Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.
Previously, using a synthetic peptide strategy, we determined that four distinct regions of human beta-thyrotropin (beta TSH) 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 beta 101-112, which inhibited binding of radiolabeled beta TSH to receptor in radioreceptor assay with an IC50 of approximately 100 microM. In the current studies, we systematically substituted the native amino acids in region beta 101-112 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 Lys110 with 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 beta 101-112, 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 beta 101-112 sequence significantly enhanced the inhibitory activity of the region.(ABSTRACT TRUNCATED AT 250 WORDS)
Leinung
M C
MC
Division of Endocrinology and Metabolism, Mayo Clinic and Medical School, Rochester, Minnesota 55901.
Bergert
E R
ER
McCormick
D J
DJ
Morris
J C
JC
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D004563:Electrochemistry; D006801:Humans; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000409:Alanine