THYROID Volume 7, Number 6, 1997 Mary Ann Liebert, Inc. Thyrotropin Receptor Expression in Adrenal, Kidney, and Thymus CM. DUTTON,1 W. JOBA,2 C. SPITZWEG,2 A.E. HEUFELDER,2 and R.S. BAHN,1 ABSTRACT Because the thyrotropin receptor (TSHR) has long been considered a thyroid-specific protein, its presence in ex¬ trathyroidal tissues has been controversial. In this study, we sought to detect and quantify this potentially low abundance mRNA in various extrathyroidal tissues using liquid hybridization analysis (LHA) and to detect pro¬ tein with immunohistochemical studies. Strongly positive protected bands, indicating the presence of both intact (2.4 kb) and variant (1.3 kb) TSHr mRNA, were apparent in LHA gel lanes corresponding to normal thyroid, Graves' thyroid, and thymus. Less abundant protected bands of the same sizes were present in lanes corresponding to normal adrenal, and samples from normal kidney were faintly positive. The full-length transcriptrvariant tran¬ script ratio was approximately 1:1 in all positive tissues. Immunohistochemical analysis of TSHR-like reactivity in paraffin-embedded thymus, adrenal, and kidney revealed specific staining in each of these tissues. No TSHR mRNA or TSHR-like immunoreactivity was detected in samples from several other normal human tissues. We conclude that measurable TSHR mRNA and protein expression is not restricted to the thyroid gland. Further study is warranted to determine whether these extrathyroidal receptors play a role in normal physiology or in disease. INTRODUCTION Because the thyrotropin receptor (TSHR) has long been considered a thyroid-specific protein, its presence in extrathyroidal tissues has been controversial. Because this receptor is considered a candidate antigen involved in the extrathyroidal manifestations of Graves' disease, its presence has been sought in human orbital and skin tis¬ sues (1). Both full-length TSHR mRNA and a 1.3-kb vari¬ ant TSHR transcript have been detected in normal and Graves' orbital connective tissues using reverse-transcrip- tase polymerase chain reaction (RT-PCR) (2-7), and thy¬ rotropin (TSH)-like immunoreactivity has been reported in these tissues (8,9). In addition, two recent studies have demonstrated TSHR mRNA in normal thymus using Northern blotting or RT-PCR (10,11). In this study, we sought to detect and quantify this mRNA in various extrathyroidal tissues. Previous studies of extrathyroidal TSHR expression have been difficult to interpret because low abundance transcripts detected only by PCR-amplification of cDNA may have limited physio¬ logical significance (12), and immunohistochemical stud¬ ies may lack specificity. Therefore, we used the direct, semi- quantitative, and specific method of liquid hybridization analysis (LHA) to complement our immunohistochemical studies of extrathyroidal TSHR expression. MATERIALS AND METHODS RNA preparation Total RNA was isolated directly from normal or Graves' human thyroid tissue, cultured Chinese hamster ovary (CHO) cells that had been transfected with plasmid con¬ taining the human TSHR (JP09 line), or a negative con¬ trol counterpart (JP02 line) (13) using the Totally RNA Kit (Ambion, Austin, TX). Total RNA samples isolated from various other normal human tissues (including thy¬ mus, kidney, adrenal testis, brain, liver and skeletal mus¬ cle) were obtained commercially (Clontech, Palo Alto, CA). 'Division of Endocrinology, Mayo Clinic/Foundation, Rochester, Minnesota. 2Medizinische Klinik, Klinikum Innenstadt, University of Munich, Germany. 879