Construction of Stable Expression of Human Thyrotropin Receptor α-Subunits on HEK 293T Cells_Journal of Biomedical Engineering

Authors：

 TANGGongshun

Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu 610041, China;

Corresponding author：

TANGGongshun, Email: tgs010@163.com

Keywords：

thyroid stimulating hormone receptor; lentiviral vector; HEK 293T; graves disease

DOI：

10.7507/1001-5515.20140029

Video：

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The aim of this study was to establish stable expression of human thyroid stimulating hormone receptor (TSHR) α-subunit (hTSHRA) on human embryonic kidney 293T (HEK 293T). HEK 293T cell lines with stable expression of hTSHRA could be used for detecting affinity between hTSHRA and potential TSHR blocking-peptide. We firstly constructed hTSHRA gene into lentiviral vectors GV218. The sequence comparison indicated that we had constructed GV218-hTSHRAA. Western blot demonstrated the 52 kD aim band of hTHSRA on over-expressed HEK 293T cells. GV218-hTSHRA constructions and pHelper were then co-transfected into HEK 293T cells to form packaging plasmid. The HEK 293T cells that stably expressed hTSHRA could also express green fluorescent protein. The titer of lentiviral packaging vector is 2×10⁸ TU/mL with qPCR. The lentiviral packaging vector thereafter was transfected into HEK 293T cells again. The hTSHRA expressed on the HEK 293T cells. Human TSHRA stably expressed on HEK 293T upon continuously passaging. Therefore, we established hTSHRA stable expression on HEK 293T cells by constructing GV218-hTHSR lentiviral packaging vector. It is a useful tool for studying TSHR affinity with anti-thyroid peptide.

Citation： TANGGongshun. Construction of Stable Expression of Human Thyrotropin Receptor α-Subunits on HEK 293T Cells. Journal of Biomedical Engineering, 2014, 31(1): 146-151. doi: 10.7507/1001-5515.20140029 Copy

1.	BJÖRKHOLM M, PISA P, ARVER S, et al. Haematologic effects of granulocyte-macrophage colony stimulating factor in a patient with thiamazole-induced agranulocytosis[J]. J Intern Med, 1992, 232(5):443-445.
2.	BISWAS N, AHN Y H, GOLDMAN J M, et al. Aplastic anemia associated with antithyroid drugs[J]. Am J Med Sci, 1991, 301(3):190-194.
3.	MEMI E, KARRAS S, TZOTZAS T, et al. Propylthiouracil hepatitis:report of a case and extensive review of the literature[J]. J Pediatr Endocrinol Metab, 2012, 25(3-4):331-343.
4.	SANDERS J, JEFFREYS J, DEPRAETERE H, et al. Characteristics of a human monoclonal autoantibody to the thyrotropin receptor:sequence structure and function[J]. Thyroid, 2004, 14(8):560-570.
5.	N AU'U ÑEZ MIGUEL R, SANDERS J, CHIRGADZE D Y, et al. Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain:a comparative structural study of protein-protein interactions[J]. J Mol Endocrinol, 2009, 42(5):381-395.
6.	NEUMANN S, KLEINAU G, COSTANZI S, et al. A low-molecular-weight antagonist for the human thyrotropin receptor with therapeutic potential for hyperthyroidism[J]. Endocrinology, 2008, 149(12):5945-5950.
7.	HAAS A K, KLEINAU G, HOYER I, et al. Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor[J]. Cell Mol Life Sci, 2011, 68(1):159-167.
8.	CHEN C R, MCLACHLAN S M, RAPOPORT B. Identification of key amino acid residues in a thyrotropin receptor monoclonal antibody epitope provides insight into its inverse agonist and antagonist properties[J]. Endocrinology, 2008, 149(7):3427-3434.
9.	CHEN C R, MCLACHLAN S M, RAPOPORT B. Suppression of thyrotropin receptor constitutive activity by a monoclonal antibody with inverse agonist activity[J]. Endocrinology, 2007, 148(5):2375-2382.
10.	PENNA-MARTINEZ M, WINTEN C, FICHTEL T, et al. Isolation of thyroid cells obtained by fine-needle aspiration biopsy[J]. Thyroid, 2005, 15(9):989-995.
11.	TSUI S, NAIK V, HOA N, et al. Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors:a tale of two antigens implicated in Graves' disease[J]. J Immunol, 2008, 181(6):4397-4405.
12.	FUKUSHIMA H, MIZUTANI M, IMAMURA K, et al. Development of a novel preparation method of recombinant proteoliposomes using baculovirus gene expression systems[J]. J Biochem, 2008, 144(6):763-770.
13.	AGRETTI P, CHIOVATO L, DE MARCO G, et al. Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues[J]. Eur J Endocrinol, 2002, 147(6):733-739.
14.	DUTTON C M, JOBA W, SPITZWEG C, et al. Thyrotropin receptor expression in adrenal, kidney, and thymus[J]. Thyroid, 1997, 7(6):879-884.
15.	SELLITTI D F, AKAMIZU T, DOI S Q, et al. Renal expression of two 'thyroid-specifić genes:thyrotropin receptor and thyroglobulin[J]. Exp Nephrol, 2000, 8(4-5):235-243.
16.	BAHN R S, DUTTON C M, NATT N, et al. Thyrotropin receptor expression in Graves' orbital adipose/connective tissues:potential autoantigen in Graves' ophthalmopathy[J]. J Clin Endocrinol Metab, 1998, 83(3):998-1002.
17.	BHATTACHARYYA K K, COENEN M J, BAHN R S. Thyroid transcription factor-1 in orbital adipose tissues:potential role in orbital thyrotropin receptor expression[J]. Thyroid, 2005, 15(5):422-426.
18.	LU S, GUAN Q, LIU Y, et al. Role of extrathyroidal TSHR expression in adipocyte differentiation and its association with obesity[J]. Lipids Health Dis, 2012, 11:17.

1. BJÖRKHOLM M, PISA P, ARVER S, et al. Haematologic effects of granulocyte-macrophage colony stimulating factor in a patient with thiamazole-induced agranulocytosis[J]. J Intern Med, 1992, 232(5):443-445.
2. BISWAS N, AHN Y H, GOLDMAN J M, et al. Aplastic anemia associated with antithyroid drugs[J]. Am J Med Sci, 1991, 301(3):190-194.
3. MEMI E, KARRAS S, TZOTZAS T, et al. Propylthiouracil hepatitis:report of a case and extensive review of the literature[J]. J Pediatr Endocrinol Metab, 2012, 25(3-4):331-343.
4. SANDERS J, JEFFREYS J, DEPRAETERE H, et al. Characteristics of a human monoclonal autoantibody to the thyrotropin receptor:sequence structure and function[J]. Thyroid, 2004, 14(8):560-570.
5. N AU'U ÑEZ MIGUEL R, SANDERS J, CHIRGADZE D Y, et al. Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain:a comparative structural study of protein-protein interactions[J]. J Mol Endocrinol, 2009, 42(5):381-395.
6. NEUMANN S, KLEINAU G, COSTANZI S, et al. A low-molecular-weight antagonist for the human thyrotropin receptor with therapeutic potential for hyperthyroidism[J]. Endocrinology, 2008, 149(12):5945-5950.
7. HAAS A K, KLEINAU G, HOYER I, et al. Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor[J]. Cell Mol Life Sci, 2011, 68(1):159-167.
8. CHEN C R, MCLACHLAN S M, RAPOPORT B. Identification of key amino acid residues in a thyrotropin receptor monoclonal antibody epitope provides insight into its inverse agonist and antagonist properties[J]. Endocrinology, 2008, 149(7):3427-3434.
9. CHEN C R, MCLACHLAN S M, RAPOPORT B. Suppression of thyrotropin receptor constitutive activity by a monoclonal antibody with inverse agonist activity[J]. Endocrinology, 2007, 148(5):2375-2382.
10. PENNA-MARTINEZ M, WINTEN C, FICHTEL T, et al. Isolation of thyroid cells obtained by fine-needle aspiration biopsy[J]. Thyroid, 2005, 15(9):989-995.
11. TSUI S, NAIK V, HOA N, et al. Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors:a tale of two antigens implicated in Graves' disease[J]. J Immunol, 2008, 181(6):4397-4405.
12. FUKUSHIMA H, MIZUTANI M, IMAMURA K, et al. Development of a novel preparation method of recombinant proteoliposomes using baculovirus gene expression systems[J]. J Biochem, 2008, 144(6):763-770.
13. AGRETTI P, CHIOVATO L, DE MARCO G, et al. Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues[J]. Eur J Endocrinol, 2002, 147(6):733-739.
14. DUTTON C M, JOBA W, SPITZWEG C, et al. Thyrotropin receptor expression in adrenal, kidney, and thymus[J]. Thyroid, 1997, 7(6):879-884.
15. SELLITTI D F, AKAMIZU T, DOI S Q, et al. Renal expression of two 'thyroid-specifić genes:thyrotropin receptor and thyroglobulin[J]. Exp Nephrol, 2000, 8(4-5):235-243.
16. BAHN R S, DUTTON C M, NATT N, et al. Thyrotropin receptor expression in Graves' orbital adipose/connective tissues:potential autoantigen in Graves' ophthalmopathy[J]. J Clin Endocrinol Metab, 1998, 83(3):998-1002.
17. BHATTACHARYYA K K, COENEN M J, BAHN R S. Thyroid transcription factor-1 in orbital adipose tissues:potential role in orbital thyrotropin receptor expression[J]. Thyroid, 2005, 15(5):422-426.
18. LU S, GUAN Q, LIU Y, et al. Role of extrathyroidal TSHR expression in adipocyte differentiation and its association with obesity[J]. Lipids Health Dis, 2012, 11:17.

Journal of Biomedical Engineering

Construction of Stable Expression of Human Thyrotropin Receptor α-Subunits on HEK 293T Cells

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