Previously, using a synthetic peptide strategy, we determined that four distinct regions of human β-thyrotropin (βTSH) were responsible for interaction of TSH with the TSH receptor. The most potent of these four regions was the carboxyl-terminus of the subunit, represented by the peptide sequence β101-112, which inhibited binding of radiolabeled βTSH to receptor in radioreceptor assay with an IC50 of approximately 100 µM. In the current studies, we systematically substituted the native amino acids in region β101-112 with alanine, and we have determined which residues within this span are important to the binding activity of TSH to its receptor. Substitution of Lys101, Asn103, Tyr104, Cys105, Lys107, and Lys110with alanine each caused a significant fall in activity as compared to the native sequence, whereas substitution at the remaining positions had little or no effect. Because three of these residues are positively charged at physiologic pH, we hypothesized that this charge may be important to the binding activity of the sequence. We modified the charge characteristics of the region by synthesizing two series of analogs in which the residues identified in the alanine substitution studies were substituted with Arg, D-Lys, and D-Arg at each position. In addition, a series of analogs containing basic residues, either added to or substituted for nonbasic residues in the sequence β101-112, was synthesized. Substitution of Arg, D-Lys, and D-Arg for Lys101, Lys107, and Lys110 had little effect on activity; however, inclusion of additional basic residues in the β101-112 sequence significantly enhanced the inhibitory activity of the region. Substitution of Ala, Ser, Lys or D-Lys for Cys105 resulted in marked reduction in activity. In order to determine if the activity of the region was sequence specific or, rather, due to the amino acid composition of the region, we reversed and scrambled the amino acid residues in an additional series of peptides. The peptide with reversed sequence as well as four peptides with scrambled sequence possessed equal activity to the native peptide, suggesting that the amino acid composition and the net charge of the region is more important than its specific sequence. We concluded that a net positive charge of region 101-112 of βTSH is an important factor in the inhibitory activity of peptides representing this portion of the hormone. Enhancement of the charge, by addition of basic residues, may increase the potency of interaction of TSH with its receptor. However, positive charge is not all important, as removal of Cys105 results in marked loss in activity even though the resulting peptide may have overall significantly greater charge.
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