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Abstract
The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.
Highlights
The receptors for glycoprotein hormones (thyrotropin receptor (TSHR),1 lutropin receptor (LHR), and follitropin receptor (FSH)) are members of a subfamily of G protein-coupled receptors, characterized by an extremely large amino-terminal extracellular domain, which is the high affinity binding site for the respective ligands [1, 2] and is, in the case of TSHR, the primary autoantigen in autoimmune thyroid disease, including Graves’ disease [1]
Immunoprecipitation and Immunoblot of TSHR in the Clonal Cell Lines Stably Expressing TSHR—Using clonal cell lines stably expressing the highest levels of receptors, we determined the molecular masses and glycosylation patterns of TSHR expressed in Chinese hamster ovary (CHO) cells treated with and without tunicamycin and in a series of CHO-Lec cells
In immunoprecipitation experiments (2-h pulse followed by 2-h chase), TSHR protein expressed in parental CHO cells (CHO-TSHR) was visualized as an ϳ95-kDa precursor and an ϳ120-kDa mature protein (Fig. 2A, lane 1) as previously reported [27]
Summary
The receptors for glycoprotein hormones (thyrotropin receptor (TSHR), lutropin receptor (LHR), and follitropin receptor (FSH)) are members of a subfamily of G protein-coupled receptors, characterized by an extremely large amino-terminal extracellular domain, which is the high affinity binding site for the respective ligands [1, 2] and is, in the case of TSHR, the primary autoantigen in autoimmune thyroid disease, including Graves’ disease [1]. Subsequent studies on the functional role of Nlinked oligosaccharides of TSHR have been performed with the truncated form of TSHR ectodomain expressed in mammalian cells and insect cells, yielding controversial data [15, 17, 18]. Intact TSHR protein showing high affinity TSH binding has so far been expressed only in mammalian cells as the full-length receptor or the ectodomain fused to a membrane-anchoring peptide (19 –21). A dolichol pyrophosphate precursor (Glc3Man9GlcNAc2) is at first transferred to Asn side chain of Asn-X-Ser/Thr consensus sequence for Nlinked oligosaccharides in a nascent polypeptide in the endoplasmic reticulum. Tunicamycin inhibits transfer of dolichol pyrophosphate precursor to Asn in the consensus sequence for N-linked glycosylation (Asn-X-Ser/Thr)
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