Chemokine Receptor Signaling &HIV Nef Receptor Defects

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Abstract

Our efforts are focused on 1) mechanistic studies of vesicular trafficking and cell signaling defects induced by HIV and SIV encoded Nef proteins and 2) structure-function correlation of chemokine receptors and mechanisms of G-protein coupled receptor (GPCR) signaling in primary leukocytes. Among the accessory proteins encoded by HIV and SIV, the membrane associated myristoylated Nef proteins, which are critical for maintaining viral virulence, modulate multiple vesicular trafficking and signal transduction pathways. Among the many cellular receptors modulated by Nef, CD4 and HLA-I are the most extensively investigated. Genetic and biochemical studies have suggested two different mechanisms for intra-cellular trapping of CD4 and HLA-I induced by Nef. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. 1) However, we have shown that the related AP-1 and AP-3 adaptors also interact with Nef, hinting role(s) for these complexes in intracellular retention of CD4; 2) by using genetic inhibitors of endocytosis and siRNA induced knock down of AP-2, we showed that accelerated CD4 endocytosis was not a dominant mechanism of HIV-1 Nef in epithelial cells, T lymphocyte cell lines or peripheral blood lymphocytes (PBLs). Furthermore, we showed that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef expressing cells. In contrast, AP-2 mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef induced downregulation of CD4; 3) HIV and SIV Nef proteins induced different defects in the trafficking of HLA-I receptors depending on the cell type and in a Nef and HLA-I allele specific manner. Both the plasma membrane associated receptor and de novo synthesized receptor during post-Golgi itinerary were diverted to lysosomes in AP-1 dependent manner; 4) during the search for other lymphocyte receptors that may be susceptible to HIV Nef, we discovered signaling defects from chemokine receptors, CCR5 and CXCR4 that were disproportionate with Nef?s effect on receptor expression. The signaling defects resulted from subversion of secondary messenger regulating leukocyte chemotaxis and obligatory cytoskeletal changes. Chemokine receptors are members of the GPCR superfamily and share a common 3-dimensional structure composed of seven transmembrane (TM) domains. Some members of the human chemokine receptor family serve as coreceptors for HIV entry besides their essential roles in regulating leukocyte chemotaxis in inflammation. M-tropic and T-tropic viruses preferentially use CCR5 (R5 strain) and CXCR4 (X4 strain) respectively. Naturally occurring mutations in the coreceptors and their ligands influence HIV transmission and AIDS progression. Analogous to other GPCRs, ligand binding to the chemokine receptors induces conformational change that recruits G-alpha subunit of trimeric G protein followed by GTP hydrolysis. This activation sets up a cascade of events leading to polarized cellular motility and other cellular activation pathways. However, many chemokine receptors differ from this general paradigm in a cell and receptor specific manner. In recent years, 1) we have identified the structural requirements of CC and CXC chemokine receptors for the biological function and HIV usage; 2) investigated the effects of naturally occurring CCR5 mutants impaired for surface expression on the function of wt receptor and its use by HIV; 3) addressed the mechanistic differences between the CC and CXC chemokine receptors in the agonist-driven receptor signaling, desensitization and internalization. In particular, we showed that CCR5 resides mostly in plasma membrane rafts and that trafficking of agonist-bound CCR5 follows a predominantly non-clathrin itinerary that may be facilitated by caveolin expression; 4) showed that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were much higher than those needed for maximal chemotactic responses. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest; 5) evaluated the role(s) organized lipid domains (also known as rafts) in the various steps of leukocyte chemokine receptor activation including chemotaxis, degranulation, MAP kinase activation and receptor trafficking; 6) we showed further that most of the chemokine receptors are excluded from Triton X-100 insoluble lipid rafts in primary leukocytes, and at high agonist concentrations are rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. However, the chemotactic response mediated by these receptors is critically dependent on lipid raft integrity, which is required for amplification of PI3K mediated signaling events at the leading edge of polarized leukocytes. In contrast, late signaling events such as degranulation, src and MAP kinase activation that are linked to arrestin recruitment during receptor endocytosis do not require raft integrity irrespective of the lipid microdomain distribution of the signaling receptors. Thus, chemotactic signaling is a rapid and reversible local response to low agonist inputs that is spatially constrained by polarized raft assemblies and asymmetric recruitment of secondary messengers, while the late signaling events resulting from global activation at high agonist inputs are linked to receptor endocytosis, which may or may not be raft-associated.