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      The F 0F 1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells

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          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 F 0F 1 ATP synthase complex. The catalytic subunits of the F 1 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 F 1 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.

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          Author and article information

          Journal
          Mol Endocrinol
          Mol. Endocrinol
          mend
          mend
          Molecular Endocrinology
          Endocrine Society (Washington, DC )
          0888-8809
          1944-9917
          September 2016
          2 August 2016
          : 30
          : 9
          : 996-1011
          Affiliations
          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
          Author notes
          Address all correspondence and requests for reprints to: Mark S. Roberson, PhD, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, T4-018 Veterinary Research Tower, Ithaca, NY 14853. E-mail: msr14@ 123456cornell.edu .
          [*]

          K.A.-W. and J.X. are cofirst authors.

          Article
          PMC5414608 PMC5414608 5414608 ME-15-1324
          10.1210/me.2015-1324
          5414608
          27482602
          151ab0a2-8f79-474a-ba70-c2e53b87001b
          Copyright © 2016 by the Endocrine Society
          History
          : 18 December 2015
          : 29 July 2016
          Funding
          Funded by: National Institutes of Health/National Institute of Child Health and Human Development
          Award ID: R01 HD34772
          Award ID: R21 HD082780
          This work was supported by National Institutes of Health/National Institute of Child Health and Human Development Grants R01 HD34772 and R21 HD082780.
          Categories
          Original Research

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