title: The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors.
abstract: OBJECTIVE
  Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells.
  
  
  METHODS
  Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding.
  
  
  RESULTS
  LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange.
  
  
  CONCLUSIONS
  Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.
authors.affiliation: Department of Molecular and Cellular Pharmacology and Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.
authors.forename: K Saidas
authors.initials: KS
authors.lastname: Nair
authors.forename: Ana
authors.initials: A
authors.lastname: Mendez
authors.forename: Joe B
authors.initials: JB
authors.lastname: Blumer
authors.forename: Derek H
authors.initials: DH
authors.lastname: Rosenzweig
authors.forename: Vladlen Z
authors.initials: VZ
authors.lastname: Slepak
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C102754:GPSM2 protein, human; C498915:Gpsm1 protein, mouse; D020730:Guanine Nucleotide Dissociation Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; C498511:Rasd1 protein, mouse; D015293:Transducin; D018631:ras Proteins