A recombinant extracellular domain of the thyrotropin (TSH) receptor binds tsh in the absence of membranes

Gattadahalli S. Seetharamaiah, Alexander Kurosky, Rajesh K. Desai, John S. Dallas, Bellur S. Prabhakar

Research output: Contribution to journalArticle

Abstract

We produced large quantities of the extracellular domain of the human TSH receptor (ETSHR) using the baculovirus expression system. Insect cells containing the ETSHR protein were sequentially extracted using lysis, nuclease, and high salt buffers to enrich for recombinant protein. The ETSHR protein was purified to homogeneity on a C4 reverse phase semipreparative column using HPLC. The recombinant protein was identified as ETSHR by immunoreactivity with antibodies prepared against TSHR-derived synthetic peptides. The identity of the ETSHR was further confirmed by amino acid compositional analyses, which agreed with the amino acid composition predicted from reported cDNA sequence analyses. Protein sequence analyses confirmed that the first 26 amino acids of the N-terminal region and the C-terminal amino acid were identical to the predicted amino acid sequence. The purified ETSHR was refolded in the presence of 1.5 M guanidine-HCl and 1 mM each of cystine and cysteine. [125I] TSH bound to the refolded ETSHR in vitro in a dose-dependent manner and was specifically blocked by unlabeled TSH, but not by LH or FSH. It was notable that a membrane requirement was not essential for TSH to bind to ETSHR.

Original languageEnglish (US)
Pages (from-to)549-554
Number of pages6
JournalEndocrinology
Volume134
Issue number2
DOIs
StatePublished - 1994

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Thyrotropin Receptors
Membranes
Amino Acids
Recombinant Proteins
Cystine
Baculoviridae
Guanidine
Protein Sequence Analysis
Cysteine
Sequence Analysis
Insects
Amino Acid Sequence
Buffers
Proteins
Complementary DNA
Salts
High Pressure Liquid Chromatography
Peptides
Antibodies

ASJC Scopus subject areas

  • Endocrinology

Cite this

Seetharamaiah, G. S., Kurosky, A., Desai, R. K., Dallas, J. S., & Prabhakar, B. S. (1994). A recombinant extracellular domain of the thyrotropin (TSH) receptor binds tsh in the absence of membranes. Endocrinology, 134(2), 549-554. https://doi.org/10.1210/endo.134.2.8299554

A recombinant extracellular domain of the thyrotropin (TSH) receptor binds tsh in the absence of membranes. / Seetharamaiah, Gattadahalli S.; Kurosky, Alexander; Desai, Rajesh K.; Dallas, John S.; Prabhakar, Bellur S.

In: Endocrinology, Vol. 134, No. 2, 1994, p. 549-554.

Research output: Contribution to journalArticle

Seetharamaiah, GS, Kurosky, A, Desai, RK, Dallas, JS & Prabhakar, BS 1994, 'A recombinant extracellular domain of the thyrotropin (TSH) receptor binds tsh in the absence of membranes', Endocrinology, vol. 134, no. 2, pp. 549-554. https://doi.org/10.1210/endo.134.2.8299554
Seetharamaiah, Gattadahalli S. ; Kurosky, Alexander ; Desai, Rajesh K. ; Dallas, John S. ; Prabhakar, Bellur S. / A recombinant extracellular domain of the thyrotropin (TSH) receptor binds tsh in the absence of membranes. In: Endocrinology. 1994 ; Vol. 134, No. 2. pp. 549-554.
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