Specific Interaction of Gai3 with the Oa1 G-Protein Coupled Receptor Controls the Size and Density of Melanosomes in Retinal Pigment Epithelium Alejandra Young 1,2 , Meisheng Jiang 3 , Ying Wang 3 , Novruz B. Ahmedli 1 , John Ramirez 1 , Benjamin E. Reese 4 , Lutz Birnbaumer 5 , Debora B. Farber 1,2 * 1 Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States of America, 2 Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America, 3 Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America, 4 Department of Psychological and Brain Sciences, University of California Santa Barbara, Santa Barbara, California, United States of America, 5 Laboratory of Neurobiology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America Abstract Background: Ocular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gai subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gai subfamily has three highly homologous members, Gai1, Gai2 and Gai3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gai3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gai interaction. Methodology: By using the genetic mouse models Gai1-/-, Gai2-/-, Gai3-/- and the double knockout Gai1-/-, Gai3-/- that lack functional Gai1, Gai2, Gai3, or both Gai1 and Gai3 proteins, respectively, we show that Gai3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST- pull-down and immunoprecipitation assays conclusively demonstrate that Gai3 is the only Gai that binds to Oa1. Western blots show that Gai3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gai3 in melanosomal biogenesis. Conclusion: Our results identify the Oa1 transducer Gai3 as the first downstream component in the Oa1 signaling pathway. Citation: Young A, Jiang M, Wang Y, Ahmedli NB, Ramirez J, et al. (2011) Specific Interaction of Gai3 with the Oa1 G-Protein Coupled Receptor Controls the Size and Density of Melanosomes in Retinal Pigment Epithelium. PLoS ONE 6(9): e24376. doi:10.1371/journal.pone.0024376 Editor: David E. Clapham, Harvard Medical School, United States of America Received July 6, 2011; Accepted August 5, 2011; Published September 8, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the National Institutes of Health (NIH) Grant EY015141 to DBF, BER, and MJ and by The Vision of Children Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: farber@jsei.ucla.edu Introduction Hypopigmentation mutations affecting melanin synthesis or melanosomal biogenesis in the retinal pigment epithelium (RPE) of mammals are known to have profound effects on the developing retina and visual pathways, including abnormal crossing of the optic axons, nystagmus, strabismus, foveal hypoplasia, and reduced visual acuity [1]. Two forms of albinism are commonly recognized: oculocutaneous albinism (OCA), in which neither the eye nor the skin or hair are pigmented and ocular albinism (OA), which affects primarily the eye pigmentation. Ocular albinism type 1 (OA1, also called Nettleship-Falls type), is the most common form of ocular albinism. It has an estimated prevalence of 1/50,000 in the general population of the United States [2]. Although the cutaneous manifestations of OA1 are very mild, affected patients present abnormal macromelanosomes in both the RPE and skin [3]. In contrast to other forms of albinism, melanin is not dramatically reduced in OA1; in fact, this disease is characterized by the unusual coexistence of the typical albino visual defects with a substantial amount of melanin in the eyes [4]. Different types of mutations in the OA1 gene have been associated with ocular albinism type 1 (http://albinismdb.med.umn.edu/ oa1mut.html#mutations). OA1, the protein product of the OA1 gene, is a G protein-coupled receptor localized to RPE melanosomal membranes and the initiator of the observed abnormal visual phenotype in ocular albinism. The position of OA1 within these membranes, with its N-terminal towards the lumen of the melanosome and C-terminal towards the cytoplasm, suggests that it may function as a novel intracellular GPCR activated by the binding of a melanosomal ligand. This ligand could thus regulate melanosomal biogenesis through activation of specific G-proteins found in the RPE cytoplasm [5]. In addition, PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24376