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University of Maryland Baltimore

University of Maryland Baltimore
Experimental Therapeutics Symposium 2009

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  • 337 amino acids Calculated Molecular weight: 45kD N-term 27AA C-term: 37AA 5 serines, 2threo., 1 tyrosine 3 rd loop: 13AA’s 3 serines

Penn Penn Presentation Transcript

  • Lab Focus
    • Characterizing G protein-coupled receptor signaling, and regulation by kinases (2 nd messenger, GRKs, arrestins)
    • Linking signaling, regulation to cell function
    • a) differentiated cell functions
    • b) growth, survival
    • Stepping up the integrative scale: from cell to tissue and organ system function, disease
  • Example: Arrestins desensitize  2 ARs in ASM to mitigate relaxant effect of beta-agonists
  • GPCR Regulation
  • β arr2-KO Targeting arrestins increases ASM  2 AR signaling in ASM cells Murine: Human:
  • Tissue function: airway relaxation ex vivo
  • Organ system function
  • Application to Cancer Biology
    • Role of different GPCRs in cell growth survival, metastasis, tumor progression
    • Of interest:  2 ARs, EP receptors, chemokine receptors, proton-sensing receptors
    • Regulators of GPCRs or ligand generation:
    • NSAIDs, glucocorticoids
  • PKA as a GPCR effector
    • PKA is anti-mitogenic in numerous mesenchymal cell types
    • PKA is pro-mitogenic and anti-apoptotic in cardiac myocytes, epithelial cells (Kulik)
  • Antiapoptotic effects of epinephrine and forskolin depend on PKA activation Sastry, K. S. R. et al. J. Biol. Chem. 2007;282:14094-14100 LNCaP cells
  • 1. Can other G s -coupled receptors: EP2 prostaglandin IP prostacyclin A2a or A2b adenosine GPR4 proton-sensing similarly regulate cancer cell survival? 2. What are effects of: a. Stress (increased circulating EPI/NE); b. Inflammation and anti-inflammatory agents NSAIDS and Glucocorticoids ( ↓ inflammatory prostanoids) Questions:
  • Inflammation begets PKA activating agents
    • Cytokines and hypoxia important stimuli
    • Induced prostaglandins most important, activate EP receptors on numerous cells types
    • EP2 receptors stimulated PKA activity in almost all cell types
    • PKA effects on: a) intracellular signaling; and b) gene transcription often significant
    • Other products of inflammation include chemokines, proteases
  • Extent, mechanisms by which NSAIDs or anti-inflammatory agents (steroids) reduce cancer incidence or progression are unclear
    • Reduced inflammatory cell influx
    • Δ product generation by any of the participating cells (direct and indirect)
    • Direct effects on cancer cells affecting survival or migration/metastasis
    • Reduced stem cell recruitment?
  • EPITHELIAL CELLS FIRST HIT PRE- MALIGNANT CELLS
  • STEM CELLS PRE- MALIGNANT CELLS STROMAL CELLS STEM CELLS INFLAMM BLOOD CELLS + + ? ? + TUMOR CELLS STROMAL CELLS +
  • Approaches, Tools
    • 1 º cells, acutely dissociated or in culture:
    • smooth muscle, fibroblast, epithelial, immune
    • Tissue ex vivo
    • TG and -/- mice
    • Retro and lenti viral infection of cells, tissue
    • Recombinant protein expression in 1 º cells:
    • mutant receptors, regulators
    • 2 nd messenger, signaling assays: cell/cell-free
    • Real-time trafficking (GFP chimeras)
  •  
  • TUMOR CELLS STROMAL CELLS STEM CELLS INFLAMM BLOOD CELLS
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  •  
  •  
  • Example 2: PKC desensitizes the CysLT1R to alter LTD4-regulated ASM and monocyte function
  • Human Cysteinyl Leukotriene CysLT1 Receptor PKC PKC PKC PKC PKA PKA PKA C C C C A A A
  • A B C D E F Fig. 7
  • A B C D E STRUCTURE-FUNCTION APPROACH: RECEPTOR SIGNALING, TRAFFICKING
  • Fresh Human Monocytes THP-1 Monocyte cell functions
  • Airways ex vivo CysLT1R m3mAChR
  • A B C D
  • Products induced by fibroblasts, smooth muscle during inflammation
    • Chemokines, cytokines
    • Proteases, matrix turnover proteins
    • Eicosanoids, COX-2 products
    • Prostanoids capable of activating cellular PKA
    • Others too numerous to make sense of