Studies Of Chemokine Receptors And Receptor Modulation

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Abstract

A major focus of my laboratory is the structure-function correlation of chemokine receptors and mechanisms of G-protein coupled receptor (GPCR) signaling. Chemokine receptors are members of the GPCR superfamily and share a common three-dimensional structure composed of seven trans-membrane (TM) domains. Some members of the human chemokine receptor family serve as co-receptors 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 co-receptors 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 determined 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 on the function of wt receptor and its use by HIV; and 3) addressed the mechanistic differences between the CC and CXC chemokine receptors in 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. In the past year, we have evaluated the significance of agonist driven endocytosis to cell motility and other leukocyte functions and the role(s) of plasma membrane lipid rafts in these processes. We show that in primary human leukocytes, the threshold agonist levels for endocytosis of the chemotactic receptors CCR2B, CXCR1, CXCR2 and CXCR4 are much higher than those needed for maximal chemotactic response. Moreover, CXCR1, CXCR2 and CXCR4 in neutrophils, CCR2B in monocytes and CXCR4 in PBLs could be reactivated in response to repeated application of increasing dose of the same agonist up to a threshold. Thus leukocyte migration in response to chemokines does not appear to be dependent on endocytosis of receptors. 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. 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. But, 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. The cytoplasmic domains of plasma membrane receptors contain sequence determinants that regulate orderly protein sorting during anterograde transport, specify organelle targeting and determine the intracellular fate of internalized receptors after agonist binding or cell activation. Animal viruses have evolved several strategies to subvert mechanisms of protein sorting, resulting in aberrant trafficking, intracellular trapping and degradation of targeted receptors. Among viral modulators of receptor expression and trafficking, the 27-32 kDal myristoylated Nef proteins of HIV and SIV have been most extensively characterized for their ability to downregulate CD4 and HLA-I receptors. Genetic and biochemical studies have outlined 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. However, we showed previously that [DE]XXXL[LI]-type peptide signals from HIV Nef interacted in a bipartite manner with combinations of gamma-sigma1 of AP-1 and delta-sigma3 subunit heterodimers of the AP-1 and AP-3 vesicles respectively. We have extended the above studies to show that the loss of CD4 at the cell surface in HIV-1 Nef expressing cells did not result exclusively from accelerated endocytosis of CD4. However, both the recycling CD4 and the nascent receptor in transit to the plasma membrane were susceptible to intra-cellular retention and degradation in HIV-1 Nef expressing cells. In contrast, SIV Nef induced CD4 downregulation resulted mostly from enhanced endocytosis and was dramatically reversed by genetic inhibitors of endocytosis or siRNA driven knock down of AP-2 complexes. In contrast to the mechanisms outline above, Nef induced a predominant AP-1 dependent vesicular trapping of the HLA-I receptor during its transit from the Golgi vesicles to the plasma membrane and during receptor recycling from the cell surface.