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J. Biol. Chem., Vol. 275, Issue 35, 27457-27465, September 1, 2000

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Bioengineering of Human Thyrotropin Superactive Analogs by Site-directed "Lysine-scanning" Mutagenesis
COOPERATIVE EFFECTS BETWEEN PERIPHERAL LOOPS^*

Holger Leitolf, Kim Phuong T. Tong, Mathis Grossmann^§, Bruce D. Weintraub, and Mariusz W. Szkudlinski^¶

From the Laboratory of Molecular Endocrinology, Division of Endocrinology, Diabetes, and Nutrition, University of Maryland School of Medicine and Division of Basic Science, Institute of Human Virology, Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the L3 loop of the common -subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent L3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the L3 loop analog compared with both the L1 loop analog and the hTSH-L3 loop analog was observed. We demonstrated that spatially distinct regions of the common -subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.

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