A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor.
A mutational strategy is presented that allowed us to identify hormone-binding determinants in the extracellular portion of the human growth hormone receptor (hGHbp), a 238-residue protein with sequence homology to a number of cytokine receptors. By systematically replacing side chains with alanine we probed the importance of charged residues (49 total, typically located on the surface), aromatic residues (9 total), and neighbors of these (26 total). The alanine substitutions that were most disruptive to hormone binding are located predominantly in four segments of a cysteine-rich domain in the hGHbp, and collectively they form a patch when mapped upon a structural model proposed for cytokine receptors. Control experiments with monoclonal antibodies confirmed that most of these alanine substitutions do not disrupt the overall antigenic structure of the hGHbp. This high-resolution functional analysis will complement structural studies and provides a powerful basis for evaluating and engineering the energetics of hormone-receptor interactions. Moreover, the hormone-binding determinants identified here may be similarly located in other, homologous, receptors.
Bass
S H
SH
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.
Mulkerrin
M G
MG
Wells
J A
JA
D000409:Alanine; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine; D005453:Fluorescence; D014644:Genetic Variation; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014364:Tryptophan; D014443:Tyrosine
D000911:Antibodies, Monoclonal; D011986:Receptors, Somatotropin; D014443:Tyrosine; D014364:Tryptophan; D013006:Growth Hormone; D003545:Cysteine; D000409:Alanine