An investigation of the membrane topology of the ionotropic glutamate receptor subunit GluR1 in a cell-free system.
We have utilized cell-free translation in rabbit-reticulocyte lysate supplemented with canine pancreatic microsomal membranes to study the processing and membrane topology of the rat ionotropic glutamate receptor subunit GluR1. In vitro-synthesized RNA encoding GluR1 was translated to yield a primary translation product with an apparent molecular mass of 99 kDa. In the presence of microsomal membranes this protein was processed to give a band of 107 kDa. Treatment with peptide-N-glycosidase F showed that this increase in molecular mass was due to N-linked glycosylation. Incubation of the processed receptor with proteinase K revealed the presence of a 68 kDa protease-resistant band which decreased to 56 kDa following deglycosylation. A deletion mutant (GluR1M1) lacking the predicted transmembrane domains was fully translocated across the microsomal membrane and protected from the action of the protease. The mutant and wild-type receptor could be immunoprecipitated by anti-peptide antibodies directed against the C-terminus. Following translocation of the wild-type and mutant receptor across the microsomal membrane and treatment with proteinase K the antibody binding to GluR1 was abolished, but was retained for GluR1M1. These data allow identification of the orientation of the N- and C-termini of GluR1 within the microsome; results which are consistent with an extracellular N-terminal and intracellular C-terminal localization following incorporation into the plasma membrane.
Department of Pharmacology, Medical School, Edgbaston, Birmingham, U.K.
A J
AJ
Seal
G L
GL
Collingridge
J M
JM
Henley
D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D004285:Dogs; D006031:Glycosylation; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D008961:Models, Structural; D008970:Molecular Weight; D016296:Mutagenesis; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011817:Rabbits / Q000276:immunology; D051381:Rats; D018091:Receptors, AMPA / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D018091:Receptors, AMPA; D011994:Recombinant Proteins