The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters.

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

Mapping of the extracellular RBP4 ligand binding domain on the RBPR2 receptor for Vitamin A transport.

The distribution of dietary vitamin A/all-trans retinol/ROL throughout the body is critical for maintaining retinoid function in peripheral tissues and for retinoid delivery to the eye in the support of visual function. In the circulation, all-trans-retinol bound to the RBP4 protein is transported and sequestered into target tissues for long-term storage. Two membrane receptors that facilitate all-trans retinol uptake from RBP4 have been proposed. While it is well established that the membrane receptor, STRA6, binds to circulatory RBP4 for ROL transport into the eye, the second vitamin A receptor, RBPR2, which is expressed in non-ocular tissues, is less characterized. Based on the structural homology between these two RBP4 receptors, published literature, and from our recent work in Rbpr2 -/- deficient mice, we hypothesized that RBPR2 might also have high-binding affinity for RBP4 and this mechanism facilitates ROL transport. Herein, we aimed to elucidate the membrane topology and putative RBP4 binding residues on RBPR2 to understand its physiological function for retinoid homeostasis. Using in silico analysis and site-directed mutagenesis, we identified a potential RBP4 binding domain on RBPR2. We employed an in vitro cell-based system and confirmed that mutations of these residues on RBPR2 affected its binding to exogenous RBP4 and subsequently vitamin A uptake. Using Surface Plasmon Resonance assays, we analyzed both the binding affinities and kinetic parameters of wild-type RBPR2 and individual mutants affecting the RBPR2-RBP4 binding domain with its physiological ligand RBP4. These studies not only revealed a putative RBP4 binding domain on RBPR2 but also provided new structural, biochemical, and critical information on its proposed role in RBP4 binding for ROL transport and retinoid homeostasis.

Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function.

The systemic transport of dietary vitamin A/all-trans retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant rbpr2 zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body Rbpr2 knockout mouse (Rbpr2-/-) model. These mice were viable on both vitamin A-sufficient and -deficient diets. Rbpr2-/- mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when Rbpr2-/- mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

The role of motor proteins in photoreceptor protein transport and visual function.

BACKGROUND: Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, wherein the perception of vision is initiated when these neurons respond to photons in the light stimuli. The photoreceptor cell is structurally studied as outer segments (OS) and inner segments (IS) where proper protein sorting, localization, and compartmentalization are critical for phototransduction, visual function, and survival. In human retinal diseases, improper protein transport to the OS or mislocalization of proteins to the IS and other cellular compartments could lead to impaired visual responses and photoreceptor cell degeneration that ultimately cause loss of visual function. RESULTS: Therefore, studying and identifying mechanisms involved in facilitating and maintaining proper protein transport in photoreceptor cells would help our understanding of pathologies involving retinal cell degeneration in inherited retinal dystrophies, age-related macular degeneration, and Usher Syndrome. CONCLUSIONS: Our mini-review will discuss mechanisms of protein transport within photoreceptors and introduce a novel role for an unconventional motor protein, MYO1C, in actin-based motor transport of the visual chromophore Rhodopsin to the OS, in support of phototransduction and visual function.

Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye.

Vitamins are essential compounds obtained through diet that are necessary for normal development and function in an organism. One of the most important vitamins for human physiology is vitamin A, a group of retinoid compounds and carotenoids, which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in the vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids such as ß-carotene from plants or preformed vitamin A such as retinyl esters from animal sources are absorbed into the body via the small intestine and converted into all-trans retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carotenoid cleavage oxygenases (CCOs), such as Beta-carotene 15,15'-monooxygenase (BCO1), to produce all-trans retinal that subsequently gets converted into all-trans retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein 4 receptor 2 (RBPR2) in systemic tissues (e.g., in the retina and the liver, respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all-trans retinol into the blood for peripheral cell use. This article aims to review literature regarding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.

Molecular determinant-based typing of KIR alleles and KIR ligands.

Killer cell immunoglobulin-like receptors (KIRs) regulate NK cell function. KIRs and their HLA ligands are highly polymorphic in nature with substantial allelic polymorphism. At present, there is a lack of an expedient method for KIR and HLA allele typing with relevant functional information. Here, we developed a single-nucleotide polymorphism (SNP) assay to type various allele groups of KIR2DL1 with distinct functional properties based on polymorphism at position 245. We also established a SNP assay to type different KIR ligands based on polymorphism at position 77 in HLA-C and position 83 in HLA-B and -A. Our SNP assays for KIR and KIR ligand typing are much cheaper and faster than existing high-resolution typing. Importantly, our high-throughput methods provide readouts that are informative in predicting NK cell activity in health, disease, and transplantation.