A recombinant extracellular domain of the thyrotropin (TSH) receptor binds TSH in the absence of membranes.
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.
Seetharamaiah
G S
GS
Department of Microbiology, University of Texas Medical Branch, Galveston 77555.
Kurosky
A
A
Desai
R K
RK
Dallas
J S
JS
Prabhakar
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007986:Luteinizing Hormone / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D000906:Antibodies; D018076:DNA, Complementary; D006146:Guanidines; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D003553:Cystine; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D013972:Thyrotropin; D019791:Guanidine; D003545:Cysteine