Studies Of Chemokine Receptors And Receptor Modulation B

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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. They possess extra-cellular N-termini of variable length and a C-terminal cytoplasmic tail containing unique domains critical for ligand dependent receptor signaling, internalization and desensitization. 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; 3) other studies 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. 4) In primary cells and lymphoid cell lines, we identified crosstalk between CCR5 and CXCR4 and other T cell receptors that may be co-modulated during chemokine signaling. 5) In primary leukocytes and expression systems, CXCR1and CXCR2 chemokine receptors mediating innate immunity, display rapid endocytic trafficking kinetics and do not segregate in plasma membrane rafts. 6) And, by contrast, in monocytes CCR2B & CXCR4 that mediate innate as well as acquired immunity in addition to homeotic response display activation dependent changes in plasma membrane raft distribution. Among the accessory proteins encoded by HIV-1, the 27 kDal membrane associated myristoylated Nef down-modulates the surface expression of many cellular receptors, notably CD4 and HLA-I. Genetic and biochemical studies have outlined two different mechanisms for intra-cellular trapping of CD4 and HLA-I induced by Nef. In collaboration with Juan Bonifacino's laboratory, we identified the protein trafficking pathways used by CD4 and HLA-I that are subverted by Nef. We used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from HIV Nef interacted in a bipartite manner with combinations of the gamma & sigma1 of AP-1 and delta & sigma3 of AP-3. The analogous alpha & sigma2 of AP-2 and epsilon & sigma4 subunits of AP-4 did not interact with Nef. The interactions with gamma & sigma1 and delta & sigma3 were dependent on the [DE] and L[LI] residues of the signals but not on other residues. These sequence requirements were similar to those for binding to the whole complexes in vitro and for function of the signals in vivo. These observations uncovered a novel mode of recognition of sorting signals by AP complexes, and demonstrated a role for AP-1 in the down-regulation of CD4 by Nef in vivo. We have extended the above studies to show that the loss of CD4 at the cell surface in Nef expressing cells did not result from enhanced endocytosis of CD4. As a matter of fact, it was irrelevant whether CD4 underwent endocytosis. Nef subverted both the anterograde and retrograde CD4 traffic by interacting with AP1 complexes. In contrast, Nef effect on HLA-I required ongoing endocytosis of this receptor. In spite of the apparent divergent mechanisms, interaction with the AP1 and AP3 vesicles constituted a common theme of Nef effect on both receptors.