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The Presence of a Leu-Gly-Asn Repeat–Enriched Protein (LGN), a Putative Binding Partner of Transducin, in ROD Photoreceptors

¹From the Department of Molecular and Cellular Pharmacology and Neuroscience Program, University of Miami School of Medicine, Miami, Florida; the ²Zilkha Neurogenetic Institute, The Mary D. Allen Laboratory for Vision Research, Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California; and the ³Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana.

PURPOSE. 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 GTPS 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 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 subunit of transducin and potentially regulate its function.

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