Abstract
We investigated the transmembrane topology of the GluR3 subunit that was translated in rabbit reticulocytes supplemented with microsomal membranes. A prolactin reporter epitope was fused to GluR3 at six locations, bracketing each of the proposed transmembrane domains. The sidedness of the epitope in the microsomal membrane was then assessed by proteinase K sensitivity. The N terminus and the entire region between M3 and M4 was extracellular, and the C terminus was intracellular by this method. Four native N-linked glycosylation sites in the amino terminus and one introduced site between M3 and M4 were utilized, confirming the extracellular location of these regions. Epitopes inserted upstream and downstream of M2 were protease sensitive and thus intracellular. Our results support a topological model for glutamate receptor subunits that consists of three transmembrane domains, M1, M3, and M4, and another domain, the proposed channel-lining M2, which forms a reentrant membrane segment with both ends facing the cytoplasm.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Base Sequence
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Cell Membrane / metabolism
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Cell Membrane / ultrastructure
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Female
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Intracellular Membranes / metabolism
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Kainic Acid / pharmacology
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Macromolecular Substances
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Membrane Potentials / drug effects
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Microsomes / metabolism
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Models, Structural
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Mutagenesis
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Mutagenesis, Insertional
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Mutagenesis, Site-Directed
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Oocytes / drug effects
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Oocytes / physiology
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Prolactin / chemistry
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Prolactin / isolation & purification
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Protein Biosynthesis
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Protein Structure, Secondary*
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RNA, Messenger / metabolism
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Rabbits
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Receptors, Glutamate / biosynthesis
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Receptors, Glutamate / chemistry*
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Receptors, Glutamate / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / isolation & purification
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Restriction Mapping
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Reticulocytes / metabolism
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Sequence Deletion
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Transcription, Genetic
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Xenopus laevis
Substances
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Macromolecular Substances
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RNA, Messenger
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Receptors, Glutamate
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Recombinant Fusion Proteins
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Prolactin
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Kainic Acid