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Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles

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Abstract

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγ) and is regulated by the α subunit of transducin (Tα). Here, we show an overall mechanism for PDE regulation by identifying Pγ complexes in OS homogenates prepared with an isotonic buffer. Before Tα activation, three Pγ complexes exist in the soluble fraction. Complex a, a minor complex, contains Pαβ, Tα, and a protein named Pδ. Complex b, Pαβγγb, has a PDE activity similar to that of membranous Pαβγγ, PαβγγM, and its level, although its large portion is Pδ-free, is estimated to be 20–30% of the total Pαβγγ. Complex c, (Pγ·GDP-Tα) c2 , appears to be a dimer of Pγ·GDP-Tα. Upon Tα activation, (1) complex a stays unchanged, (2) Pαβγγb binds to membranes, (3) the level of (Pγ·GDP-Tα) c2 is reduced as its GTP-form is produced, (4) complex d, Pγ·GTP-Tαd, is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Pαβγγ, PαβγγM and/or Pαβγγb, becomes Pγ-depleted. These observations indicate that Pγ as a complex with GTP-Tα dissociates from Pαβγγ on membranes and is released to the soluble fraction and that Pγ-depleted PDE is the GTP-Tα-activated PDE. After GTP hydrolysis, both (Pγ·GDP-Tα) c2 and Pγ·GDP-Tαd, without liberating Pγ, deactivate Pγ-depleted PDE. The preferential order to be used for the deactivation is membranous Pγ·GDP-Tαd, solubilized Pγ·GDP-Tαd and (Pγ·GDP-Tα) c2 . Release of Pγ·GTP-Tα complexes to the soluble fraction is relevant to light adaptation.

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Acknowledgments

We thank Dr. Richard Needleman, Mr. Masaomi Matsumoto, and Ms. Marta Matsumoto for critical reading of the manuscript. We also thank Drs. N. Li and W. Baehr (University of Utah) for providing of the plasmid for expression of recombinant Pδ. This work was supported in part by National Institute of Health Grants EY07546, EY09631, and EY13877, Jules and Doris Stein Professorship and an unrestricted grant from Research to Prevent Blindness, Grants-in Aids for Scientific Research from Japan Society for the promotion of Science 18310088 and 18370062, and Iketani Foundation for science and Engineering 0191093-A.

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Authors and Affiliations

  1. Department of Ophthalmology, Kresge Eye Institute, Wayne State University, 4717 St. Antoine St., Detroit, MI, 48201-1423, USA

    Akio Yamazaki

  2. Department of Pharmacology, Wayne State University, 4717 St. Antoine St, Detroit, MI, 48201, USA

    Akio Yamazaki & Russell K. Yamazaki

  3. College of Osteopathic Medicine, Touro University, Henderson, NV, 89014, USA

    Vladimir A. Bondarenko

  4. Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Taiwan

    Isao Matsuura

  5. Department of Intellectual Properties, Niigata University, Niigata, 950-2181, Japan

    Masahiro Tatsumi

  6. Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73190, USA

    Sadamu Kurono, Naoka Komori & Hiroyuki Matsumoto

  7. Department of Biology, Graduate School of Science, Kobe University, Kobe, 657-8501, Japan

    Fumio Hayashi

  8. Division of Integrated Project, EcoTopia Science Institute, Nagoya University, Nagoya, 464-8603, Japan

    Jiro Usukura

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  1. Akio Yamazaki
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Yamazaki, A., Bondarenko, V.A., Matsuura, I. et al. Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles. Mol Cell Biochem 339, 215–233 (2010). https://doi.org/10.1007/s11010-010-0387-8

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