Regulation of thyrotropin receptor protein expression in insect cells.
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.
Seetharamaiah
G S
GS
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Kaithamana
S
S
Desai
R K
RK
Prabhakar
B S
BS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000737:chemistry / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation; D006031:Glycosylation; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D008970:Molecular Weight; D009230:Myxedema / Q000276:immunology; D009500:Neutralization Tests; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D018411:Spodoptera / Q000166:cytology* / Q000378:metabolism / Q000821:virology; D014158:Transcription, Genetic / Q000235:genetics; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C094372:thyrotropin-binding inhibitory immunoglobulin