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

Background: Human macrophages (M phi) 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 M phi, 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 Findings: We performed large-scale anti-CD4 immunoprecipitations in human primary M phi 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 M phi 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/Significance: This is the first time that the anti-CD4 co-immunoprecipitation sub-proteome has been analysed in human primary M phi. Our data have identified important M phi 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 M phi and provide candidates/targets for further biochemical studies.