The Membrane Receptor for Plasma Retinol-Binding Protein, A New Type of Cell-Surface Receptor

Members of the lipocalin superfamily share a common structural fold, but differ from each other with respect to the molecules with which they interact. They all contain eight β-strands (A—H) that fold to form a well-defined β-barrel, which harbours a binding pocket for hydrophobic ligands. These strands are connected by loops that vary in size and structure and make up the closed and open ends of the pocket. In addition to binding ligands, some members of the family interact with other macromolecules, the specificity of which is thought to be associated with the variable loop regions. Here, we have investigated whether the macromolecular-recognition properties can be transferred from one member of the family to another. For this, we chose the prototypical lipocalin, the plasma retinol-binding protein (RBP) and its close structural homologue the epididymal retinoic acid-binding protein (ERABP). RBP exhibits three molecular-recognition properties: it binds to retinol, to transthyretin (TTR) and to a cell-surface receptor. ERABP binds retinoic acid, but whether it interacts with other macromolecules is not known. Here, we show that ERABP does not bind to TTR and the RBP receptor, but when the loops of RBP near the open end of the pocket (L-1, L-2 and L-3, connecting β-strands A—B, C—D and E—F, respectively) were substituted into the corresponding regions of ERABP, the resulting chimaera acquired the ability to bind TTR and the receptor. L-2 and L-3 were found to be the major determinants of the receptor- and TTR-binding specificities respectively. Thus we demonstrate that lipocalins serve as excellent scaffolds for engineering novel biological functions.

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STRA6, a Cell-Surface Receptor for Retinol-Binding Protein: The Plot Thickens

Cell Metabolism ◽

10.1016/j.cmet.2007.02.006 ◽

2007 ◽

Vol 5 (3) ◽

pp. 164-166 ◽

Cited By ~ 49

Author(s):

William S. Blaner

Keyword(s):

Cell Surface ◽

Binding Protein ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Surface Receptor ◽

A Cell

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Structure-function studies on human retinol-binding protein using site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3000437 ◽

1994 ◽

Vol 300 (2) ◽

pp. 437-442 ◽

Cited By ~ 24

Author(s):

A Sivaprasadarao ◽

J B Findlay

Keyword(s):

Cell Surface ◽

Specific Binding ◽

Binding Protein ◽

Specific Interaction ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Directed Mutagenesis ◽

Surface Receptor

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

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Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector

Biochemical Journal ◽

10.1042/bj2960209 ◽

1993 ◽

Vol 296 (1) ◽

pp. 209-215 ◽

Cited By ~ 13

Author(s):

A Sivaprasadarao ◽

J B C Findlay

Keyword(s):

Escherichia Coli ◽

Signal Sequence ◽

Binding Protein ◽

Protein A ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Serum Retinol ◽

T7 Promoter ◽

Secretion Vector ◽

Surface Receptor

In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1. However, this construct did not generate detectable expression of RBP in E. coli. When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm. Deletion of the 3′ untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx. 20-fold). After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin. The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e. it interacted with retinol, transthyretin and its cell-surface receptor. Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher. This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.

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Specific binding of fluorescein labelled serum retinol-binding protein to its cell surface receptor in isolated, purified bovine pigment epithelial cells

Experimental Eye Research ◽

10.1016/0014-4835(80)90033-0 ◽

1980 ◽

Vol 30 (5) ◽

pp. 489-499 ◽

Cited By ~ 3

Author(s):

Patricia G. Jones ◽

Joram Heller

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Specific Binding ◽

Binding Protein ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Serum Retinol ◽

Pigment Epithelial ◽

Surface Receptor ◽

Pigment Epithelial Cells

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SUN-LB130 Identification of IGSF1 Ligands

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.2113 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Courtney L Smith ◽

Andrew N Bayne ◽

Jean-François Trempe ◽

Daniel J Bernard

Keyword(s):

Cell Surface ◽

Transmembrane Domain ◽

Protein Complexes ◽

Protein A ◽

Negative Control ◽

Cell Surface Receptor ◽

Immunoglobulin Superfamily ◽

Central Hypothyroidism ◽

Surface Receptors ◽

Surface Receptor

Abstract Immunoglobulin superfamily, member 1 (IGSF1), is an X-linked, type 1 transmembrane glycoprotein that is highly expressed in the anterior pituitary gland and testes. Mutations in the IGSF1 gene cause congenital central hypothyroidism, variable hypoprolactinemia, growth hormone dysregulation, and macroorchidism. Igsf1 knockout mice exhibit reduced pituitary TRH receptor (Trhr1) expression with an associated impairment in TRH-stimulated TSH secretion. The mechanism through which IGSF1 loss leads to reductions in Trhr1 levels is unresolved, at least in part because IGSF1’s cellular functions are unknown. The mature form of the IGSF1 protein consists of seven extracellular Ig loops, a single transmembrane domain containing a positively charged arginine, and a short intracellular carboxy-tail devoid of known functional motifs. Recently, IGSF1 was argued to be a member of the leukocyte receptor cluster (LRC) family. LRC proteins act as cell surface receptors for soluble or membrane-bound proteins. We therefore hypothesized that IGSF1 is a cell surface receptor for a presently undescribed ligand that regulates Trhr1 expression in pituitary thyrotrope cells. To identify candidate IGSF1 ligands, we implemented a new ligand trapping method, Ecto-Fc MS. We fused the extracellular (Ecto) domain of IGSF1 to the fragment crystallizable (Fc) region of human IgG, creating an Ecto-Fc fusion protein. Secreted IGSF1-Fc was purified and used as a ligand trap for bait proteins extracted from rat testes. The protein complexes were affinity purified with protein A beads, trypsin digested into peptides, subjected to orthogonal high-pH fractionation, and identified by tandem LC-MS/MS. More than 700 proteins were enriched in IGSF1-Fc preparations compared to an Fc-only negative control. Several secreted ligands and plasma-membrane proteins were identified, many of which are also expressed in pituitary thyrotrope cells. Identifying the ligand or ligands will enable us to determine IGSF1 function, and may lead to the discovery of novel causes of central hypothyroidism and macroorchidism.

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