Profiling of the CD4 receptor complex proteins

Krotov, Grigory; Krutikova, M. P.; Згода, В. Г.; Filatov, Alexander

doi:10.1134/s0006297907110077

Cited by 9 publications

(11 citation statements)

References 32 publications

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“…A rather low amount of this pool can also significantly impact quantitative aspects of biochemical reactions initiating T cell activation (Chakraborty and Das, 2010). Moreover, both pY394Lck and a sizeable pool of CD45 were found in heavy DRMs suggesting that kinase activity of pY394Lck is spatially restricted and controlled by its membrane colocalization with this phosphatase (Krotov et al, 2007; Ballek et al, 2012). This observation found its support in a recent demonstration that a fraction of CD45 and CD4 are colocalized by a virtue of membrane confinement accounting for their enormously enhanced association rate (Haugh and Lauffenburger, 1997; James et al, 2011).…”

Section: Incorporating Membrane Compartmentalization Into the Model Omentioning

confidence: 99%

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

Filipp¹,

Ballek²,

Manning³

2012

Front. Immun.

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In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering.

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Section: Incorporating Membrane Compartmentalization Into the Model Omentioning

confidence: 99%

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

Filipp¹,

Ballek²,

Manning³

2012

Front. Immun.

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“…9, 20 To test this possibility, we used a derivative of the Jurkat cell line deficient for Lck, J.CaM1.6 (Figure 5a). As both Jurkat and J.CaM1.6 cells demonstrated identical spot patterns in the resting and activated states (Figure 5b), we concluded that Lck was not essential for LPAP phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

“…It is tempting to speculate that Lck kinase, which has been reported to associate with LPAP, 9, 20 might be involved in LPAP phosphorylation. However, our data do not support this hypothesis because J.CaM1.6, a Lck-deficient Jurkat cell line, expressed identical phosphoforms of LPAP that we registered on 2D-PAGE compared with wt Jurkat cells.…”

Section: Discussionmentioning

confidence: 99%

“…The protein structure and topology are not resolved for LPAP and have been described only in theoretical models. LPAP starts with a signal peptide (amino acid [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], which is cleaved during protein maturation. The hydrophobic portion of the protein (amino acid represents the transmembrane domain.…”

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confidence: 99%

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Lymphocyte phosphatase‐associated phosphoprotein proteoforms analyzed using monoclonal antibodies

2015

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Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified.

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“…Most reports to date have analysed CD4 interaction complexes in lymphoid cell lines, revealing some of the well-known associating proteins, such as LCK, CD45, transferrin receptor (CD71), CD98, myosins, vimentin, tubulins, actins, annexin II and lymphocyte phosphatase associated phosphoprotein (LPAP) [29], [30], [31], [32]. However, little is known about how CD4 antigen is arranged at the surface of Mφ, which notably lack LCK expression.…”

Section: Introductionmentioning

confidence: 99%

Proteomic-Based Identification of CD4-Interacting Proteins in Human Primary Macrophages

et al. 2011

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BackgroundHuman macrophages (Mφ) express low levels of CD4 glycoprotein, which is constitutively recycled, and 40–50% of its localization is intracellular at steady-state. Although CD4-interacting proteins in lymphoid cells are well characterised, little is known about the CD4 protein interaction-network in human Mφ, which notably lack LCK, a Src family protein tyrosine kinase believed to stabilise CD4 at the surface of T cells. As CD4 is the main cellular receptor used by HIV-1, knowledge of its molecular interactions is important for the understanding of viral infection strategies.Methodology/Principal FindingsWe performed large-scale anti-CD4 immunoprecipitations in human primary Mφ followed by high-resolution mass spectrometry analysis to elucidate the protein interaction-network involved in induced CD4 internalization and degradation. Proteomic analysis of CD4 co-immunoisolates in resting Mφ showed CD4 association with a range of proteins found in the cellular cortex, membrane rafts and components of clathrin-adaptor proteins, whereas in induced internalization and degradation CD4 is associated with components of specific signal transduction, transport and the proteasome.Conclusions/SignificanceThis is the first time that the anti-CD4 co-immunoprecipitation sub-proteome has been analysed in human primary Mφ. Our data have identified important Mφ cell surface CD4-interacting proteins, as well as regulatory proteins involved in internalization and degradation. The data give valuable insights into the molecular pathways involved in the regulation of CD4 expression in Mφ and provide candidates/targets for further biochemical studies.

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Profiling of the CD4 receptor complex proteins

Cited by 9 publications

References 32 publications

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

Lymphocyte phosphatase‐associated phosphoprotein proteoforms analyzed using monoclonal antibodies

Proteomic-Based Identification of CD4-Interacting Proteins in Human Primary Macrophages

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