High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.
A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.
Cunningham
B C
BC
Department of Biomolecular Chemistry, Genentech, South San Francisco, CA 94080.
Wells
J A
JA
D000409:Alanine; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes / Q000276:immunology; D013006:Growth Hormone / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010928:Placental Lactogen; D011388:Prolactin; D011487:Protein Conformation; D011986:Receptors, Somatotropin / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D011986:Receptors, Somatotropin; D011388:Prolactin; D013006:Growth Hormone; D010928:Placental Lactogen; D000409:Alanine