Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis.
A strategy, termed homolog-scanning mutagenesis, was used to identify the epitopes on human growth hormone (hGH) for binding to its cloned liver receptor and eight different monoclonal antibodies (Mab's). Segments of sequences (7 to 30 residues long) that were derived from homologous hormones known not to bind to the hGH receptor or Mab's, were systematically substituted throughout the hGH gene to produce a set of 17 chimeric hormones. Each Mab or receptor was categorized by a particular subset of mutant hormones was categorized by a particular subset of mutant hormones that disrupted binding. Each subset of the disruptive mutations mapped within close proximity on a three-dimensional model of hGH, even though the residues changed within each subset were usually distant in the primary sequence. The mapping analysis correctly predicted those Mab's which could or could not block binding of the receptor to hGH and further suggested (along with other data) that the folding of these chimeric hormones is like that of HGH. By this analysis, three discontinuous polypeptide determinants in hGH--the loop between residues 54 and 74, the central portion of helix 4 to the carboxyl terminus, and to a lesser extent the amino-terminal region of helix 1--modulate binding to the liver receptor. Homolog-scanning mutagenesis should be of general use in identifying sequences that cause functional variation among homologous proteins.
Cunningham
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BC
Department of Biomolecular Chemistry, Genentech, Inc., South San Francisco, CA 94080.
Jhurani
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Ng
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Wells
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JA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002678:Chimera; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis*; D005796:Genes; D013006:Growth Hormone / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011986:Receptors, Somatotropin / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011986:Receptors, Somatotropin; D013006:Growth Hormone