title: Detection of the human GPR50 orphan seven transmembrane protein by polyclonal antibodies mapping different epitopes.
abstract: GPR50 is an orphan seven transmembrane protein related to the melatonin receptor subfamily comprising MT(1) and MT(2) receptors. In the absence of any known ligand for GPR50, other tools are critical for the characterization of this protein. Here, we describe the generation, purification and characterization of the first rabbit polyclonal antibodies generated against peptides corresponding to the N-terminus, C-terminus and two additional regions within the intracellular tail of GPR50. Immune sera were purified on peptide-antigen affinity columns. Antibodies specifically recognized a GPR50-YFP fusion protein on the plasma membrane of HEK 293 cells in immunofluorescence experiments. In Western blot experiments, the monomeric and dimeric forms of GPR50 were detected as proteins of 66 and 130 kDa, respectively. In addition, these new antibodies were sufficiently sensitive to detect GPR50 in brain slices of the rat pituitary and human hippocampus. In conclusion, we successfully produced antibodies against the orphan GPR50 protein that will become valuable tools for functional studies of this protein.
authors.affiliation: Institut Cochin, Department of Cell Biology, Paris, France.
authors.forename: Hassina Ould
authors.initials: HO
authors.lastname: Hamouda
authors.forename: Patty
authors.initials: P
authors.lastname: Chen
authors.forename: Angélique
authors.initials: A
authors.lastname: Levoye
authors.forename: Nejla
authors.initials: N
authors.lastname: Sözer-Topçular
authors.forename: Avais M
authors.initials: AM
authors.lastname: Daulat
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Guillaume
authors.forename: Rivka
authors.initials: R
authors.lastname: Ravid
authors.forename: Egemen
authors.initials: E
authors.lastname: Savaskan
authors.forename: Gilles
authors.initials: G
authors.lastname: Ferry
authors.forename: Jean A
authors.initials: JA
authors.lastname: Boutin
authors.forename: Philippe
authors.initials: P
authors.lastname: Delagrange
authors.forename: Ralf
authors.initials: R
authors.lastname: Jockers
authors.forename: Pascal
authors.initials: P
authors.lastname: Maurice
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism*; D001666:Binding Sites, Antibody; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D000906:Antibodies; D000939:Epitopes; C509754:GPR50 protein, human; D009419:Nerve Tissue Proteins; D043562:Receptors, G-Protein-Coupled