The use of the amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyrotrophin receptor.
In order to produce significant quantities of the human thyrotrophin (TSH) receptor we have investigated the use of two eukaryotic high expression systems. DNA encoding the receptor was obtained by the polymerase chain reaction (PCR) applied to thyroid cDNA. Receptor DNA was inserted into the baculovirus system; despite high mRNA levels there was little or no demonstrable protein production. However, using a novel amplifiable glutamine synthetase system, clones of transfected Chinese hamster ovary (CHO) cells expressed a high affinity TSH receptor (KD 0.225 +/- 0.046 nM, Bmax 20,000-45,000 sites/cell for individual clones). This was coupled to adenylate cyclase as measured by a TSH-stimulatable increase in extracellular cyclic AMP (cAMP), a detectable response being noted at 1 microU/ml TSH with half-maximal at around 25-50 microU/ml. The high expression allowed detection of both TSH binding inhibition and adenylate cyclase stimulation by autoantibodies in unfractionated sera from patients with Graves' disease.
Harfst
E
E
Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK.
Johnstone
A P
AP
Gout
I
I
Taylor
A H
AH
Waterfield
M D
MD
Nussey
S S
SS
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D005822:Genetic Vectors; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis; D013972:Thyrotropin / Q000494:pharmacology
D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases