title: Generation and characterization of an antagonistic monoclonal antibody against an extracellular domain of mouse DP2 (CRTH2/GPR44) receptors for prostaglandin D2.
abstract: Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.
authors.affiliation: International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.|Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Furuedai, Suita, Osaka, Japan.
authors.forename: Nanae
authors.initials: N
authors.lastname: Nagata
authors.affiliation: Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Iwanari
authors.affiliation: Department of Advanced Clinical Science and Therapeutics, The University of Tokyo, Tokyo, Japan.|Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Kumagai
authors.affiliation: Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Kusano-Arai
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.|Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Furuedai, Suita, Osaka, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Aritake
authors.affiliation: Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.affiliation: International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.|Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Furuedai, Suita, Osaka, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Urade
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004195:Disease Models, Animal; D018604:Epitope Mapping; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007114:Immunization; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D054448:Precursor Cells, B-Lymphoid / Q000276:immunology; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology*; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000276:immunology*; D014517:Ureteral Obstruction / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D000071557:beta-Arrestins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; D000071557:beta-Arrestins; C470438:15-methylprostaglandin D2; D000242:Cyclic AMP; D015230:Prostaglandin D2; C036293:prostaglandin D2 receptor