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Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.
We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.
Amino Acid Sequence, Animals, Binding Sites, Blotting, Western, Cell Line, Cell Membrane/metabolism, Cricetinae, DNA, Complementary/metabolism, Electrophoresis, Polyacrylamide Gel, Glycosylation, Green Fluorescent Proteins, Inositol Phosphates/metabolism, Kinetics, Luminescent Proteins/metabolism, Microscopy, Fluorescence, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Palmitic Acids/chemistry, Phosphorylation, Precipitin Tests, Protein Binding, Protein Biosynthesis, Protein Conformation, Protein Processing, Post-Translational, Receptors, Adrenergic, alpha/chemistry, Receptors, Adrenergic, alpha/metabolism, Recombinant Fusion Proteins/metabolism, Semliki forest virus/metabolism, Sequence Homology, Amino Acid, Time Factors, Transfection, Tunicamycin/pharmacology
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