Membrane Receptor For Retinol-binding Protein Research Articles

Vitamin A Transport and the Transmembrane Pore in the Cell-Surface Receptor for Plasma Retinol Binding Protein

Vitamin A and its derivatives (retinoids) play diverse and crucial functions from embryogenesis to adulthood and are used as therapeutic agents in human medicine for eye and skin diseases, infections and cancer. Plasma retinol binding protein (RBP) is the principal and specific vitamin A carrier in the blood and binds vitamin A at 1∶1 ratio. STRA6 is the high-affinity membrane receptor for RBP and mediates cellular vitamin A uptake. STRA6 null mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 null humans have a wide range of severe pathological phenotypes in many organs including the eye, brain, heart and lung. Known membrane transport mechanisms involve transmembrane pores that regulate the transport of the substrate (e.g., the gating of ion channels). STRA6 represents a new type of membrane receptor. How this receptor interacts with its transport substrate vitamin A and the functions of its nine transmembrane domains are still completely unknown. These questions are critical to understanding the molecular basis of STRA6′s activities and its regulation. We employ acute chemical modification to introduce chemical side chains to STRA6 in a site-specific manner. We found that modifications with specific chemicals at specific positions in or near the transmembrane domains of this receptor can almost completely suppress its vitamin A transport activity. These experiments provide the first evidence for the existence of a transmembrane pore, analogous to the pore of ion channels, for this new type of cell-surface receptor.

Differential and Isomer-Specific Modulation of Vitamin A Transport and the Catalytic Activities of the RBP Receptor by Retinoids

Retinoids are vitamin A derivatives with diverse biological functions. Both natural and artificial retinoids have been used as therapeutic reagents to treat human diseases, but not all retinoid actions are understood mechanistically. Plasma retinol binding protein (RBP) is the principal and specific carrier of vitamin A in the blood. STRA6 is the membrane receptor for RBP that mediates cellular vitamin A uptake. The effects of retinoids or related compounds on the receptor's vitamin A uptake activity and its catalytic activities are not well understood. In this study, we dissected the membrane receptor-mediated vitamin A uptake mechanism using various retinoids. We show that a subset of retinoids strongly stimulates STRA6-mediated vitamin A release from holo-RBP. STRA6 also catalyzes the exchange of retinol in RBP with certain retinoids. The effect of retinoids on STRA6 is highly isomer-specific. This study provides unique insights into the RBP receptor's mechanism and reveals that the vitamin A transport machinery can be a target of retinoid-based drugs.

Oral Vitamin A and Retinoic Acid Supplementation Stimulates Antibody Production and Splenic Stra6 Expression in Tetanus Toxoid–Immunized Mice ,

Coadministration of retinoic acid (RA) and polyinosinic acid:polycytidylic acid (PIC) has been shown to cooperatively enhance the anti–tetanus toxoid (anti-TT) vaccine response in adult mice. Germinal center formation in the spleen is critical for a normal antibody response. Recent studies have identified Stimulated by Retinoic Acid-6 (Stra6) as the cell membrane receptor for retinol-binding protein (RBP) in many organs, including spleen. The objectives of the present studies were to test whether orally administered vitamin A (VA) itself, either alone or combined with RA, and/or treatment with PIC regulates Stra6 gene expression in mouse spleen and, concomitantly, antibody production. Eight-week-old C57BL/6 mice were immunized with TT. In an initial kinetic study, oral VA (6 mg/kg) increased anti-TT IgM and IgG production as well as splenic Stra6 mRNA expression. In treatment studies that were analyzed 9 d postimmunization, retinoids including VA, RA, VA and RA combined, and PIC significantly increased plasma anti-TT IgM and IgG (P < 0.05) and splenic Stra6 mRNA (P < 0.05). Treatments that included PIC elevated plasma anti-TT IgM and IgG concentrations >20-fold (P < 0.01). Immunohistochemistry of STRA6 protein in mouse spleen confirmed its increase after immunization and retinoid treatment. In conclusion, retinoid treatments that included VA, RA, VA and RA combined, and the combination of retinoid and PIC stimulated the expression of Stra6 in spleen, which potentially could increase the local uptake of retinol. Concomitantly, these treatments increased the systemic antigen-specific antibody response. The ability of oral retinoids to stimulate systemic immunity has implications for public health and therapeutic use of VA.

An Essential Ligand-binding Domain in the Membrane Receptor for Retinol-binding Protein Revealed by Large-scale Mutagenesis and a Human Polymorphism

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.

Solubilization and purification of the retinol-binding protein receptor from human placental membranes

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.