It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-β subunit, designated TSH-βv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-βv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-β, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

Background: Novel small molecular ligands (SMLs) to the thyrotropin receptor (TSHR) have potential as improved molecular probes and as therapeutic agents for the treatment of thyroid dysfunction and thyroid cancer. Methods: To identify novel SMLs to the TSHR, we developed a transcription-based luciferase-cAMP highthroughput screening system and we screened 48,224 compounds from a 100K library in duplicate. Results: We obtained 62 hits using the cut-off criteria of the mean -three standard deviations above the baseline. Twenty molecules with the greatest activity were rescreened against the parent CHO-luciferase cell for nonspecific activation, and we selected two molecules (MS437 and MS438) with the highest potency for further study. These lead molecules demonstrated no detectible cross-reactivity with homologous receptors when tested against luteinizing hormone (LH)/human chorionic gonadotropin receptor and follicle stimulating hormone receptor-expressing cells. Molecule MS437 had a TSHR-stimulating potency with an EC 50 of 13 · 10 -8 M, and molecule MS438 had an EC 50 of 5.3 · 10 -8 M. The ability of these small molecule agonists to bind to the transmembrane domain of the receptor and initiate signal transduction was suggested by their activation of a chimeric receptor consisting of an LHR ectodomain and a TSHR transmembrane. Molecular modeling demonstrated that these molecules bound to residues S505 and E506 for MS438 and T501 for MS437 in the intrahelical region of transmembrane helix 3. We also examined the G protein activating ability of these molecules using CHO cells co-expressing TSHRs transfected with luciferase reporter vectors in order to measure G sa , G bc , G aq , and G a12 activation quantitatively. The MS437 and MS438 molecules showed potent activation of G sa , G aq , and G a12 similar to TSH, but neither the small molecule agonists nor TSH showed activation of the G bc pathway. The small molecules MS437 and MS438 also showed upregulation of thyroglobulin (Tg), sodium iodine symporter (NIS), and TSHR gene expression. Conclusions: Pharmacokinetic analysis of MS437 and MS438 indicated their pharmacotherapeutic potential, and their intraperitoneal administration to normal female mice resulted in significantly increased serum thyroxine levels, which could be maintained by repeated treatments. These molecules can therefore serve as lead molecules for further development of powerful TSH agonists.

Metropolis Monte Carlo (MMC) loop refinement has been performed on the three extracellular loops (ECLs) of rhodopsin and opsin-based homology models of the thyroid-stimulating hormone receptor transmembrane domain, a class A type G protein-coupled receptor. The Monte Carlo sampling technique, employing torsion angles of amino acid side chains and local moves for the six consecutive backbone torsion angles, has previously reproduced the conformation of several loops with known crystal structures with accuracy consistently less than 2 Å. A grid-based potential map, which includes van der Waals, electrostatics, hydrophobic as well as hydrogen-bond potentials for bulk protein environment and the solvation effect, has been used to significantly reduce the computational cost of energy evaluation. A modified sigmoidal distance-dependent dielectric function has been implemented in conjunction with the desolvation and hydrogen-bonding terms. A long high-temperature simulation with 2 kcal/mol repulsion potential resulted in extensive sampling of the conformational space. The slow annealing leading to the low-energy structures predicted secondary structure by the MMC technique. Molecular docking with the reported agonist reproduced the binding site within 1.5 Å. Virtual screening performed on the three lowest structures showed that the ligand-binding mode in the inter-helical region is dependent on the ECL conformations.