Human alpha-subunit analogs act as partial agonists to the thyroid-stimulating hormone receptor: differential effects of free and yoked subunits.

The alpha-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 alpha analogs can serve as agonists or antagonists to the human thyroid-stimulating hormone (TSH) receptor (TSHR), a potent alpha mutant, obtained by replacing four amino acid residues with lysine (alpha4K), was assayed and compared with the wild-type alpha-subunit. When added to CHO cells expressing TSHR, alpha4K, and to a very limited extent the fused homodimer, alpha4K-alpha4K, but not alpha, exhibited agonist activity as judged by cAMP production. When yoked to TSHR to yield fusion proteins, neither alpha, alpha4K, alpha-alpha, nor alpha4K-alpha4K activated TSHR, although yoked alpha4K and alpha4K-alpha4K 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 TSHbeta and alpha-TSHR, alpha4K-TSHR, alpha-alpha-TSHR, and alpha4K-alpha4K-TSHR. These results demonstrate that agonists to TSHR can be obtained with alpha-subunit analogs and suggest that rational protein engineering may lead to more potent alpha-based derivatives. The differences found between the experimental paradigms of adding free alpha analogs to TSHR and covalent attachment are attributed to con-formational constraints imposed by fusion of the alpha-subunit analog and receptor, and may suggest an important role for a free (C-terminal) alpha-carboxyl in the absence of the beta-subunit.