ABSTRACT
The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.
Subject(s)
Fusion Regulatory Protein 1, Heavy Chain/chemistry , Large Neutral Amino Acid-Transporter 1/chemistry , Cryoelectron Microscopy , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Fusion Regulatory Protein 1, Heavy Chain/ultrastructure , Humans , Large Neutral Amino Acid-Transporter 1/metabolism , Large Neutral Amino Acid-Transporter 1/ultrastructure , Models, Molecular , Protein Conformation , Protein Folding , Protein MultimerizationABSTRACT
The L-type amino acid transporter 1 (LAT1; also known as SLC7A5) catalyses the cross-membrane flux of large neutral amino acids in a sodium- and pH-independent manner1-3. LAT1, an antiporter of the amino acid-polyamine-organocation superfamily, also catalyses the permeation of thyroid hormones, pharmaceutical drugs, and hormone precursors such as L-3,4-dihydroxyphenylalanine across membranes2-6. Overexpression of LAT1 has been observed in a wide range of tumour cells, and it is thus a potential target for anti-cancer drugs7-11. LAT1 forms a heteromeric amino acid transporter complex with 4F2 cell-surface antigen heavy chain (4F2hc; also known as SLC3A2)-a type II membrane glycoprotein that is essential for the stability of LAT1 and for its localization to the plasma membrane8,9. Despite extensive cell-based characterization of the LAT1-4F2hc complex and structural determination of its homologues in bacteria, the interactions between LAT1 and 4F2hc and the working mechanism of the complex remain largely unknown12-19. Here we report the cryo-electron microscopy structures of human LAT1-4F2hc alone and in complex with the inhibitor 2-amino-2-norbornanecarboxylic acid at resolutions of 3.3 Å and 3.5 Å, respectively. LAT1 exhibits an inward open conformation. Besides a disulfide bond association, LAT1 also interacts extensively with 4F2hc on the extracellular side, within the membrane, and on the intracellular side. Biochemical analysis reveals that 4F2hc is essential for the transport activity of the complex. Together, our characterizations shed light on the architecture of the LAT1-4F2hc complex, and provide insights into its function and the mechanisms through which it might be associated with disease.
Subject(s)
Cryoelectron Microscopy , Fusion Regulatory Protein 1, Heavy Chain/chemistry , Fusion Regulatory Protein 1, Heavy Chain/ultrastructure , Large Neutral Amino Acid-Transporter 1/chemistry , Large Neutral Amino Acid-Transporter 1/ultrastructure , Amino Acids/metabolism , Binding Sites , Biological Transport , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Disulfides/chemistry , Disulfides/metabolism , Fusion Regulatory Protein 1, Heavy Chain/antagonists & inhibitors , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Humans , Large Neutral Amino Acid-Transporter 1/genetics , Large Neutral Amino Acid-Transporter 1/metabolism , Models, Molecular , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Norbornanes/chemistry , Norbornanes/pharmacology , Protein Binding , Protein ConformationABSTRACT
Angiogenesis is essential for tumor growth, and an enhanced vasculature supplying nutrients and oxygen might reflect malignant potential. L-type amino acid transporter 1 (LAT1/4F2hc) comprises a major nutrient transport system responsible for the Na+-independent transport of large neutral amino acids. Seventy five to seventy eight percent N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinoma cells showed high LAT1/4F2hc expression. While the intracarcinoma microvasculatures of fenestrated endothelial cells highly expressing LAT1/4F2hc might progressively transport essential amino acids from the microvasculatures to the extracellular matrix, non-fenestrated endothelial cells and pericytes did not. The present study revealed that the tumor angiogenesis is one of target anti-L-type amino acid transporter 1 drug.
