Human α-subunit analogs act as partial agonists to the thyroid-stimulating hormone receptor

Abstract

The α-subunit is common to the heterodimeric glycoprotein hormones and has been highly conserved throughout vertebrate evolution. In an effort to determine if wild-type and engineered human α analogs can serve as agonists or antagonists to the human thyroid-stimulating hormone (TSH) receptor (TSHR), a potent α mutant, obtained by replacing four amino acid residues with lysine (α4K), was assayed and compared with the wild-type α-subunit. When added to CHO cells expressing TSHR, α4K, and to a very limited extent the fused homodimer, α4K-α4K, but not α, exhibited ago-nistactivity as judged by cAMP production. When yoked to TSHR to yield fusion proteins, neither α, α4K, α-α, nor α4K-α4K activated TSHR, although yoked α4K and α4K-α4K were weak inhibitors of TSH binding to TSHR. The yoked subunit-receptor complexes were, however, functional as evidenced by increased cAMP production in cells co-expressing human TSHβ and α-TSHR, α4K-TSHR, α-α-TSHR, and α4K-α4K-TSHR. These results demonstrate that agonists to TSHR can be obtained with α-subunit analogs and suggest that rational protein engineering may lead to more potent α-based derivatives. The differences found between the experimental paradigms of adding free α analogs to TSHR and covalent attachment are attributed to conformational constraints imposed by fusion of the α-subunit analog and receptor, and may suggest an important role for a free (C-terminal) α-carboxyl in the absence of the β-subunit.