Further characterization of the receptor-binding region of the thyroid-stimulating hormone a subunit: A comprehensive synthetic peptide study of the α-subunit 26-46 sequence

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone a subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of ¹²⁵I-labeled bovine TSH to porcine thyroid membranes. Removal, either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys³¹, Cys³², Phe³³, Arg³⁵, Arg⁴², Lys⁴⁴, and Lys⁴⁵, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly³⁰ and Ser³⁴, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg³⁵ (leaving the α-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the α-subunit 26-46 sequence. The first lies between residues Gly³⁰ and Arg³⁵ and includes Cys³¹, Cys³², and Phe³³ as important constituents, and the second region lies between residues Arg⁴² and Lys⁴⁵ and includes Lys⁴⁴ as an important residue and Ser⁴³ as a less important component.