Abstract
Abstract: :
Purpose: Mutations in the human Ocular Albinism 1 (OA1) gene cause ocular but not cutaneous albinism. OA1 may function as a G–protein–coupled receptor (GPCR), but no ligand for OA1 has been identified, and the intracellular distribution of the protein suggests OA1 as the first identified intracellular GPCR. In this study we use recombinant OA1 expression to begin to assess OA1 interactions Methods: The coding sequence for OA1 was cloned from Human cDNA, and inserted into pDS2 Red such that the fluorescent protein was linked to the OA1 carboxyl terminus, creating OA1–RFP. Beta–arrestin–GFP was obtained from R. Lefkowitz, (Duke University). COS, MCF7 and human retinal pigment epithelial cells (RPE) were transfected to express both the red OA1 and the green ß–arrestin. Cells were also transfected with each vector alone and with the native RFP and GFP vectors as controls. Confocal microscopy was used to determine the distribution of fluorescent proteins. Results: OA1–RFP was localized to the Golgi apparatus and in the cytoplasm in a punctate/vesicular pattern. Beta–arrestin GFP was localized diffusely in the cytoplasm, similar to GFP. In cells co–transfected to express both proteins, ß–arrestin co–localized with OA1. No significant difference in distribution was observed among the three cell types tested. While OA1–RFP localized to the secretory system, there is no way to distinguish between the three likely interpretations of this distribution, OA1 in transit to the cell surface, OA1 as a permanent resident in the cytoplasm, or OA1 recycled from the cell surface. Conclusions: Co–transfection of ß–arrestin with OA1 altered the distribution of ß–arrestin resulting in co–localization of the two proteins, suggesting that OA1 may interact with ß–arrestin. Our results support the hypothesis that OA1 may function as a GPCR, and provide for a model system useful for identifying the ligand for OA1.
Keywords: receptors: pharmacology/physiology • retinal pigment epithelium • signal transduction: pharmacology/physiology