Abstract
The beta-adrenergic receptor (beta AR) contains significant amounts of N-linked carbohydrate. Deletion mutants spanning the four consensus glycosylation sites on the receptor and single amino acid substitutions within the two amino-terminal consensus glycosylation sites reveal that both the amino-terminal sites are utilized. None of the glycosylation-defective beta AR mutants exhibited altered ligand binding in transient expression assays. In addition, the mutant beta ARs which were completely devoid of carbohydrate were capable of coupling to Gs and stimulating adenylyl cyclase in stable L cell lines. In contrast to the wild-type beta AR, the glycosylation-deficient beta ARs expressed in these cells showed a 50% decrease in the level of accumulation on the cell surface. Therefore, while glycosylation of the beta AR does not appear to be essential for receptor function, it is important for correct trafficking of the beta AR protein through the cell.
Highlights
BAR mutants exhibited altered ligand binding in transient expression assays
The mutant BARS which were completely devoid of carbohydrate were capable of coupling to G. and stimulating adenylyl cyclase in stable
The wild-type and single substitution mutants were affected by tunicamycin treatment, while the amino-terminal deletion and the double substitution mutant were not. These results suggest that all of the N-linked carbohydrate is contained at the amino terminus of the protein and that both of the consensus glycosylation sites are utilized
Summary
From the Departments of Molecular Biology and $Biochemistry, Pennsyluania I9486 and Rahway, New Jersey 07065. Site-directed mutagenesis of the PAR has determined that the ligand binding site of the receptor involves residues within the proposed transmembrane domains of the protein [14,15,16,17]. Likewise treatment of cells containing the PAR with inhibitors of Nlinked glycosylation had no effect on the ability of the newly synthesized receptor to bind ligands [25,26,27]. Using tunicamycin treatment of cells, George et al [26] reported no effect on the coupling of the PAR to adenylyl cyclase. We demonstrate that, while not required for ligand binding, glycosylation is important for the accumulation of the PAR at the cell surface and for complete coupling of the receptor to the endogenous adenylyl cyclase system
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