An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism

Abstract

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.

FIGURE 1.

Outline of experimental procedures for an unbiased approach to study the structure and function of STRA6.

FIGURE 2.

Vitamin A uptake activities from holo-RBP of 902 random bovine STRA6 mutants. Wild-type (WT) STRA6 activity is defined as 100%. Mutant names are shown below the x axis.

FIGURE 3.

Characterization of STRA6 mutants generated by random mutagenesis. A, cell surface expression of STRA6 mutants: two defective mutants with normal cell surface expression (mutants 444 and 595), one mutant with weak cell surface expression (mutant 678), and one mutant with complete loss of cell surface expression (mutant 69). The specific mutations are indicated. B, vitamin A uptake activity of STRA6 mutants 69, 444, 595, and 678. C, RBP binding activity of STRA6 mutants 69, 444, 595, and 678. WT, wild type.

FIGURE 4.

Alanine-scanning mutagenesis of the putative RBP-binding region in STRA6 that was identified by random mutagenesis. Glycine and alanine residues were mutated to leucine. A, sequence alignment of the region from different species. Residues where mutations lead to significant loss of vitamin A uptake are indicated by asterisks. B, vitamin A uptake activity from holo-RBP for this series of STRA6 mutants. Mutants with significant loss of vitamin A uptake are indicated by asterisks (D333A, Y336A, G340L, and G342L). WT, wild type.

FIGURE 5.

Characterization of four defective STRA6 mutants identified through alanine-scanning mutagenesis. A, cell surface expression of STRA6 mutants D333A, Y336A, G340L, and G342L. All mutants maintained cell surface expression except D333A. B, quantitation of cell surface expression of the four STRA6 mutants. C, RBP binding activity of the four STRA6 mutants. WT, wild type.

FIGURE 6.

Vitamin A uptake activity from holo-RBP for human STRA6 variants. Human STRA6 variants were produced in both the untagged (gray bars) and Myc-tagged (white bars) human STRA6 background. A, vitamin A uptake activity from holo-RBP of human STRA6 missense mutants associated with birth defects (P90L, P293L, T321P, P90L/T321P, R655C, and T644M). B, vitamin A uptake activity of human STRA6 polymorphisms (D26N, G339S, L517F, and I527M). The activity of wild-type (WT) STRA6 (tagged or untagged) is defined as 100%.

FIGURE 7.

Analysis of protein expression of human STRA6 variants (P90L, T321P, P90L/T321P, P293L, T644M, R655C, and G339S). A, live cell staining of human STRA6 variants to examine cell surface expression (upper panels) and fixed and permeabilized cell staining of human STRA6 variants (lower panels). B, Western blot analysis of the expression of human STRA6 variants. WT, wild type.

FIGURE 8.

Quantitation of cell surface expression and RBP binding activities of human STRA6 variants (P90L, T321P, P90L/T321P, P293L, T644M, R655C, and G339S). A, quantitation of cell surface expression. The expression level of wild-type (WT) STRA6 is defined as 100%. B, RBP binding activities. The activity of wild-type STRA6 is defined as 100%.

FIGURE 9.

Locations of key STRA6 mutations and polymorphisms analyzed in this study in the transmembrane topology model of STRA6. Transmembrane domains are indicated with Roman numerals. Thickened lines mark the region of STRA6 subjected to random mutagenesis. Single bovine STRA6 mutants with severe functional defects (defined as vitamin A uptake activity <10% of the wild type) are shown. The three essential STRA6 residues involved in RBP interaction (Tyr³³⁶, Gly³⁴⁰, and Gly³⁴² in bovine STRA6) are drawn as circled Y, G, and G, respectively. Natural human mutations and polymorphisms are shown in boldface and marked with asterisks (residue numbers according to human STRA6). The residue affected by human polymorphism G339S is equivalent to bovine Gly³⁴⁰ (marked with an asterisk). The diagram of holo-RBP is based on code 1HBP in the Protein Data Bank.