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

Abstract:

: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.

journal_name

Endocrine

journal_title

Endocrine

authors

Angelova K,Fremont V,Jain R,Zhang M,Puett D,Narayan P,Szkudlinski MW

doi

10.1385/ENDO:24:1:025

subject

Has Abstract

pub_date

2004-06-01 00:00:00

pages

25-31

issue

1

eissn

1355-008X

issn

1559-0100

pii

ENDO:24:1:025

journal_volume

24

pub_type

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