In vitro translation and membrane topology of rat recombinant mGluR1 alpha.
The structure and post-translational processing of the metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was analysed by in vitro cell-free translation, protease protection and deglycosylation. We show that mGluR1 alpha can be synthesized in the rabbit-reticulocyte translation system to yield a predominant polypeptide product with an apparent molecular weight of 142 kDa. In the presence of dog-pancreatic microsomes this polypeptide was processed to an apparent molecular weight of 147 kDa. Treatment with the enzyme peptide-N-glycosidase F (PNGF) demonstrated that the increase in the apparent molecular weight of the processed translation product was due to N-linked glycosylation. Addition of the non-selective protease, proteinase K; resulted in the loss of this 147 kDa band and the appearance of a protected fragment of approx 92 kDa. A carboxy-terminal deletion mutant of mGluR1 alpha was almost completely protected from protease action. These data show that the amino terminal of mGluR1 alpha is translocated into the lumen of the endoplasmic reticulum and will consequently be located extracellularly when targeted to the plasma membrane. The data presented here on mGluR1 alpha indicates the potential of in vitro translation and protease protection in the study of the molecular structure and processing of glutamate receptors.
Department of Pharmacology, Medical School, University of Birmingham, Edgbaston, U.K.
A J
AJ
Seal
G L
GL
Collingridge
J M
JM
Henley
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002474:Cell-Free System; D003001:Cloning, Molecular; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins; D010450:Endopeptidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D005947:Glucose