2B4 Is Constitutively Associated with Linker for the Activation of T Cells in Glycolipid-Enriched Microdomains: Properties Required for 2B4 Lytic Function

Klem, Jennifer; Verrett, Pamela C.; Kumar, Vinay; Schatzle, John D.

doi:10.4049/jimmunol.169.1.55

Cited by 44 publications

(44 citation statements)

References 38 publications

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“…Although one study reported that CD244 coprecipitated with Grb2 (40), we did not detect binding of these two molecules in our system. That interaction may be mediated by the linker for the activation of T cells, which has been reported to associate with CxC cysteine motif found in the transmembrane region of CD244 (41). The cytoplasmic tail of CS1 has a similar, putative Grb2 binding to the one found in CD229, but we were unable to detect any interaction between these two proteins.…”

Section: Discussionmentioning

confidence: 53%

Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229

Martı́n

Valle

Saborit-Villarroya

et al. 2005

The Journal of Immunology

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Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.

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Section: Discussionmentioning

confidence: 53%

Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229

Martı́n

Valle

Saborit-Villarroya

et al. 2005

The Journal of Immunology

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“…CD244-mediated signaling is also influenced by the transmembrane domain of the receptor through its constitutive association with LAT in humans (18) and mice (19). However, CD244-mediated signaling is not influenced by the transmembrane or extracellular domains of the receptor, but rather it is defined solely by tyrosine-based motifs in the cytoplasmic tail (33).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, it has been reported that CD244 and SAP colocalize to the immunological synapse upon the formation of conjugates between NK cells and target B cells and this finding highlights the role of CD244-SAP complex during the NK cellmediated control of EBV infection (17). CD244 has also been found to associate constitutively with the adaptor molecule LAT via a dicysteine motif (CxC) located in the transmembrane domain of CD244 (18,19). In this paper, we describe a novel interaction between CD244 and the adaptor protein 3BP2 in NK cells and show that 3BP2 links CD244 to downstream events regulating the cytotoxic function of CD244 without affecting IFN-␥ secretion.…”

mentioning

confidence: 86%

The Adaptor Protein 3BP2 Binds Human CD244 and Links this Receptor to Vav Signaling, ERK Activation, and NK Cell Killing

Saborit-Villarroya¹,

Valle²,

Romero³

et al. 2005

The Journal of Immunology

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Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-γ secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-γ release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-γ release involve distinct NK pathways.

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“…Indeed, two previous studies describe an interaction of human 2B4 with LAT [40,41]. Another study detected an association of mouse 2B4 with LAT and described the importance of the CXC motif for this interaction [36]. CSS-PALM 3.0 recognized the CXC motif in 2B4 as a likely site of palmitoylation [37].…”

Section: Discussionmentioning

confidence: 94%

“…4E and G). A previous study of mouse 2B4 suggested that the "CXC" motif contained within the LACFCV sequence is important for an association with LAT and for 2B4-mediated effector functions [36]. Additionally, this "CXC" motif is recognized as a potential palmitoylation site by the palmitoylation software CSS-PALM 3.0 [37].…”

Section: Resultsmentioning

confidence: 99%

Recruitment of activating NK‐cell receptors 2B4 and NKG2D to membrane microdomains in mammalian cells is dependent on their transmembrane regions

et al. 2015

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Membrane microdomains play an important role in the regulation of natural killer (NK) cell activities. These cholesterol-rich membrane domains are enriched at the activating immunological synapse and several activating NK-cell receptors are known to localize to membrane microdomains upon receptor engagement. In contrast, inhibitory receptors do not localize in these specialized membrane domains. In addition, the functional competence of educated NK cells correlates with a confinement of activating receptors in membrane microdomains. However, the molecular basis for this confinement is unknown. Here, we investigate the structural requirements for the recruitment of the humanactivating NK-cell receptors NKG2D and 2B4 to detergent-resistant membrane fractions in the murine BA/F3 cell line and in the human NK-cell line NKL. This stimulation-dependent recruitment occurred independently of the intracellular domains of the receptors. However, either interfering with the association between NKG2D and DAP10, or mutating the transmembrane region of 2B4 impacted the recruitment of the receptors to detergentresistant membrane fractions and modulated the function of 2B4 in NK cells. Our data suggest a potential interaction between the transmembrane region of NK-cell receptors and membrane lipids as a molecular mechanism involved in determining the membrane confinement of activating NK-cell receptors.Keywords: 2B4 r Activating receptor r Lipid rafts r Membrane microdomains r NK cells r NKG2D r Signal transduction Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction NK cells are large, granular lymphocytes of the innate immune system that contribute to successful immune responses by killing virally infected or transformed cells and by producing various cytokines [1][2][3]. The function of NK cells is controlled by the integration of signals from activating and inhibitory surface receptors Correspondence: Prof. Carsten Watzl e-mail: watzl@ifado.de [4]. Although many inhibitory receptors recognize MHC class I or MHC-related molecules on target cells, activating receptors interact with a range of ligands, whose expression can be induced by infection, transformation, or other forms of cellular stress [5]. Contact with a susceptible target cell induces NK-cell adherence and the formation of the immunological synapse (NKIS) with the target cell. This is followed by the polarization of surface receptors and signaling molecules and the clustering of membrane microdomains at the NKIS [6]. Subsequently, the NK cells polarize their lytic granules and kill the attached target cell by directed degranulation and they secrete cytokines such as 8].C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 1258-1269 Leukocyte signaling 1259Membrane microdomains, also called lipid rafts, are defined as cholesterol-and sphingolipid-rich membrane domains that affect various cellular functions, such as signaling, protein sorting, or endocy...

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2B4 Is Constitutively Associated with Linker for the Activation of T Cells in Glycolipid-Enriched Microdomains: Properties Required for 2B4 Lytic Function

Cited by 44 publications

References 38 publications

Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229

Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229

The Adaptor Protein 3BP2 Binds Human CD244 and Links this Receptor to Vav Signaling, ERK Activation, and NK Cell Killing

Recruitment of activating NK‐cell receptors 2B4 and NKG2D to membrane microdomains in mammalian cells is dependent on their transmembrane regions

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