BMSEHA15
Published online 15 January 2010
(Haematologica 2010, 10.3324/haematol.2009.018572)
Copyright © 2010 by Ferrata Storti Foundation
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Article

L718P mutation in the membrane-proximal cytoplasmic tail of β3 promotes abnormal {alpha}IIbβ3 clustering and lipid microdomain coalescence, and associates with thrombasthenia-like phenotype

Asier Jayo1,*, Isabel Conde1,*, Pedro Lastres1, Constantino Martínez2, José Rivera2, Vicente Vicente2, Consuelo González-Manchón1,3

1 Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain
2 Centro de Hemodonación, Universidad de Murcia, Murcia, Spain
3 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)

Correspondence: Consuelo González-Manchón, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. Phone: international +34-91-8373112; Fax: international 34-91-5360432. E-mail: cgmanchon{at}cib.csic.es

ABSTRACT

Background: Support for the role of transmembrane and membrane-proximal domains of {alpha}IIbβ3 integrin in maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bidirectional transmembrane signaling.

Design and methods: We report the functional characterization of mutant {alpha}IIbβ3 integrin carrying the Leu718Pro mutation in the membrane-proximal region of the β3 cytoplasmic domain, identified in heterozygosis in a patient with severe bleeding phenotype and defective platelet aggregation and adhesion.

Results: Transiently transfected cells expressed similar levels of normal and mutant {alpha}IIbβ3, but surface expression of mutant {alpha}vβ3 was reduced due to its retention in intracellular compartments. Cells stably expressing mutant {alpha}IIbβ3 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}IIbβ3 clusters underneath the entire cell body and the presence of atypical high-density patches of clustered {alpha}IIbβ3 containing encircled areas devoid of integrin that showed decreased affinity for the fluorescent lipid analog DiIC16 and were disrupted in cholesterol-depleted cells.

Conclusions: These findings are consistent with an important role of the membrane-proximal region of β3 in modulating {alpha}IIbβ3 clustering and lateral redistribution of membrane lipids. Since the β3 mutant associated to thrombasthenic phenotype in a patient carrying one normal β3 allele, the results support a dominant role of clustering in regulating integrin {alpha}IIbβ3 functions in vivo.

Key words: {alpha}IIbβ3 mutation, integrin clustering, lipid phase separation, thrombasthenia.


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J. S. Bennett and D. T. Moore
Regulation of platelet {beta}3 integrins
Haematologica, July 1, 2010; 95(7): 1049 - 1051.
[Full Text] [PDF]