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Science 2 June 1989:
Vol. 244. no. 4908, pp. 1081 - 1085
DOI: 10.1126/science.2471267
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Articles

Science, Vol 244, Issue 4908, 1081-1085
Copyright © 1989 by American Association for the Advancement of Science

articles

High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis

BC Cunningham and JA Wells

Department of Biomolecular Chemistry, Genentech, South San Francisco, CA 94080.

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.


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X. Gu, R. Lawrence, and M. Krieger (2000)
J. Biol. Chem. 275, 9120-9130
   Abstract »    Full Text »    PDF »
Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic Escherichia coli Is Essential for Binding of 987P Fimbriae to Sulfatide.
B.-K. Choi and D. M. Schifferli (1999)
Infect. Immun. 67, 5755-5761
   Abstract »    Full Text »    PDF »
Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants.
D. K. Gavin, S. M. Young Jr., W. Xiao, B. Temple, C. R. Abernathy, D. J. Pereira, N. Muzyczka, and R. J. Samulski (1999)
J. Virol. 73, 9433-9445
   Abstract »    Full Text »    PDF »
A Novel Immunological Approach Establishes That the Auxin-binding Protein, Nt-abp1, Is an Element Involved in Auxin Signaling at the Plasma Membrane.
N. Leblanc, K. David, J. Grosclaude, J.-M. Pradier, H. Barbier-Brygoo, S. Labiau, and C. Perrot-Rechenmann (1999)
J. Biol. Chem. 274, 28314-28320
   Abstract »    Full Text »    PDF »
Biological Properties of Human Prolactin Analogs Depend Not Only on Global Hormone Affinity, but Also on the Relative Affinities of Both Receptor Binding Sites.
S. Kinet, S. Bernichtein, P. A. Kelly, J. A. Martial, and V. Goffin (1999)
J. Biol. Chem. 274, 26033-26043
   Abstract »    Full Text »    PDF »
Dissecting the Role of a Conserved Motif (the Second Region of Homology) in the AAA Family of ATPases. SITE-DIRECTED MUTAGENESIS OF THE ATP-DEPENDENT PROTEASE FtsH.
K. Karata, T. Inagawa, A. J. Wilkinson, T. Tatsuta, and T. Ogura (1999)
J. Biol. Chem. 274, 26225-26232
   Abstract »    Full Text »    PDF »
Mapping Interactions of Escherichia coli GreB with RNA Polymerase and Ternary Elongation Complexes.
N. Loizos and S. A. Darst (1999)
J. Biol. Chem. 274, 23378-23386
   Abstract »    Full Text »    PDF »
Dissection of Individual Functions of the Sendai Virus Phosphoprotein in Transcription.
M. C. Bowman, S. Smallwood, and S. A. Moyer (1999)
J. Virol. 73, 6474-6483
   Abstract »    Full Text »
A Small Region in HMG I(Y) Is Critical for Cooperation with NF-kappa B on DNA.
X. M. Zhang and G. L. Verdine (1999)
J. Biol. Chem. 274, 20235-20243
   Abstract »    Full Text »    PDF »
Mutational Analysis of the PapB Transcriptional Regulator in Escherichia coli. REGIONS IMPORTANT FOR DNA BINDING AND OLIGOMERIZATION.
Y. Xia and B. E. Uhlin (1999)
J. Biol. Chem. 274, 19723-19730
   Abstract »    Full Text »    PDF »
Circular Permutation Analysis as a Method for Distinction of Functional Elements in the M20 Loop of Escherichia coli Dihydrofolate Reductase.
T. Nakamura and M. Iwakura (1999)
J. Biol. Chem. 274, 19041-19047
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The Transmembrane Domain of the E5 Oncoprotein Contains Functionally Discrete Helical Faces.
A. J. Adduci and R. Schlegel (1999)
J. Biol. Chem. 274, 10249-10258
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Structural Features of the Ligand-binding Domain of the Serotonin 5HT3 Receptor.
D. Yan, M. K. Schulte, K. E. Bloom, and M. M. White (1999)
J. Biol. Chem. 274, 5537-5541
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Identification of Three Distinct Receptor Binding Sites of Murine Interleukin-11.
V. A. Barton, K. R. Hudson, and J. K. Heath (1999)
J. Biol. Chem. 274, 5755-5761
   Abstract »    Full Text »    PDF »
Identification of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Binding Site on the Hemopexin Carboxyl Domain of Human Gelatinase A by Site-directed Mutagenesis. THE HIERARCHICAL ROLE IN BINDING TIMP-2 OF THE UNIQUE CATIONIC CLUSTERS OF HEMOPEXIN MODULES III AND IV.
C. M. Overall, A. E. King, D. K. Sam, A. D. Ong, T. T. Y. Lau, U. M. Wallon, Y. A. DeClerck, and J. Atherstone (1999)
J. Biol. Chem. 274, 4421-4429
   Abstract »    Full Text »    PDF »
Alf1p, a CLIP-170 Domain-containing Protein, Is Functionally and Physically Associated with alpha -Tubulin.
B. Feierbach, E. Nogales, K. H. Downing, and T. Stearns (1999)
J. Cell Biol. 144, 113-124
   Abstract »    Full Text »    PDF »


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