Abstract
The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.
Highlights
Higher eukaryotic organisms depend upon the dietary supply of vitamin A for a range of essential biological processes including vision, reproduction, growth, and development [1]
Transfer of Retinol from retinol-binding protein (RBP) to cellular retinol-binding protein (CRBP) Is Mediated by the Placental RBP Receptor—To examine the hypothesis that the membrane-bound receptor might mediate the transfer of retinol from extracellular RBP to cytoplasmic CRBP, we have developed a simple assay using human placental membranes as the source of the receptor and the E. coli-expressed recombinant CRBP-Strep fusion protein
This paper reports an investigation into the process by which retinol from the extracellular RBP is transported to intracellular CRBP across the plasma membrane
Summary
Higher eukaryotic organisms depend upon the dietary supply of vitamin A for a range of essential biological processes including vision, reproduction, growth, and development [1]. Dietary vitamin A is first transported into the liver, where it forms a complex with a specific 21-kDa carrier protein, called retinol-binding protein (RBP).. Within the cytoplasm of vitamin A-requiring cells, retinol is transported by cellular retinol-binding protein (CRBP), a 15-kDa protein that is structurally distinct from RBP [4]. Work with retinal pigment epithelial cells [11] and placental brush-border membranes [12] suggested that, following the binding of RBP to the receptor, retinol is delivered to the cell, and the resultant apo-RBP remains in the extracellular compartment. Based on the knowledge of the three-dimensional structures of RBP [17] and CRBP [18] complexed with retinol, and the currently available information on RBP-receptor interactions, we have put forward a hypothesis according to which the RBP receptor directly mediates the transfer of retinol from extracellular RBP to intracellular CRBP [19]. In this paper, using the recombinant binding proteins and human placental membranes, we provide experimental support for this hypothesis
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