Mechanisms of internalization and recycling of the chemokine receptor, CCR5

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C-3 exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner

Activation of Matrix-Metalloproteinase-2 and Membrane-Type-1-Matrix-Metalloproteinase in Endothelial Cells and Induction of Vascular Permeability In Vivo by Human Immunodeficiency Virus-1 Tat Protein and Basic Fibroblast Growth Factor

Previous studies indicated that the Tat protein of human immunodeficiency virus type-1 (HIV-1) is a progression factor for Kaposi's sarcoma (KS). Specifically, extracellular Tat cooperates with basic fibroblast growth factor (bFGF) in promoting KS and endothelial cell growth and locomotion and in inducing KS-like lesions in vivo. Here we show that Tat and bFGF combined increase matrix-metalloproteinase-2 (MMP-2) secretion and activation in endothelial cells in an additive/synergistic manner. These effects are due to the activation of the membrane-type-1-matrix-metalloproteinase and to the induction of the membrane-bound tissue inhibitor of metalloproteinase-2 (TIMP-2) by Tat and bFGF combined, but also to Tat-mediated inhibition of both basal or bFGF-induced TIMP-1 and -2 secretion. Consistent with this, Tat and bFGF promote vascular permeability and edema in vivo that are blocked by a synthetic MMP inhibitor. Finally, high MMP-2 expression is detected in acquired immunodeficiency virus syndrome (AIDS)-KS lesions, and increased levels of MMP-2 are found in plasma from patients with AIDS-KS compared with HIV-uninfected individuals with classic KS, indicating that these mechanisms are operative in AIDS-KS. This suggests a novel pathway by which Tat can increase KS aggressiveness or induce vasculopathy in the setting of HIV-1 infection

The Interaction of Activated Integrin Lymphocyte Function-associated Antigen 1 with Ligand Intercellular Adhesion Molecule 1 Induces Activation and Redistribution of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 in T Lymphocytes

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the β2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the β2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2