Abstract
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.
Highlights
Mass spectrometry (MS)-based identification of the components of purified protein complexes has become one of the most powerful and routinely used technologies for high-throughput detection of protein interactions [1,2]
In order to effectively demonstrate the induction of CD4 internalization and degradation, we detected the expression of CD4 in Mw before and after treatment with conditioned media from activated T cells by flow cytometry
In control resting Mw, several cell surface proteins associated with CD4 were identified, including CD9, a tetraspanin-family member involved in cell adhesion, cell motility and IL-16 signalling [36,37,38,39]; CD163, involved in the clearance and endocytosis of hemoglobin/haptoglobin complexes [40,41]; integrin subunit beta (CD18), involved in cell surface adhesion and reported to interact with integrins alpha-M and alpha-X [42]; protein S100, a calcium binding protein known to be involved in phagocyte migration and infiltration at sites of wounding [43]; chemokine receptor 1 (CCR-1), a G protein-coupled receptor [44]; adaptor protein 2 (AP-2), a known adaptor protein which functions in protein transport via transport vesicles in different membrane trafficking pathways [25,45], and HLA class I, involved in antigen presentation [46]
Summary
Mass spectrometry (MS)-based identification of the components of purified protein complexes has become one of the most powerful and routinely used technologies for high-throughput detection of protein interactions [1,2]. CD4 is the main cellular receptor used by human immunodeficiency viruses HIV-1, HIV-2 and simian immunodeficiency virus [9,10,11]. It is a type I transmembrane glycoprotein of 55 kDa expressed on the surface of Regulatory and Helper subsets of T lymphocytes and interacts with MHC class-II carrying cells [12]. Endocytosis of CD4 can occur, through clathrin-coated pits, when the cytoplasmic domain becomes serine phosphorylated, leading to its dissociation from LCK [17,18,19] In myeloid cells, such as Mw, which do not express LCK, CD4 is constitutively internalized and 40–50% is intracellular at steady-state [16]. 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
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