We have continued to study the cellular and molecular processes that control chemokine/receptor structure and function with an emphasis on chemokines that effect HIV-1 replication. Natural forms of chemokines secreted from normal lymphocytes and macrophages under different cell activation conditions are being purified from cells and culture supernatants for further characterization. The fine structure of these proteins are then analyzed using several techniques including gel electrophoresis, peptide mapping and mass spectrometry. Activated lymphocytes synthesize and secrete significant amounts of anti-HIV-1 beta chemokines MIP-1a and MIP-1b. We have found that native MIP-1b secreted by activated peripheral blood lymphocytes (PBLs) is a truncated form missing two amino-terminal amino acids {MIP-1b(3-69)} by mass spectrometric analysis. Functional studies of natural MIP-1b(3-69) revealed that it retained potent CCR5-specific activities of anti-HIV-1 entry inhibition and down-modulation of CCR5 receptors on T cells. Characterization of chemokine receptor specificity showed that MIP-b(3-69) not only had the ability of intact MIP-1b to induce calcium signals through CCR5 but also gained calcium signaling activity via CCR1 and CCR2. Our results demonstrate that N-terminally truncated MIP-1b acts as a chemokine agonist, with altered receptor specificity, which may constitute an important regulatory mechanism for cell recruitment during inflammatory and antiviral responses. CXCR4 and its ligand stromal cell-derived factor (SDF) -1 play important roles in HIV-1 pathophysiology, leukocyte trafficking, inflammation, hematopoiesis, embryogenesis, angiogenesis and cancer metastasis. We have investigated the effects of cytokines on regulation of CXCR4 functions in monocytes/macrophages. We demonstrate that monocytes/macrophages express functional surface CXCR4 as assessed by ligand induced calcium mobilization, chemotaxis, and receptor endocytosis. Infection of macrophages by primary X4 viruses was inhibited by IL-4, IL-13 or GM-CSF which was associated with down-modulation of CXCR4. Modulation of CXCR4 by cytokines occurs at post-translation steps in the absence of protein degradation and is mediated by endocytosis. Endocytosis induced by cytokine is independant of actin filament polymerization in contrast to SDF stimulated endocytosis. GM-CSF enhanced expression of GRK-3, b-arrestin1, Pyk2, and FAK. Cytokine treatment increased total and tyrosine-specific phosphorylation of CXCR4 and phospho-FAK on Y-397. Complexes of GRK3/CXCR4 and FAK/CXCR4 were induced by inhibitory cytokines. Tyrosine phosphorylation was critical in the regulation of cell surface expression of CXCR4 by cytokines but not by SDF-1 and was essential for infection of macrophages by X4, R5, and X4R5 HIV-1 types. Our data indicate that ligand dependent and ligand independent endocytosis of chemokine receptors are mediated by different sorting mechanisms. Cytokines may target the focal adhesion molecules, the cytoskeleton and components of sequestration machinery of G-protein coupled receptors, thereby regulating endocytosis of chemokine receptors and migration of cells toward chemokines. Cytokine induced ligand-independent endocytosis of chemokine receptors may have therapeutic value in HIV-1 infection, inflammation, metastasis and hematopoiesis.
