Molecular Basis for the Interaction of the Mammalian Amino Acid Transporters B0AT1 and B0AT3 with Their Ancillary Protein Collectrin

Fairweather, Stephen J.; Bröer, Angelika; Subramanian, Nandhitha; Tümer, Emrah; Cheng, Qi; Schmoll, Dieter; O’Mara, Megan L.; Bröer, Stefan

doi:10.1074/jbc.m115.648519

Cited by 43 publications

(47 citation statements)

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“…The catalytic domain of ACE2 is irrelevant for B 0 AT1 expression (Fairweather et al, 2015), we therefore chose collectrin due to its smaller size. CHO cells do not express detectable amounts of ACE2 (Iwata et al, 2009) to replace collectrin.…”

Section: Discussionmentioning

confidence: 99%

“…These results suggested that simple substrate analogues can be used as inhibitors, but are unlikely to be specific. We have previously reported that LeuT can serve as a highly suitable template for the generation of a homology model of B 0 AT1 (O'Mara et al, 2006;Fairweather et al, 2015). The homology model is likely to be reliable in the regions around the binding site in the core of the transporter, which show high sequence similarity, while loops are less conserved (Broer and Gether, 2012).…”

Section: Figurementioning

confidence: 99%

“…In the intestine where collectrin is missing, the exopeptidase angiotensin converting enzyme 2 (ACE2) carries out the same role (Kowalczuk et al, 2008). The peptidase domain of ACE2 is not required for trafficking and catalytic function of B 0 AT1 (Fairweather et al, 2015). Mice lacking ACE2 or collectrin each exhibit some, but not all, aspects of the global B 0 AT1 knockout phenotype (Malakauskas et al, 2009;Bernardi et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…Accordingly, substrate-dependent membrane depolarisation is observed upon expression in Xenopus laevis oocytes. Similar to many other transporters, B 0 AT1 requires ancillary proteins for trafficking to the cell surface and for catalytic function (Fairweather et al, 2015). In the kidney, this role is fulfilled by the type I membrane protein collectrin (TMEM27) (Danilczyk et al, 2006;Malakauskas et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Cheng

Shah

Bröer

et al. 2017

British J Pharmacology

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BACKGROUND AND PURPOSEThe neutral amino acid transporter B 0 AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B 0 AT1 mediates the Na + -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B 0 AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B 0 AT1. EXPERIMENTAL APPROACHA CHO-based cell line was generated, stably expressing collectrin and B 0 AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B 0 AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B 0 AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter. KEY RESULTSWe characterized a series of novel inhibitors of the B 0 AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC 50 of 44 ± 9 μM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine. CONCLUSION AND IMPLICATIONSThe tools established in this study can be widely used to identify new transport inhibitors. Using these tools, we were able to identify compounds that can be used to study epithelial transport, to induce protein restriction, or be developed further through medicinal chemistry. AbbreviationsCHO-BC, CHO cells stably transfected with B 0 AT1 and collectrin; FGF21, fibroblast growth factor 21; GIP, gastric inhibitory peptide; GLP-1, glucagon-like peptide 1; MW, molecular weight; NMDG, N-methyl-D-glucamine

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Section: Discussionmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Cheng

Shah

Bröer

et al. 2017

British J Pharmacology

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“…Collectrin/Tmem27 and ACE2 play important roles in renal and intestinal amino acid transport by acting as binding partners of amino acid transporters, regulating their trafficking and expression on the cell surface, and involving in their catalytic activities. [59][60][61] Collectrin/Tmem27 has been shown to bind to protein complexes involved in intracellular and ciliary movement of vesicles and membrane proteins. 62 Collectrin/Tmem27 in pancreatic beta cells was proteolytically processed in the extracellular region.…”

Section: Putative Novel Sea Domains In Other Cell Surface Proteins Rementioning

confidence: 99%

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54

Pei

Grishin²

2017

Protein Science

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SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan-like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single-cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, a/ e-sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin-like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin-like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans.

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Loss of CLTRN function produces a neuropsychiatric disorder and a biochemical phenotype that mimics Hartnup disease

Pillai

Yubero

Shayota

et al. 2019

American J of Med Genetics Pt A

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Hartnup disease is an autosomal recessive condition characterized by neutral aminoaciduria and behavioral problems. It is caused by a loss of B 0 AT1, a neutral amino acid transporter in the kidney and intestine. CLTRN encodes the protein collectrin that functions in the transportation and activation of B 0 AT1 in the renal apical brush bordered epithelium. Collectrin deficient mice have severe aminoaciduria. However, the phenotype associated with collectrin deficiency in humans has not been reported.Here we report two patients, an 11-year-old male who is hemizygous for a small, interstitial deletion on Xp22.2 that encompasses CLTRN and a 22-year-old male with a deletion spanning exons 1 to 3 of CLTRN. Both of them present with neuropsychiatric phenotypes including autistic features, anxiety, depression, compulsions, and motor tics, as well as neutral aminoaciduria leading to a clinical diagnosis of Hartnup disease and treatment with niacin supplementation. Plasma amino acids were normal in both patients. One patient had low 5-hydroxyindoleacetic acid levels, a serotoninergic metabolite. We explored the expression of collectrin in the murine brain and found it to be particularly abundant in the hippocampus, brainstem, and cerebellum.We propose that collectrin deficiency in humans can be associated with aminoaciduria and a clinical picture similar to that seen in Hartnup disease. Further studies are needed to explore the role of collectrin deficiency in the neurological phenotypes. Nishitha R. Pillai and Delia Yubero shared joint first authorship.

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Molecular Basis for the Interaction of the Mammalian Amino Acid Transporters B0AT1 and B0AT3 with Their Ancillary Protein Collectrin

Abstract: Background: Collectrin is required for membrane expression of the broad neutral amino acid transporters (B 0 AT1 and -3). Results: Collectrin activates B

Cited by 43 publications

References 86 publications

Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54

Loss of CLTRN function produces a neuropsychiatric disorder and a biochemical phenotype that mimics Hartnup disease

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Molecular Basis for the Interaction of the Mammalian Amino Acid Transporters B0AT1 and B0AT3 with Their Ancillary Protein Collectrin

Abstract: Background: Collectrin is required for membrane expression of the broad neutral amino acid transporters (B 0 AT1 and -3). Results: Collectrin activates B

Cited by 43 publications

References 86 publications

Identification of novel inhibitors of the amino acid transporter B0AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Identification of novel inhibitors of the amino acid transporter B0AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54

Loss of CLTRN function produces a neuropsychiatric disorder and a biochemical phenotype that mimics Hartnup disease

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Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes