IN-VITRO TRANSLATION AND MEMBRANE TOPOLOGY OF RAT RECOMBINANT MGLUR1-ALPHA

The structure and post-translational processing of the metabotropic gl utamate receptor 1 alpha (mGluR1 alpha) was analysed by in vitro cell- free translation, protease protection and deglycosylation. We show tha t mGluR1 alpha can be synthesized in the rabbit-reticulocyte translati on system to yield a predominant polypeptide product with an apparent molecular weight of 142 kDa. In the presence of dog-pancreatic microso mes this polypeptide was processed to an apparent molecular weight of 147 kDa. Treatment with the enzyme peptide-N-glycosidase F (PNGF) demo nstrated that the increase in the apparent molecular weight of the pro cessed 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 a mino terminal of mGluR1 alpha is translocated into the lumen of the en doplasmic reticulum and will consequently be located extracellularly w hen targeted to the plasma membrane. The data presented here on mGluR1 alpha indicates the potential of in vitro translation and protease pr otection in the study of the molecular structure and processing of glu tamate receptors.