Takashi Akamizu

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: Correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.

Thyrotropin receptor processing and interaction with thyrotropin

In vitro transcription/translation, using rat thyrotropin receptor cDNA, results in the formation of nonglycosylated proteins able to bind thyrotropin, one of which approximates the 87 Kd size predicted for the receptor. In the presence of canine pancreatic microsomal membranes, putative glycosylation sites are modified as evidenced by digestion with endoglycosidase H. Using a deletion mutant, the presence of a hydrophobic peptide after the initiation signal is established as a signal peptide critical to post translational processing by the canine pancreatic membranes but not to binding thyrotropin.

A microsequencing approach to identify proteins which appear to interact with thyrotropin in rat FRTL-5 thyroid cells

In order to resolve questions concerning the in situ structure of the thyrotropin (TSH) receptor, [35S]methionine-labeled thyroid cell preparations were detergent solubilized and proteins exhibiting TSH-dependent binding to TSH-Sepharose were identified. Two such proteins, 43 and 70 kd, are identified in this report as gamma-actin and a member of the heat shock 70 protein family, respectively, based on the microsequence of two peptides from each. Identification of the former was confirmed by Western blotting and immunostaining using anti-actin, the latter by its ability to bind [32P]ATP, a characteristic feature of this family of proteins. The results suggest that TSH-cross linking reports defining TSH receptor subunits should be viewed with caution in the absence of comparative sequence data; consideration must, however, be given to the existence of receptor associated proteins.

The Cloning Road to the TSH Receptor and Autoimmune Graves’ Disease

Graves’ disease, the major form of autoimmune thyroid disease, is believed to be caused by autoantibodies to the thyrotropin (TSH) receptor. How these antibodies develop is still unknown; it is also not clear whether this is a thyroid disease or one of abnormal lymphocyte control. It can be presumed that the TSH receptor is structurally related to the receptors for other glycoprotein hormones; yet its relatively unique role as an autoantigen is unexplained. Further, it is increasingly evident that the sera of patients with Graves’ disease have a multiplicity of antibodies to thyroid antigens in addition to the TSH receptor and that these may be relevant to understanding the expression of the disease or its pathogenesis.