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

    74 Citations (Scopus)

    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

    Fingerprint

    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
    • Endocrinology, Diabetes and Metabolism

    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/en.134.2.549

    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, 02.1994, 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/en.134.2.549
    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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