An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown., applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out. outcome and demonstrates the importance of the innate immune system in the development of tumors

Random mutagenesis of G protein alpha subunit G(o)alpha. Mutations altering nucleotide binding

Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G(o)alpha for GTP gamma S or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype

Gα12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF

Heterotrimeric G proteins, G12 and G13, have been shown to transduce signals from G protein-coupled receptors to activate Rho GTPase in cells. Recently, we identified p115RhoGEF, one of the guanine nucleotide exchange factors (GEFs) for Rho, as a direct link between Gα13 and Rho [Kozasa, T., et al. (1998) Science 280, 2109–2111; Hart, M. J., et al. (1998) Science 280, 2112–2114]. Activated Gα13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, Gα12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from Gα12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with Gα12 and Gα13. RGS domain of LARG demonstrated Gα12- and Gα13-specific GAP activity. LARG synergistically stimulated SRF activation by Gα12 and Gα13 in HeLa cells, and the SRF activation by Gα12-LARG was further stimulated by coexpression of Tec tyrosine kinase. It was also found that LARG is phosphorylated on tyrosine by Tec. In reconstitution assays, the RhoGEF activity of nonphosphorylated LARG was stimulated by Gα13 but not Gα12. However, when LARG was phosphorylated by Tec, Gα12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through Gα12 and that the regulation of RhoGEFs by heterotrimeric G proteins G12/13 is further modulated by tyrosine phosphorylation

A new non-canonical pathway of Gαq protein regulating mitochondrial dynamics and bioenergetics

Contrary to previous assumptions, G proteins do not permanently reside on the plasma membrane, but are constantly monitoring the cytoplasmic surfaces of the plasma membrane and endomembranes. Here, we report that the Gαq and Gα11 proteins locate at the mitochondria and play a role in a complex signaling pathway that regulates mitochondrial dynamics. Our results provide evidence for the presence of the heteromeric G protein (Gαq/11βγ) at the outer mitochondrial membrane and for Gαq at the inner membrane. Both localizations are necessary to maintain the proper equilibrium between fusion and fission; which is achieved by altering the activity of mitofusin proteins, Drp1, OPA1 and the membrane potential at both the outer and inner mitochondrial membranes. As a result of the absence of Gαq/11, there is a decrease in mitochondrial fusion rates and a decrease in overall respiratory capacity, ATP production and OXPHOS-dependent growth. These findings demonstrate that the presence of Gαq proteins at the mitochondria serves as a physiological function: stabilizing elongated mitochondria and regulating energy production in Drp1 and Opa1 dependent mechanisms. This thereby links organelle dynamics and physiology. © 2014 Elsevier Inc.This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (BFU2011-30080, SAF2009-08007 & CSD2007-00020), and the Comunidad de Madrid regional authorities (S2011/BMD-2402). C. B. was supported by a JAE-Pre fellowship (CSIC)Peer Reviewe