Nelly Kieffer

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a β3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either αIIb or β3 allowed normal expression, heterodimerization, processing and surface exposure of αIIb_GFPβ3 and αIIbβ3_GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced αIIbβ3 capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged αIIbβ3. GFP-tagged αIIbβ3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125^FAK tyrosine phosphorylation similar to wild-type αIIbβ3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to β3, but not to αIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of αIIbβ3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged αIIbβ3 during the early stages of cell attachment and spreading, starting with αIIbβ3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of αIIbβ3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either αIIb or β3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the β3 integrin cytoplasmic tail, rather than the αIIb subunit, plays a major role in αIIbβ3 affinity modulation. With the successful direct visualization of functional αIIbβ3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor αvβ3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on 3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire 3 cytoplasmic domain induced constitutive LIBS exposure on αvβ3 and IIb β3. Deletion of the C-terminal NITY⁷⁵⁹ sequence or disruption of the NPLY⁷⁴⁷ motif by a Y747A substitution impaired extracellular conformational changes on αvβ3 following RGDS, echistatin or Mn²⁺ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn²⁺-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY⁷⁴⁷ motif in the β3 cytoplasmic domain, rather than potential phosphorylation of Tyr⁷⁴⁷ or Tyr⁷⁵⁹, is a prerequisite for conformational changes within the αvβ3 ectodo-main, and suggest that two different mechanisms are responsible for RGDS- and Mn²⁺-dependent conformational changes.

We have investigated the effect of a new Leu¹⁹⁶Pro mutation, identified in the MIDAS-like domain of the β3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on β3 integrin receptor function. Expression of the mutant β3Pro¹⁹⁶ subunit in CHO cells, either associated with recombinant human αIIb or αv, resulted in normal biosynthesis of β3 and heterodimerization with αv or IIb, but selectively interfered with αIIbβ3 maturation and transport to the cell surface. Functional analysis of the β3 mutant receptors revealed strong inhibition of αvβ3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125^FAK phosphorylation and fibrin clot retraction, as opposed to normal αIIbβ3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant αIIbβ3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL¹⁹⁶T motif in the β3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in β3 is not involved in αIIbβ3-mediated cell adhesion to immobilized fibrinogen, while it is essential for αvβ3-mediated interaction with this ligand. ^* This work was supported by grants from Centre de Recherche Public-Santé (CRP-Santé, Luxembourg), and CNRS (France). ⁺MC. M-K is a recipient of a European Community Marie Curie fellowship (ERBFMBICT 961531). ⁺ MC. M-K is a recipient of a European Community Marie Curie fellowship (ERBFMBICT 961531). ⁺⁺ P.C is a recipient of a fellowship from the Ministère de la Coopération, Luxembourg.

A naturally occurring point mutation (Ser⁷⁵²Pro substitution) in the β subunit cytoplasmic domain of the platelet fibrinogen receptor GPIIb-IIIa (integrin αIIβ3), causing Glan-zmann's thrombasthenia, has been shown to abrogate bidirectional transmembrane signaling of GPIIb-IIla when expressed in heterologous cells (Chen YP. 1994, Blood 84, 1857–1865). As the vitronectin receptor αvβ3 constitutively mediates cell attachment to RGD containing extracellular matrix proteins, the purpose of this study was to explore the regulatory role of Ser⁷⁵² in αvβ3 vitronectin receptor function, by cotransfecting recombinant human αv cDNA together with human β3 mutant cDNA into Chinese hamster ovary (CHO) cells. CHO cells expressing wild type human αvβ3 acquired the ability to attach and spread on fibrinogen and von Willebrand factor, in contrast to non transfected CHO cells that only bound to vitronectin and fibronectin. Overexpression of a truncated recombinant β3 subunit (β3δ744) generated αv(hamster)β3(human) ehimers that mediated attachment but lost the ability to promote cell spreading on vitronectin, von Willebrand factor and fibrinogen, and to concentrate in focal contact sites, demonstrating a negative effect of β3δ744 on αvβ3 dependent postreceptor occupancy events. Transfection of β3Ser⁷⁵²Pro reproduced the same negative effect as β3δ744, whereas β3Ser⁷⁵²Ala restored normal receptor function by allowing pronounced attachment and spreading on fibrinogen and von Willebrand factor. Our results provide evidence that (1) the C-terminal cytoplasmic domain of $3 (amino acids 744–762) is essential for av(33 integrin postreceptor occupancy events; (2) within this domain, the Ser⁷⁵²Pro mutation affects αvβ3 postreceptor occupancy events by preventing cell spreading and focal contact localization; (3) the defective receptor function of the vitronectin receptor avB3 is due to the presence of Pro⁷⁵², rather than the absence of Ser⁷⁵², as a Ser to Ala substitution at position 752 restores normal (33 integrin cell spreading and adhesive plaque formation.