Author(s): PetajaRepo UE, Hogue M, Laperriere A, Walker P, Bouvier M
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Abstract Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes that include protein folding, post-translational modifications, and transport through distinct cellular compartments. Relatively little is known about the nature and kinetics of specific steps involved in these processes. Here, the human delta opioid receptor expressed in human embryonic kidney 293S cells is used as a model to delineate these steps and to establish the kinetics of receptor synthesis, glycosylation, and transport. We found that the receptor is synthesized as a core-glycosylated M(r) 45,000 precursor that is converted to the fully mature M(r) 55,000 receptor with a half-time of about 120 min. In addition to trimming and processing of two N-linked oligosaccharides, maturation involves addition of O-glycans containing N-acetylgalactosamine, galactose, and sialic acid. In contrast to N-glycosylation, which is initiated co-translationally and is completed when the protein reaches the trans-Golgi network, O-glycosylation was found to occur only after the receptor exits from the endoplasmic reticulum (ER) and was terminated as early as the trans-Golgi cisternae. Once the carbohydrates are fully processed and the receptor reaches the trans-Golgi network, it is transported to the cell surface in about 10 min. The exit from the ER was found to be the limiting step in overall processing of the receptor. This indicates that early events in the folding of the receptor are probably rate-limiting and that receptor folding intermediates are retained in the ER until they can adopt the correct conformation. The overall low efficiency of receptor maturation, less than 50\% of the precursor being processed to the fully glycosylated protein, further suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER. This indicates that folding and ER export are key events in control of receptor cell surface expression. Whether or not the low efficiency of the ER export is a general feature among G protein-coupled receptors remains to be investigated.
This article was published in J Biol Chem
and referenced in Journal of Clinical & Experimental Pharmacology