Most important, there was no difference in TBI levels between the Ad-TSHR-289C and the Ad-TSHR-D1NETCimmunized animals, despite the greater incidence of thyrotoxicosis and goiter in the former group. Open in a separate window Figure 5 Comparable high levels of TBI immunoglobulins are induced in mice immunized with adenovirus expressing different structural forms of the TSHR. free A subunit as opposed to a genetically altered TSHR that cleaves minimally into PGC1A subunits. These data show that shed A subunits Engeletin induce or amplify the immune response leading to hyperthyroidism and provide new insight into the etiology of Graves disease. Introduction Graves disease is usually a common organ-specific autoimmune disease with an incidence of approximately 4 in 10,000 people per year (1). A syndrome including goiter and thyrotoxicosis, the disease differs from all other autoimmune diseases in being associated with target organ hyperfunction rather than organ damage. Thyrotoxicosis is directly caused by autoantibodies that activate the thyrotropin (TSH) receptor (TSHR) (examined in ref. 2). Genetic factors play an important role in Graves disease (for example, observe ref. 3). A concordance rate of 0.35 between identical twins (4), however, indicates that a substantial contribution derives from nongenetic factors. Of the latter, there is the intriguing possibility that this molecular structure of the target antigen contributes to the development of thyroid-stimulatory autoantibodies (TSAbs). The TSHR is unique among the glycoprotein hormone receptors in undergoing intramolecular cleavage into disulfide-linked subunits (Physique ?(Figure1a)1a) (5, 6). A consequence of TSHR cleavage is usually shedding of some of the extracellular, autoantibody-binding A subunits (7, 8). Even though physiological relevance of TSHR cleavage is not known, it is noteworthy that functional autoantibodies do not arise to the noncleaving glycoprotein hormone receptors. Open in a separate window Physique 1 (a) Schematic representation of the WT TSHR that undergoes cleavage into an extracellular A subunit and a serpentine, membrane-spanning B subunit. The cleavage process involves excision of a C peptide region (examined in ref. 2). (b) TSHR altered by deletion of amino acid residues 317C366 and substitution of GQE367-369NET. This receptor (TSHR-D1NET) undergoes minimal cleavage into subunits (15). (c) TSHR-289 is the TSHR truncated after amino acid 289, at the approximate Engeletin region of spontaneous intramolecular cleavage into subunits (14). Consequently, TSHR-289 approximates the free, or shed, A subunit. (Adapted from ref. 9.) Recently, we observed that the epitope for functionally important TSAbs in Graves disease is partially obscured in the WT TSHR but is exposed on the TSHR ectodomain tethered to the cell surface by a glycosylphosphatidylinositol anchor (9). These findings raised the possibility that the shed A subunit, not the full-length TSHR expressed on the cell surface, initiates or enhances the immune response to the TSHR that leads to hyperthyroidism. Testing this hypothesis requires an animal model of Graves disease. No spontaneous model exists, and immunization with TSHR protein plus adjuvant does not replicate the disease (reviewed in ref. 2). In contrast, processing and presentation of TSHR expressed in vivo induces Abs to the TSHR epitope(s) necessary for receptor activation (10C13). One of these approaches, Engeletin intramuscular injection into mice of adenovirus particles containing the cDNA for the human TSHR (13), provides an ideal system for testing the above hypothesis. We constructed adenoviruses encoding either free A subunits (Figure ?(Figure1c)1c) or TSHR engineered to not cleave into subunits (TSHR-D1NET; Figure ?Figure1b).1b). The data obtained confirm the hypothesis and further our understanding of the pathogenesis of Graves disease. Methods Adenovirus constructs and immunization of mice. The cDNA for TSHR-289 (14) and TSHR-D1NET (15) in the vector pECE-neo were digested with EcoRI (New England Biolabs Inc., Beverly, Massachusetts, USA), blunted with DNA polymerase Klenow fragment (USB Corp., Cleveland, Ohio, USA), digested with XbaI (New England Biolabs Inc.), and ligated into the transfer vector pHMCMV6 (16). Insert-positive plasmids were digested with I-CeuI and PI-SceI (both from BD Biosciences Clontech, Palo Alto, California, USA) and ligated into the same sites of pAdHM4CMV (17). Adenoviruses containing TSHR-289 or TSHR-D1NET were linearized.