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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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Effects of acute chemical modification on STRA6-catalyzed retinol release from holo-RBP.STRA6-catalzyed retinol release from holo-RBP causes a decline in retinol fluorescence. In all experiments, holo-RBP was added at 0 min. A and B. Effects of MTSEA-biotin treatment on the retinol release activity of STRA6-WT and STRA6-S385C, respectively. Grey traces, no treatment. Green traces, MTSEA-biotin treatment. Control membrane without STRA6 was used as the negative control in A (open circles). C. Comparison of the effects of MTSEA-biotin on STRA6-WT and STRA6-S385C. Activity of the untreated reaction for STRA6-WT is defined as 1. Grey bars, no treatment. Green bars, MTSEA-biotin treatment. D. Structures of MTSEH, MTSBS, MTSPT and MTSEA-biotin. E. Comparison of effects of MTSEH, MTSBS, MTSPT and MTSEA-biotin of retinol transport activity of S385C. The color of the trace in E matches the color of the chemical in D. Grey trace, no treatment. F. Quantitation of retinol release activity of experiments in E. Activity of the untreated reaction is defined as 1.
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pone-0073838-g001: Effects of acute chemical modification on STRA6-catalyzed retinol release from holo-RBP.STRA6-catalzyed retinol release from holo-RBP causes a decline in retinol fluorescence. In all experiments, holo-RBP was added at 0 min. A and B. Effects of MTSEA-biotin treatment on the retinol release activity of STRA6-WT and STRA6-S385C, respectively. Grey traces, no treatment. Green traces, MTSEA-biotin treatment. Control membrane without STRA6 was used as the negative control in A (open circles). C. Comparison of the effects of MTSEA-biotin on STRA6-WT and STRA6-S385C. Activity of the untreated reaction for STRA6-WT is defined as 1. Grey bars, no treatment. Green bars, MTSEA-biotin treatment. D. Structures of MTSEH, MTSBS, MTSPT and MTSEA-biotin. E. Comparison of effects of MTSEH, MTSBS, MTSPT and MTSEA-biotin of retinol transport activity of S385C. The color of the trace in E matches the color of the chemical in D. Grey trace, no treatment. F. Quantitation of retinol release activity of experiments in E. Activity of the untreated reaction is defined as 1.

Mentions: We chose reagents that differ in charge and size for cysteine modification (MTSEH, MTSBS, MTSPT, and MTSEA-biotin) (Figure 1). Of these four chemicals, MTSEH has the smallest size, MTSPT is positively charged, MTSBS is negatively charged, and MTSEA-biotin has the largest size. It was known previously that STRA6 is capable of catalyzing retinol release from holo-RBP [49]. This activity is largely responsible for endocytosis-independent uptake of vitamin A from holo-RBP. Using real-time analysis of STRA6-catalyzed retinol release and these chemicals, we initially studied residues within the seventh transmembrane domain and identified position S385 as highly sensitive to chemical modification (Figure 1). Without modification, STRA6-S385C has similar activity compared to the wild-type STRA6 (Figures 1A–1C). However, modification by MTSEA-biotin strongly suppresses STRA6-catalyzed retinol release for STRA6-S385C, although it also affects the wild-type protein to a much lesser extent (Figures 1A–1C). We also found that the inhibitory effect on STRA6-catalyzed retinol release is correlated only with the size of chemicals (Figures 1D–1F). MTSEH, which has the smallest size, has the smallest effect on blocking vitamin A release, while MTEA-biotin, which has largest size, shows strongest inhibition of STRA6-catalyzed retinol release. Positive or negative charge on the reagent has no additional effect on blocking vitamin A release, consistent with the fact that the substrate vitamin A has no charge.


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)

Effects of acute chemical modification on STRA6-catalyzed retinol release from holo-RBP.STRA6-catalzyed retinol release from holo-RBP causes a decline in retinol fluorescence. In all experiments, holo-RBP was added at 0 min. A and B. Effects of MTSEA-biotin treatment on the retinol release activity of STRA6-WT and STRA6-S385C, respectively. Grey traces, no treatment. Green traces, MTSEA-biotin treatment. Control membrane without STRA6 was used as the negative control in A (open circles). C. Comparison of the effects of MTSEA-biotin on STRA6-WT and STRA6-S385C. Activity of the untreated reaction for STRA6-WT is defined as 1. Grey bars, no treatment. Green bars, MTSEA-biotin treatment. D. Structures of MTSEH, MTSBS, MTSPT and MTSEA-biotin. E. Comparison of effects of MTSEH, MTSBS, MTSPT and MTSEA-biotin of retinol transport activity of S385C. The color of the trace in E matches the color of the chemical in D. Grey trace, no treatment. F. Quantitation of retinol release activity of experiments in E. Activity of the untreated reaction is defined as 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073838-g001: Effects of acute chemical modification on STRA6-catalyzed retinol release from holo-RBP.STRA6-catalzyed retinol release from holo-RBP causes a decline in retinol fluorescence. In all experiments, holo-RBP was added at 0 min. A and B. Effects of MTSEA-biotin treatment on the retinol release activity of STRA6-WT and STRA6-S385C, respectively. Grey traces, no treatment. Green traces, MTSEA-biotin treatment. Control membrane without STRA6 was used as the negative control in A (open circles). C. Comparison of the effects of MTSEA-biotin on STRA6-WT and STRA6-S385C. Activity of the untreated reaction for STRA6-WT is defined as 1. Grey bars, no treatment. Green bars, MTSEA-biotin treatment. D. Structures of MTSEH, MTSBS, MTSPT and MTSEA-biotin. E. Comparison of effects of MTSEH, MTSBS, MTSPT and MTSEA-biotin of retinol transport activity of S385C. The color of the trace in E matches the color of the chemical in D. Grey trace, no treatment. F. Quantitation of retinol release activity of experiments in E. Activity of the untreated reaction is defined as 1.
Mentions: We chose reagents that differ in charge and size for cysteine modification (MTSEH, MTSBS, MTSPT, and MTSEA-biotin) (Figure 1). Of these four chemicals, MTSEH has the smallest size, MTSPT is positively charged, MTSBS is negatively charged, and MTSEA-biotin has the largest size. It was known previously that STRA6 is capable of catalyzing retinol release from holo-RBP [49]. This activity is largely responsible for endocytosis-independent uptake of vitamin A from holo-RBP. Using real-time analysis of STRA6-catalyzed retinol release and these chemicals, we initially studied residues within the seventh transmembrane domain and identified position S385 as highly sensitive to chemical modification (Figure 1). Without modification, STRA6-S385C has similar activity compared to the wild-type STRA6 (Figures 1A–1C). However, modification by MTSEA-biotin strongly suppresses STRA6-catalyzed retinol release for STRA6-S385C, although it also affects the wild-type protein to a much lesser extent (Figures 1A–1C). We also found that the inhibitory effect on STRA6-catalyzed retinol release is correlated only with the size of chemicals (Figures 1D–1F). MTSEH, which has the smallest size, has the smallest effect on blocking vitamin A release, while MTEA-biotin, which has largest size, shows strongest inhibition of STRA6-catalyzed retinol release. Positive or negative charge on the reagent has no additional effect on blocking vitamin A release, consistent with the fact that the substrate vitamin A has no charge.

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