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Vitamin A transport and the transmembrane pore in the cell-surface receptor for plasma retinol binding protein.

Zhong M, Kawaguchi R, Ter-Stepanian M, Kassai M, Sun H - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jules Stein Eye Institute, and Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America.

ABSTRACT
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 mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 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.

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Effect of MTSEA-biotin modification on STRA6-mediated vitamin A uptake as measured by 3H-retinol uptake from 3H-retinol/RBP and HPLC analysis of retinyl esters.A. Positions of V320C and S385C in the transmembrane topology model of STRA6. Because MTSEA-biotin is membrane impermeable, the location of S385C close to the intracellular side of STRA6 makes it inaccessible to MTSEA-biotin from the extracellular side, while V320C is positioned on the boundary of membrane helix VI and extracellular domain and can be accessed by MTSEA-biotin from the extracellular side. B and C. Cell-free vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-S385C (S385C). B is 3H-retinol uptake assay from 3H-retinol/RBP. C is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. D and E. Live-cell vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-V320C (V320C). D is 3H-retinol uptake assay from 3H-retinol/RBP. E is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. In B and D, the amount of 3H-retinol associated with STRA6-WT without modification is defined as 100%. This activity reflects the binding of 3H-retinol/RBP to STRA6. In C and E, the activity of STRA6-WT with LRAT as measured by retinyl ester amount is defined as 100%. Grey bars, no treatment. Green bars, MTSEA-biotin treatment.
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pone-0073838-g003: Effect of MTSEA-biotin modification on STRA6-mediated vitamin A uptake as measured by 3H-retinol uptake from 3H-retinol/RBP and HPLC analysis of retinyl esters.A. Positions of V320C and S385C in the transmembrane topology model of STRA6. Because MTSEA-biotin is membrane impermeable, the location of S385C close to the intracellular side of STRA6 makes it inaccessible to MTSEA-biotin from the extracellular side, while V320C is positioned on the boundary of membrane helix VI and extracellular domain and can be accessed by MTSEA-biotin from the extracellular side. B and C. Cell-free vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-S385C (S385C). B is 3H-retinol uptake assay from 3H-retinol/RBP. C is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. D and E. Live-cell vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-V320C (V320C). D is 3H-retinol uptake assay from 3H-retinol/RBP. E is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. In B and D, the amount of 3H-retinol associated with STRA6-WT without modification is defined as 100%. This activity reflects the binding of 3H-retinol/RBP to STRA6. In C and E, the activity of STRA6-WT with LRAT as measured by retinyl ester amount is defined as 100%. Grey bars, no treatment. Green bars, MTSEA-biotin treatment.

Mentions: In addition to CRBP-I, LRAT is also known to couple to STRA6-mediated vitamin A uptake from holo-RBP. We next studied the effect of chemical modification on STRA6-mediated vitamin A uptake using both 3H-retinol-based vitamin A uptake assay and HPLC-based analysis of retinyl ester formation as a result of the coupling of STRA6-mediated vitamin A uptake to LRAT [32], [37], [48] (Figure 3). 3H-retinol/RBP can reveal both RBP's binding to STRA6 and STRA6′s coupling to LRAT, because 3H-retinol associated with STRA6 without LRAT largely reflects 3H-retinol/RBP bound to STRA6 [49]. In contrast, STRA6′s activity in the presence of LRAT reflects the coupling of STRA6 to LRAT [49].


Vitamin A transport and the transmembrane pore in the cell-surface receptor for plasma retinol binding protein.

Zhong M, Kawaguchi R, Ter-Stepanian M, Kassai M, Sun H - PLoS ONE (2013)

Effect of MTSEA-biotin modification on STRA6-mediated vitamin A uptake as measured by 3H-retinol uptake from 3H-retinol/RBP and HPLC analysis of retinyl esters.A. Positions of V320C and S385C in the transmembrane topology model of STRA6. Because MTSEA-biotin is membrane impermeable, the location of S385C close to the intracellular side of STRA6 makes it inaccessible to MTSEA-biotin from the extracellular side, while V320C is positioned on the boundary of membrane helix VI and extracellular domain and can be accessed by MTSEA-biotin from the extracellular side. B and C. Cell-free vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-S385C (S385C). B is 3H-retinol uptake assay from 3H-retinol/RBP. C is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. D and E. Live-cell vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-V320C (V320C). D is 3H-retinol uptake assay from 3H-retinol/RBP. E is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. In B and D, the amount of 3H-retinol associated with STRA6-WT without modification is defined as 100%. This activity reflects the binding of 3H-retinol/RBP to STRA6. In C and E, the activity of STRA6-WT with LRAT as measured by retinyl ester amount is defined as 100%. Grey bars, no treatment. Green bars, MTSEA-biotin treatment.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815300&req=5

pone-0073838-g003: Effect of MTSEA-biotin modification on STRA6-mediated vitamin A uptake as measured by 3H-retinol uptake from 3H-retinol/RBP and HPLC analysis of retinyl esters.A. Positions of V320C and S385C in the transmembrane topology model of STRA6. Because MTSEA-biotin is membrane impermeable, the location of S385C close to the intracellular side of STRA6 makes it inaccessible to MTSEA-biotin from the extracellular side, while V320C is positioned on the boundary of membrane helix VI and extracellular domain and can be accessed by MTSEA-biotin from the extracellular side. B and C. Cell-free vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-S385C (S385C). B is 3H-retinol uptake assay from 3H-retinol/RBP. C is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. D and E. Live-cell vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-V320C (V320C). D is 3H-retinol uptake assay from 3H-retinol/RBP. E is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. In B and D, the amount of 3H-retinol associated with STRA6-WT without modification is defined as 100%. This activity reflects the binding of 3H-retinol/RBP to STRA6. In C and E, the activity of STRA6-WT with LRAT as measured by retinyl ester amount is defined as 100%. Grey bars, no treatment. Green bars, MTSEA-biotin treatment.
Mentions: In addition to CRBP-I, LRAT is also known to couple to STRA6-mediated vitamin A uptake from holo-RBP. We next studied the effect of chemical modification on STRA6-mediated vitamin A uptake using both 3H-retinol-based vitamin A uptake assay and HPLC-based analysis of retinyl ester formation as a result of the coupling of STRA6-mediated vitamin A uptake to LRAT [32], [37], [48] (Figure 3). 3H-retinol/RBP can reveal both RBP's binding to STRA6 and STRA6′s coupling to LRAT, because 3H-retinol associated with STRA6 without LRAT largely reflects 3H-retinol/RBP bound to STRA6 [49]. In contrast, STRA6′s activity in the presence of LRAT reflects the coupling of STRA6 to LRAT [49].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jules Stein Eye Institute, and Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America.

ABSTRACT
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 mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 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.

Show MeSH
Related in: MedlinePlus