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

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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 homogeniety 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 - Feb 1994

ASJC Scopus subject areas

  • Endocrinology

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    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