Proton-sensing mechanisms in the immune system

Proton-sensing mechanisms in the immune system: A role for the T cell death-associated gene 8 Amar Nijagal Dr. Owen N Witte Howard Hughes Medical Institute Department of Microbiology, Immunology, and Molecular Genetics David Geffen School of Medicine University of California, Los Angeles Aerobic glycolysis in cancer Aerobic glycolysis is the conversion of glucose to lactate in the presence of oxygen. PET-[18F]FDG Acidic tumor microenvironment LACTATE H+ Robert A. Gatenby & Robert J. Gillies Nature Reviews Cancer 4, 891 -899 (2004) Generation of an acidic microenvironment facilitates tumor progression TUMOR ACIDIC MICROENVIRONMENT  Erosion of extracellular matrix  Promotion of angiogenesis  Suppression of anti-tumor immune responses TUMOR PROGRESSION Hypothesis: The cellular effects of acidic environments are mediated by specialized pH sensors. H+ Proton Receptors? Recently identified pH sensing function of GPR4 and OGR1, two related G-protein coupled receptors A novel family of orphan GPCRs OGR1 (Ovarian cancer G protein-coupled receptor 1) GPR4 (G protein-coupled receptor 4) TDAG8 (T cell death-associated gene 8) G2A (G2 accumulation receptor) ClustalW alignment TDAG8 is essential for proton-induced cAMP production by splenocytes cAMP (fmol)/1x107 cells 25000 20000 15000 10000 5000 0 WT and KO splenocytes WT TDAG8 KO G2A KO D pH* 7.4 6.4 7.4 6.4 7.4 6.4 + IBMX 30 min 37ºC w/o CO2 cAMP (fmol)/1x107 cells pH 50000 40000 30000 20000 10000 0 1 M PGE2 Measure cAMP (ELISA) *DMEM/ Hepes/EPPS/MES W T O K 2A 8 K O TD Data representative of 5 independent experiments G A G A role for TDAG8 as a regulator of T-cell activation in acidic microenvironments WT and KO splenocytes Clonal Expansion and Cytotoxic Function Activate T-cells via T-cell receptor (αCD3) 7000 6000 5000 4000 3000 2000 1000 0 D pH* Measure IL-2 (ELISA) IL-2 (pg/mL) WT TDAG8 KO 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 pH -log[H+] Data representative of 5 independent experiments Conclusions  In immune cells, TDAG8 plays a non-redundant role in acidic pHinduced production of cAMP  The GPCR dependent proton-sensing mechanism could be important for regulating cellular responses to acidic microenvironments Tumor acidic microenvironment H+ TDAG8 Suppression of anti-tumor immune responses Acknowledgements Owen N. Witte Caius G. Radu Witte Lab Members Howard Hughes Medical Institute Cancer Research Institute Putative structural requirements for pH sensing OGR1 GPR4 TDAG8 G2A OGR1 GPR4 TDAG8 G2A OGR1 GPR4 TDAG8 G2A OGR1 GPR4 TDAG8 G2A OGR1 GPR4 TDAG8 G2A OGR1 GPR4 TDAG8 G2A ---------------------MGNITADNSSMSCTIDHT ---------------------MGNHTWEG----CHVDSR ---------------------MNSTCIEE-------QHD MCPMLLKNGYNGNATPVTTTAPWASLGLSAKTCNNVSFE IHQTLAPVVYVTVLVVGFPANCLSLYFGYLQIKARNELG VDHLFPPSLYIFVIGVGLPTNCLALWAAYRQVQQRNELG LDHYLFPIVYIFVIIVSIPANIGSLCVSFLQAKKESELG ESRIVLVVVYSAVCTLGVPANCLTAWLALLQVLQGNVLA VYLCNLTVADLFYICSLPFWLQYVLQHDNWSHGDLSCQV VYLMNLSIADLLYICTLPLWVDYFLHHDNWIHGPGSCKL IYLFSLSLSDLLYALTLPLWIDYTWNKDNWTFSPALCKG VYLLCLALCELLYTGTLPLWVIYIRNQHRWTLGLLACKV KAAVGVSVVIWAKELLTSIYFLMHEEVIED-----ENQH KTAVAVSSVVWATELGANSAPLFHDELFRD-----RYNH RFALMVSLSIWILETIFNAVMLWEDETVVEYCDAEKSNF RTAILISACIFILVGIVHYPVFQTE------------DK LRAVRRSHGTQKSRKDQIQRLVLSTVVIFLACFLPYHVL LRAVRGSVSTERQEKAKIKRLALSLIAIVLVCFAPYHVL YQAVRHNKATENKEKKRIIKLLVSITVTFVLCFTPFHVM FRSIKQSMGLSAAQKAKVKHSAIAVVVIFLVCFAPYHLV LLVRSVWEAS--------CDFAKGVFNAYHFSLLLTSFN LLSRSAIYLGR----PWDCGFEERVFSAYHSSLAFTSLN LLIRCILEHAVN--FEDHSNSGKRTYTMYRITVALTSLN LLVKAAAFSYYRGDRNAMCGLEERLYTASVVFLCLSTVN 18 14 11 39 57 53 50 78 96 92 89 117 169 165 167 183 247 243 245 261 278 278 282 300 An overexpression system to evaluate GPCR-dependent pH responses Expression of the GPCR-EGFP fusions in transfected 293T cells ? H+ G protein (Heterotrimeric GTPase) G2A TDAG8 Phospholipase C Ras/Rho family GTPases GPR4 OGR1 Inositol Adenylyl Cyclase phosphates (IP) cAMP Confocal fluorescent microscopy TDAG8 and GPR4 induce pH dependent production of cAMP cAMP (nmol/10 mg protein) GPCR-EGFP transfected 293T cells 30 25 20 15 10 5 0 EGFP G2A TDAG8 GPR4 D pH* + IBMX (phosphodiesterase inhibitor) 30 min 37ºC w/o CO2 7.9 6.9 pH Measure cAMP (ELISA) *DMEM/ Hepes/EPPS/MES Data representative of 4 independent experiments OGR1 induces pH dependent production of Inositol phosphates (IP) GPCR-EGFP transfected 293T cells IP production (cpm) 6000 5000 4000 3000 2000 1000 0 D pH* EGFP G2A OGR1 45 min + LiCl (phosphatase inhibitor) 37ºC w/o CO2 7.4 6.6 pH Measure IP *DMEM/ Hepes/EPPS/MES Data representative of 4 independent experiments Knockout mouse models to study proton sensing mechanisms in primary immune cells OGR1 GPR4 TDAG8 G2A Knockout mice created THYMUS SPLEEN WT TDAG8 KO G2A KO RT-PCR TDAG8 is essential for proton-induced cAMP production by thymocytes WT and KO thymocytes cAMP (fmol)/1x107thymocytes 15000 12500 10000 7500 5000 2500 0 D pH* WT TDAG8 KO G2A KO + IBMX 30 min 37ºC w/o CO2 7.4 6.9 6.4 7.4 6.9 6.4 7.4 6.9 6.4 Measure cAMP (ELISA) *DMEM/ Hepes/EPPS/MES Data representative of 5 independent experiments