title: The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.
abstract: Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Krystal
authors.initials: K
authors.lastname: Allen-Worthington
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Xie
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Brown
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Alexa M
authors.initials: AM
authors.lastname: Edmunson
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Abigail
authors.initials: A
authors.lastname: Dowling
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Navratil
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Kurt
authors.initials: K
authors.lastname: Scavelli
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Hojean
authors.initials: H
authors.lastname: Yoon
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Do-Geun
authors.initials: DG
authors.lastname: Kim
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Bynoe
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Iain
authors.initials: I
authors.lastname: Clarke
authors.affiliation: Department of Biomedical Sciences (K.A.-W., J.X., J.L.B., A.M.E., A.D., K.S., H.Y., M.S.R.) and Microbiology and Immunology (D.-G.K., M.S.B.), College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Zoology and Physiology (A.M.N.), College of Arts and Sciences, University of Wyoming, Laramie, Wyoming 82071; and Neuroscience Program (I.C.), Monash Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Roberson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D052681:Gonadotrophs / Q000378:metabolism*; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011966:Receptors, LHRH / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D053719:Tandem Mass Spectrometry
chemical_list: D011966:Receptors, LHRH; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases