant alpha IIb beta 3 integrin carrying the Leu718Pro mutation in the membrane-proximal region of the beta 3 cytoplasmic domain, identified in heterozygosis in a patient with a severe bleeding phenotype and defective platelet aggregation and adhesion.
  
  
  RESULTS
  Transiently transfected cells expressed similar levels of normal and mutant alpha IIb beta 3, but surface expression of mutant alpha v beta 3 was reduced due to its retention in intracellular compartments. Cells stably expressing mutant alpha IIb beta 3 showed constitutive binding to soluble multivalent ligands as well as spontaneous fibrinogen-dependent aggregation, but their response to DTT was markedly reduced. Fibrinogen-adherent cells exhibited a peculiar spreading phenotype with long protrusions. Immunofluorescence analysis revealed the formation of alpha IIb beta 3 clusters underneath the entire cell body and the presence of atypical high-density patches of clustered alpha IIb beta 3 containing encircled areas devoid of integrin that showed decreased affinity for the fluorescent lipid analog DiIC(16) and were disrupted in cholesterol-depleted cells.
  
  
  CONCLUSIONS
  These findings are consistent with an important role of the membrane-proximal region of beta 3 in modulating alpha IIb beta 3 clustering and lateral redistribution of membrane lipids. Since the beta 3 mutant was associated with a thrombasthenic phenotype in a patient carrying one normal beta 3 allele, these results support a dominant role of clustering in regulating integrin alpha IIb beta 3 functions in vivo.
authors.affiliation: Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Asier
authors.initials: A
authors.lastname: Jayo
authors.forename: Isabel
authors.initials: I
authors.lastname: Conde
authors.forename: Pedro
authors.initials: P
authors.lastname: Lastres
authors.forename: Constantino
authors.initials: C
authors.lastname: Martínez
authors.forename: José
authors.initials: J
authors.lastname: Rivera
authors.forename: Vicente
authors.initials: V
authors.lastname: Vicente
authors.forename: Consuelo
authors.initials: C
authors.lastname: González-Manchón
mesh_terms: D000328:Adult; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics*; D008563:Membrane Lipids; D021962:Membrane Microdomains / Q000378:metabolism*; D020125:Mutation, Missense; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D013915:Thrombasthenia / Q000235:genetics / Q000473:pathology*
chemical_list: D039661:Integrin beta3; D008563:Membrane Lipids; D019039:Platelet Glycoprotein GPIIb-IIIa Complex