(maximum stimulation Ϸ50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular ␤-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1͞2 activation with Ϸ60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular ␤-arrestin 2 by small interfering RNA (␤-arrestin dependent). These findings imply the existence of independent G protein-and ␤-arrestin 2-mediated pathways leading to ERK1͞2 activation and the existence of distinct ''active'' conformations of a seven-membrane-spanning receptor coupled to each.

Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from G s -dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β 1 AR signaling to the EGFR. This β-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.Introduction β-Adrenergic receptors (βARs) belong to the family of 7 transmembrane receptors (7TMRs) (1) and mediate the powerful regulatory effects on cardiac function of the catecholamine neurotransmitters epinephrine and norepinephrine. β 1 ARs constitute more than 70% of the cardiac βARs. Catecholamine stimulation of β 1 ARs results in activation of heterotrimeric G proteins followed by rapid phosphorylation of the receptor, resulting in desensitization (2). Homologous desensitization of β 1 ARs is brought about by phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs), leading to the recruitment of β-arrestin, which then sterically interdicts further coupling to G proteins (3) and targets the receptor for internalization (3). In addition to β-arrestin's role in terminating G protein signaling, recent studies demonstrate that β-arrestins also function as adapter molecules, allowing for the assembly of multiprotein signaling complexes such as ERKs and tyrosine kinases (4, 5). For the angiotensin II type 1A receptor (AT 1A R), this second wave of β-arrestin-mediated signaling has recently been demonstrated to be independent of G protein signaling (6) and to require the activity of GRKs 5 and 6 (7).The signaling mechanisms that underlie the activation of the mitogenic ERK growth response by 7TMRs are complex and likely result from both classical G protein-regulated effectors such as PKA and PKC and non-G protein-mediated crosstalk, such as EGFR transactivation (8). The current paradigm of transactivation involves agonist stimulation of a 7TMR, which through a number of undefined steps leads to MMP-mediated cleavage and

The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely G q /G 11 ) or ␤-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized ␤-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SIIangiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t1 ⁄2 ϳ2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, ␤-arrestin2-dependent activation was slower (peak 5-10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via ␤-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and ␤-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints.

Autophagy is a conserved cellular process for bulk degradation of intracellular protein and organelles in lysosomes. In contrast to elegant studies of beclin1 using mouse models and cultured cells demonstrating a tumor suppression function for autophagy, knockout of other essential autophagy proteins such as ATG5, ATG7, or FIP200 (FAK family-interacting protein of 200 kDa) in various tissues did not lead to malignant tumor development in vivo. Here, we report that inhibition of autophagy by FIP200 ablation suppresses mammary tumor initiation and progression in a mouse model of breast cancer driven by the PyMT oncogene. Deletion of FIP200 resulted in multiple autophagy defects including accumulation of ubiquitinated protein aggregates and p62/ SQSTM1, deficient LC3 conversion, and increased number of mitochondria with abnormal morphology in tumor cells. FIP200 deletion did not affect apoptosis of mammary tumor cells or Ras-transformed mouse embryonic fibroblasts (MEFs), but significantly reduced their proliferation in both systems. We also observed a reduced glycolysis and cyclin D1 expression in FIP200-null mammary tumor cells and transformed MEFs. In addition, gene profiling studies revealed significantly elevated expression of interferon (IFN)-responsive genes in the early tumors of FIP200 conditional knockout mice, which was accompanied by increased infiltration of effector T cells in the tumor microenvironment triggered by an increased production of chemokines including CXCL10 in FIP200-null tumor cells. Together, these data provide strong evidence for a protumorigenesis role of autophagy in oncogeneinduced tumors in vivo and suggest FIP200 as a potential target for cancer therapy.

␤-arrestins bind to G protein-coupled receptor kinase (GRK)-phosphorylated seven transmembrane receptors, desensitizing their activation of G proteins, while concurrently mediating receptor endocytosis, and some aspects of receptor signaling. We have used RNA interference to assess the roles of the four widely expressed isoforms of GRKs (GRK 2, 3, 5, and 6) in regulating ␤-arrestinmediated signaling to the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1͞2 by the angiotensin II type 1A receptor. Angiotensin II-stimulated receptor phosphorylation, ␤-arrestin recruitment, and receptor endocytosis are all mediated primarily by GRK2͞3. In contrast, inhibiting GRK 5 or 6 expression abolishes ␤-arrestin-mediated ERK activation, whereas lowering GRK 2 or 3 leads to an increase in this signaling. Consistent with these findings, ␤-arrestin-mediated ERK activation is enhanced by overexpression of GRK 5 and 6, and reciprocally diminished by GRK 2 and 3. These findings indicate distinct functional capabilities of ␤-arrestins bound to receptors phosphorylated by different classes of GRKs.angiotensin receptor ͉ extracellular signal-regulated kinase ͉ phosphorylation ͉ small interfering RNA

The clinical success of immunotherapy that inhibits the negative immune regulatory pathway programmed cell death protein 1/PD-1 ligand (PD-1/PD-L1) has initiated a new era in the treatment of metastatic cancer. PD-L1 expression is upregulated in many solid tumors including lung cancer and functions predominantly in lactate-enriched tumor microenvironments. Here, we provided evidence for PD-L1 induction in response to lactate stimulation in lung cancer cells. Lactate-induced PD-L1 induction was mediated by its receptor GPR81. The silencing of GPR81 signaling in lung cancer cells resulted in a decrease in PD-L1 protein levels and functional inactivation of PD-L1 promoter activity. In addition, GPR81-mediated upregulation of PD-L1 in glucose-stimulated lung cancer cells that recapitulates the enhanced glycolysis in vivo was dependent on lactate dehydrogenase A (LDHA). We also demonstrated that activation of GPR81 decreases intracellular cAMP levels and inhibits protein kinase A (PKA) activity, leading to activation of the transcriptional coactivator TAZ. Interaction of TAZ with the transcription factor TEAD was essential for TAZ activation of PD-L1 and induction of its expression. Furthermore, we found that lactate-induced activation of PD-L1 in tumor cells led to reduced production of interferon-γ and induction of apoptosis of cocultured Jurkat T-cell leukemia cells. Our findings reveal an unexpected role of lactate in contributing to tumor cell protection from cytotoxic T-cell targeting and establishes a direct connection between tumor cell metabolic reprograming and tumor evasion from the immune response.

Focal adhesion kinase (FAK) has been implicated in the development of cancers, including those of the breast. Nevertheless, the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here, we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers, including aldehyde dehydrogenase, CD24, CD29, and CD61, we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their selfrenewal and migration in vitro. Finally, through transplantation in NOD-SCID mice, we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together, these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. [Cancer Res 2009;69(2):466-74]

Little is known about whether autophagic mechanisms are active in hematopoietic stem cells (HSCs) or how they are regulated. FIP200 (200-kDa FAK-family interacting protein) plays important roles in mammalian autophagy and other cellular functions, but its role in hematopoietic cells has not been examined. Here we show that conditional deletion of FIP200 in hematopoietic cells leads to perinatal lethality and severe anemia. FIP200 was cell-autonomously required for the maintenance and function of fetal HSCs. FIP200-deficient HSC were unable to reconstitute lethally irradiated recipients. FIP200 ablation did not result in increased HSC apoptosis, but it did increase the rate of HSC proliferation. Consistent with an essential role for FIP200 in autophagy, FIP200-null fetal HSCs exhibited both increased mitochondrial mass and reactive oxygen species. These data identify FIP200 as a key intrinsic regulator of fetal HSCs and implicate a potential role for autophagy in the maintenance of fetal hematopoiesis and HSCs.