Vitamin A has diverse biological functions. It is transported in the blood as a complex with retinol binding protein (RBP), but the molecular mechanism by which vitamin A is absorbed by cells from the vitamin A-RBP complex is not clearly understood. We identified in bovine retinal pigment epithelium cells STRA6, a multitransmembrane domain protein, as a specific membrane receptor for RBP. STRA6 binds to RBP with high affinity and has robust vitamin A uptake activity from the vitamin A-RBP complex. It is widely expressed in embryonic development and in adult organ systems. The RBP receptor represents a major physiological mediator of cellular vitamin A uptake.

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. Plasma retinol-binding protein (RBP)2 is the principal carrier of vitamin A in the blood (1-4). It was first proposed in the 1970s that there exists a cell surface receptor that mediates vitamin A uptake from RBP on the retinal pigment epithelium and small intestine cells (5-10). During the past 3 decades, there has been mounting evidence for the existence of RBP receptors on diverse types of tissues, including the placenta (11-13), choroid plexus (12, 14), Sertoli cells and peritubular cells of the testis (12,(15)(16)(17)(18), macrophages (19), and skin (12,20). There are also indirect pieces of evidence for the existence of an RBP receptor. For example, in an unbiased search for a serum factor that stimulates the growth of B cells, it was found that the vitamin A-RBP complex (holo-RBP) is this factor (21). Using an unbiased strategy combining photo-cross-linking, high-affinity purification, and mass spectrometry, the RBP receptor was identified as STRA6, a multi-transmembrane protein of previously unknown function (22). STRA6 binds to RBP with high affinity and specificity and facilitates the transport of vitamin A into the cell. STRA6 was originally identified as a retinoic acid-stimulated gene in cancer cell lines (23, 24).STRA6 has not been systematically characterized at the structural and functional level and is not homologous to any protein with known function. In this study, we used two complementary approaches to study the structure and functi...

STRA6 is a multi-transmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular vitamin A uptake from the vitamin A/RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in human. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A/RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope-tagging method. These studies not only revealed STRA6's extracellular, transmembrane and intracellular domains, but also implicated extracellular regions of STRA6 in RBP binding.Vitamin A and its derivatives (retinoids) are essential for diverse aspects of vertebrate physiology (1-3). Due to the hydrophobic nature of retinoids, it has been assumed that random diffusion is the primary if not the only means of transmembrane transport. However, biochemical evidence suggests that retinol uptake from the small intestine is mediated by a membrane transporter (4). There is also strong evidence for the existence of a specific mechanism to transport 11-cis retinal in the retinal pigment epithelium (RPE) that depends on interphotoreceptor retinoid-binding protein (IRBP). Apo-IRBP is much more effective in promoting the release of 11-cis retinal from the RPE than the apo-forms of other retinoid binding proteins (5). In addition, apo-IRBP is only effective when it is present on the apical, but not basal, side of the RPE (6). Another finding that challenges the assumptions about random diffusion is the identification of an ATP-dependent transporter (ABCR or ABCA4) that transports all-trans retinal released from bleached rhodopsin across membranes (7-9). Mutations in ABCR cause a wide spectrum of human vision diseases from retinitis pigmentosa to macular degeneration. Prior to the surprising discovery of ABCR's role in † This project was supported by National Institute of Health grant 1R01EY018144 (H.S.).

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries. A large number of human genetic studies have associated a common variant (Y402H) of complement factor H (CFH) with a highly significant increase in AMD risk. CFH is a modular protein with 20 homologous short consensus repeats (SCRs). The Y402H variant is located in SCR7 of both CFH and factor H-like protein 1 (FHL-1), a splice variant of CFH (containing SCR1-7) with unique biochemical properties. Because SCR7 is known to bind to heparin, C-reactive protein (CRP), and M protein from Streptococcus pyogenes, it has been hypothesized that the AMD-associated polymorphism may affect interactions with these CFH ligands. In this study, we tested this hypothesis in the context of full-length CFH (SCR1-20) and FHL-1. We systematically analyzed the interactions of the Y402 and H402 variants of CFH and FHL-1 with heparin, CRP, and several bacterial ligands: M6 protein of Streptococcus pyogenes, PspC of Streptococcus pneumoniea, and BbCRASP-1 of Borrelia burgdorferi. In comparing the Y and H variants of CFH and FHL-1, we found no significant difference in their protein secretion, cofactor activity, or interactions with heparin, BbCRASP-1, or PspC, but a significant difference in binding to CRP and M6 protein. This study reveals the fundamental properties of a common polymorphism of CFH and lays the groundwork for elucidating the role of CFH in AMD pathogenesis.