The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized and internalized. In this study we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, shRNA was used to inhibit GRK 2, 3, 5 and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6, respectively, increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization and internalization, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect in CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6−/−) displayed an increase in CXCR2-mediated G-protein activation, but in vitro exhibited a decrease in chemotaxis, receptor desensitization and internalization relative to wild type (GRK6+/+) cells. In contrast, neutrophil recruitment in vivo in GRK6−/− mice was increased in response to delivery of CXCL1 through the air-pouch model. In a wound closure assay, GRK6−/− mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure as compared to GRK6+/+ animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.
In Mycobacterium smegmatis, sigF is widely expressed during different growth stages and plays role in adaptation to stationary phase and oxidative stress. Using a sigF deletion mutant of M. smegmatis mc2155, we demonstrate that SigF is not essential for growth of bacterium. Deletion of sigF results in loss of carotenoid pigmentation which rendered increased susceptibility to H2O2 induced oxidative stress in M. smegmatis. SigF modulates the cell surface architecture and lipid biosynthesis extending the repertoire of SigF function in this species. M. smegmatis SigF regulon included variety of genes expressed during exponential and stationary phases of growth and those responsible for oxidative stress, lipid biosynthesis, energy, and central intermediary metabolism. Furthermore, we report the identification of a SigF antagonist, an anti‐sigma factor (RsbW), which upon overexpression in M. smegmatis wild type strain produced a phenotype similar to M. smegmatis mc2155 ΔsigF strain. The SigF‐anti‐SigF interaction is duly validated using bacterial two‐hybrid and pull down assays. In addition, anti‐sigma factor antagonists, RsfA and RsfB were identified and their interactions with anti‐sigma factor were experimentally validated. Identification of these proteins will help decode regulatory circuit of this alternate sigma factor.
The interleukin-8 (IL-8/CXCL8) receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We have recently shown that CXCR1 couples predominantly to the G protein-coupled receptor (GPCR) kinase-2 (GRK2) whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to GPCRs, GRKs have displayed a more diverse protein/protein interaction in cells. In this study we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. The data herein demonstrated that upon CXCR2 activation, GRK6 interacts with activator of G protein signaling 3 (AGS3) and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time-dependent and peaked at 2-3 min post-activation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent but GRK6-independent fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca2+ mobilization, phosphoinositide (PI) hydrolysis and chemotaxis. In contrast, shRNA inhibition of AGS3 enhanced CXCL8-induced Ca2+ mobilization, receptor resistance to desensitization and recycling to the cell surface with no effect in receptor internalization. Interestingly, RBL-CXCR2-AGS3−/− cells displayed a significant increase in CXCR2 expression in the cell surface, but decreased (extracellular signal-regulated kinases) ERK1/2 and P38 mitogen-activated protein kinase (MAPK) activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels including downstream effector activation, receptor trafficking and expression at the cell membrane.
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