Regulation of thyrotropin receptor protein expression in insect cells

Gattadahalli Seetharamaiah, S. Kaithamana, R. K. Desai, B. S. Prabhakar

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR- gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.

Original languageEnglish (US)
Pages (from-to)315-323
Number of pages9
JournalJournal of Molecular Endocrinology
Volume23
Issue number3
StatePublished - Dec 1999
Externally publishedYes

Fingerprint

Thyrotropin Receptors
Insects
Protein Sorting Signals
Insect Viruses
Proteins
Complementary DNA
Baculoviridae
Southern Blotting
Serum
Glycosylation
Recombinant Proteins
Northern Blotting
Autoantibodies
Polyacrylamide Gel Electrophoresis
Thyroid Gland
Western Blotting
Monoclonal Antibodies
RNA
Staining and Labeling
Viruses

ASJC Scopus subject areas

  • Endocrinology

Cite this

Regulation of thyrotropin receptor protein expression in insect cells. / Seetharamaiah, Gattadahalli; Kaithamana, S.; Desai, R. K.; Prabhakar, B. S.

In: Journal of Molecular Endocrinology, Vol. 23, No. 3, 12.1999, p. 315-323.

Research output: Contribution to journalArticle

Seetharamaiah, G, Kaithamana, S, Desai, RK & Prabhakar, BS 1999, 'Regulation of thyrotropin receptor protein expression in insect cells', Journal of Molecular Endocrinology, vol. 23, no. 3, pp. 315-323.
Seetharamaiah, Gattadahalli ; Kaithamana, S. ; Desai, R. K. ; Prabhakar, B. S. / Regulation of thyrotropin receptor protein expression in insect cells. In: Journal of Molecular Endocrinology. 1999 ; Vol. 23, No. 3. pp. 315-323.
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