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GLENN P LOBO; Identification and Functional Characterization of RBP4 binding domains on the Vitamin A transporter Rbpr2 for Vision. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4508. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We recently showed that the retinol-binding protein receptor 2 (Rbpr2) is highly expressed in tissues devoid of Stra6, and has high affinity binding for RBP4 for the intracellular uptake of vitamin A. Loss of the Rbpr2 receptor in zebrafish manifested in severe eye phenotypes. The purpose of this study was to map the extracellular sites on Rbpr2 for RBP4 binding and to confirm both in-vitro and in-vivo the functionality of RBP4 binding domains on this vitamin A transporter.
Sequence alignment of STRA6 and RBPR2 among various species was performed to identify and map previously proposed RBP4 binding sites/ domains. Site-directed mutagenesis was used to alter these sequences in zebrafish rbpr2 gene. Transient transfection of rbpr2-mutants affecting the proposed RBP4 binding sites was performed in HEK293 cells and membrane localization patterns of recombinant Rbpr2 was assessed using immunohistochemistry and confocal microscopy. Rbpr2 mutant zebrafish lines affecting the RBP4 binding sites were obtained from ZIRC to study the in-vivo functional effects on intracellular vitamin A transport for vision.
A three amino acid RBP4 binding site (Serine268-Tyrosine272-Lysine273) on STRA6 was found to be partially conserved in zebrafish Rbpr2 sequence. Immunohistochemical and confocal analysis revealed that all three rbpr2-mutants, affecting the RBP4 binding sites, had altered membrane expression patterns, when compared to WT-Rbpr2. At 5.5 days post fertilization only zebrafish rbpr2-mutants affecting the RBP4 binding domains, showed smaller eyes, cardiac edema and hydrocephaly; phenotypes associated with decreased systemic vitamin A. Histological analysis showed disrupted retinal lamination layers, and loss of rod and cone photoreceptor proteins, associated with suboptimal ocular retinoids.
In-vitro analysis of mutated RBP4 binding sites on rbpr2 resulted in improper membrane localization patterns and in-vivo led to a decrease in systemic vitamin A levels, manifesting in severe ocular phenotypes in zebrafish. Collectively our data suggests that zebrafish Rbpr2 contains functional RBP4 binding sites, which play an important role in vitamin A transport.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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