Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 10 March 1989:
Vol. 243. no. 4896, pp. 1330 - 1336
DOI: 10.1126/science.2466339
Prev | Table of Contents | Next

Articles

Science, Vol 243, Issue 4896, 1330-1336
Copyright © 1989 by American Association for the Advancement of Science

articles

Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis

BC Cunningham, P Jhurani, P Ng, and JA Wells

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

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.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Characterization of a Factor Xa Binding Site on Factor Va near the Arg-506 Activated Protein C Cleavage Site.
A. J. Gale, S. Yegneswaran, X. Xu, J.-L. Pellequer, and J. H. Griffin (2007)
J. Biol. Chem. 282, 21848-21855
   Abstract »    Full Text »    PDF »
A Long-Acting, Mono-PEGylated Human Growth Hormone Analog Is a Potent Stimulator of Weight Gain and Bone Growth in Hypophysectomized Rats.
G. N. Cox, M. S. Rosendahl, E. A. Chlipala, D. J. Smith, S. J. Carlson, and D. H. Doherty (2007)
Endocrinology 148, 1590-1597
   Abstract »    Full Text »    PDF »
Comprehensive and Quantitative Mapping of Energy Landscapes for Protein-Protein Interactions by Rapid Combinatorial Scanning.
G. Pal, J.-L. K. Kouadio, D. R. Artis, A. A. Kossiakoff, and S. S. Sidhu (2006)
J. Biol. Chem. 281, 22378-22385
   Abstract »    Full Text »    PDF »
Mapping epitopes and antigenicity by site-directed masking.
D. Paus and G. Winter (2006)
PNAS 103, 9172-9177
   Abstract »    Full Text »    PDF »
Two Wrongs Can Make a Right: Dimers of Prolactin and Growth Hormone Receptor Antagonists Behave as Agonists.
J. F. Langenheim, D. Tan, A. M. Walker, and W. Y. Chen (2006)
Mol. Endocrinol. 20, 661-674
   Abstract »    Full Text »    PDF »
Shotgun Alanine Scanning Shows That Growth Hormone Can Bind Productively to Its Receptor through a Drastically Minimized Interface.
J.-L. K. Kouadio, J. R. Horn, G. Pal, and A. A. Kossiakoff (2005)
J. Biol. Chem. 280, 25524-25532
   Abstract »    Full Text »    PDF »
Dual Function for a Unique Site within the {beta}2I Domain of Integrin {alpha}M{beta}2.
D. Ehirchiou, Y.-M. Xiong, Y. Li, S. Brew, and L. Zhang (2005)
J. Biol. Chem. 280, 8324-8331
   Abstract »    Full Text »    PDF »
Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h.
J. Chiu, P. E. March, R. Lee, and D. Tillett (2004)
Nucleic Acids Res. 32, e174
   Abstract »    Full Text »    PDF »
Increased Site 1 Affinity Improves Biopotency of Porcine Growth Hormone: EVIDENCE AGAINST DIFFUSION DEPENDENT RECEPTOR DIMERIZATION.
Y. Wan, A. McDevitt, B. Shen, M. L. Smythe, and M. J. Waters (2004)
J. Biol. Chem. 279, 44775-44784
   Abstract »    Full Text »    PDF »
Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion.
C. B. Doering, J. F. Healey, E. T. Parker, R. T. Barrow, and P. Lollar (2004)
J. Biol. Chem. 279, 6546-6552
   Abstract »    Full Text »    PDF »
The Fourth Blade within the {beta}-Propeller Is Involved Specifically in C3bi Recognition by Integrin {alpha}M{beta}2.
Y. Li and L. Zhang (2003)
J. Biol. Chem. 278, 34395-34402
   Abstract »    Full Text »    PDF »
Sequences within Domain II of the Urokinase Receptor Critical for Differential Ligand Recognition.
Y. Li, D. A. Lawrence, and L. Zhang (2003)
J. Biol. Chem. 278, 29925-29932
   Abstract »    Full Text »    PDF »
Identification of Functional Segments within the beta 2I-domain of Integrin alpha Mbeta 2.
Y.-M. Xiong, T. A. Haas, and L. Zhang (2002)
J. Biol. Chem. 277, 46639-46644
   Abstract »    Full Text »    PDF »
Functional Promiscuity of Squirrel Monkey Growth Hormone Receptor Toward both Primate and Nonprimate Growth Hormones.
S. Yi, B. Bernat, G. Pal, A. Kossiakoff, and W.-H. Li (2002)
Mol. Biol. Evol. 19, 1083-1092
   Abstract »    Full Text »    PDF »
The Amount of MUC5B Mucin in Cervical Mucus Peaks at Midcycle.
I. K. Gipson, R. Moccia, S. Spurr-Michaud, P. Argüeso, A. R. Gargiulo, J. A. Hill III, G. D. Offner, and H. T. Keutmann (2001)
J. Clin. Endocrinol. Metab. 86, 594-600
   Abstract »    Full Text »
Designing Small-Molecule Switches for Protein-Protein Interactions.
Z. Guo, D. Zhou, and P. G. Schultz (2000)
Science 288, 2042-2045
   Abstract »    Full Text »
Selection for improved subtiligases by phage display.
S. Atwell and J. A. Wells (1999)
PNAS 96, 9497-9502
   Abstract »    Full Text »    PDF »
Homologue Scanning Mutagenesis Reveals Cd66 Receptor Residues Required for Neisserial Opa Protein Binding.
M. P. Bos, D. Hogan, and R. J. Belland (1999)
J. Exp. Med. 190, 331-340
   Abstract »    Full Text »    PDF »
Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules.
E Juarez-Aguilar, F Castro-Munozledo, N. Guerra-Rodriguez, D Resendez-Perez, H. Martinez-Rodriguez, H. Barrera-Saldana, and W Kuri-Harcuch (1999)
J. Cell Sci. 112, 3127-3135
   Abstract »    PDF »
Identification and Characterization of Novel Superantigens from Streptococcus pyogenes.
T. Proft, S. Louise Moffatt, C. J. Berkahn, and J. D. Fraser (1999)
J. Exp. Med. 189, 89-102
   Abstract »    Full Text »    PDF »
Interaction of the Fungal Pathogen Candida albicans with Integrin CD11b/CD18: Recognition by the I Domain Is Modulated by the Lectin-Like Domain and the CD18 Subunit.
C. B. Forsyth, E. F. Plow, and L. Zhang (1998)
J. Immunol. 161, 6198-6205
   Abstract »    Full Text »    PDF »
Identification of Transmembrane Regions Critical for Ligand Binding to the Human D3 Dopamine Receptor Using Various D3/D1 Transmembrane Chimeras.
G. L. Alberts, J. F. Pregenzer, and W. B. Im (1998)
Mol. Pharmacol. 54, 379-388
   Abstract »    Full Text »
Homodimerization Restores Biological Activity to an Inactive Erythropoietin Mutant.
H. Qiu, A. Belanger, H.-W. P. Yoon, and H. F. Bunn (1998)
J. Biol. Chem. 273, 11173-11176
   Abstract »    Full Text »    PDF »
Structure-Function Study of the Extracellular Domain of the Human IFN-{alpha} Receptor (hIFNAR1) Using Blocking Monoclonal Antibodies: The Role of Domains 1 and 2.
J. Lu, A. Chuntharapai, J. Beck, S. Bass, A. Ow, A. M. De Vos, V. Gibbs, and K. J. Kim (1998)
J. Immunol. 160, 1782-1788
   Abstract »    Full Text »    PDF »
Dual Sets of Chimeric Alleles Identify Specificity Sequences for the bE and bW Mating and Pathogenicity Genes of Ustilago maydis.
A. R. Yee and J. W. Kronstad (1998)
Mol. Cell. Biol. 18, 221-232
   Abstract »    Full Text »    PDF »
Aspartate 171 Is the Major Primate-specific Determinant of Human Growth Hormone. ENGINEERING PORCINE GROWTH HORMONE TO ACTIVATE THE HUMAN RECEPTOR.
S. N. Behncken, S. W. Rowlinson, J. E. Rowland, B. L. Conway-Campbell, T. A. Monks, and M. J. Waters (1997)
J. Biol. Chem. 272, 27077-27083
   Abstract »    Full Text »    PDF »
Increased Proportion of Circulating Non-22-Kilodalton Growth Hormone Isoforms in Short Children: A Possible Mechanism for Growth Failure.
C. L. Boguszewski, C. Jansson, M. C. S. Boguszewski, S. Rosberg, B. Carlsson, K. Albertsson-Wikland, and L. M. S. Carlsson (1997)
J. Clin. Endocrinol. Metab. 82, 2944-2949
   Abstract »    Full Text »    PDF »
Protein-Protein Interaction Specificity of Im9 for the Endonuclease Toxin Colicin E9 Defined by Homologue-scanning Mutagenesis.
W. Li, C. A. Dennis, G. R. Moore, R. James, and C. Kleanthous (1997)
J. Biol. Chem. 272, 22253-22258
   Abstract »    Full Text »    PDF »
Identification and Reconstruction of the Binding Site within alpha Mbeta 2 for a Specific and High Affinity Ligand, NIF.
L. Zhang and E. F. Plow (1997)
J. Biol. Chem. 272, 17558-17564
   Abstract »    Full Text »    PDF »
Circulating Non-22-Kilodalton Growth Hormone Isoforms in Acromegalic Men before and after Transsphenoidal Surgery.
C. L. Boguszewski, G. Johannsson, B.-A. Bengtsson, A. Johansson, B. Carlsson, and L. M. S. Carlsson (1997)
J. Clin. Endocrinol. Metab. 82, 1516-1521
   Abstract »    Full Text »    PDF »
In vitro scanning saturation mutagenesis of an antibody binding pocket.
E. A. Burks, G. Chen, G. Georgiou, and B. L. Iverson (1997)
PNAS 94, 412-417
   Abstract »    Full Text »    PDF »
A C-terminal Mutant of the G Protein beta Subunit Deficient in the Activation of Phospholipase C-beta.
S. Zhang, O. A. Coso, R. Collins, J. S. Gutkind, and W. F. Simonds (1996)
J. Biol. Chem. 271, 20208-20212
   Abstract »    Full Text »    PDF »
Characterization of Lactogen Receptor-binding Site 1of Human Prolactin.
S. Kinet, V. Goffin, V. Mainfroid, and J. A. Martial (1996)
J. Biol. Chem. 271, 14353-14360
   Abstract »    Full Text »    PDF »
Selective Modification of Recombinant Bovine Placental Lactogen by Site-directed Mutagenesis at Its C Terminus.
D. Vashdi-Elberg, N. R. Staten, E. Sakal, R. E. McKinnie, J. Djiane, G. G. Krivi, and A. Gertler (1996)
J. Biol. Chem. 271, 5558-5564
   Abstract »    Full Text »    PDF »
The Amino-terminal One-third of alpha[IMAGE] Defines the Ligand Recognition Specificity of Integrin alpha[IMAGE]beta(3).
J. C. Loftus, C. E. Halloran, M. H. Ginsberg, L. P. Feigen, J. A. Zablocki, and J. W. Smith (1996)
J. Biol. Chem. 271, 2033-2039
   Abstract »    Full Text »    PDF »
Analysis of the Short Consensus Repeats of Human Complement Factor B by Site-directed Mutagenesis.
D. E. Hourcade, L. M. Wagner, and T. J. Oglesby (1995)
J. Biol. Chem. 270, 19716-19722
   Abstract »    Full Text »    PDF »
A Growth Hormone Agonist Produced by Targeted Mutagenesis at Binding Site 1.
S. W. Rowlinson, R. Barnard, S. Bastiras, A. J. Robins, R. Brinkworth, and M. J. Waters (1995)
J. Biol. Chem. 270, 16833-16839
   Abstract »    Full Text »    PDF »
Residues 484-508 Contain a Major Determinant of the Inhibitory Epitope in the A2 Domain of Human Factor VIII.
J. F. Healey, I. M. Lubin, H. Nakai, E. L. Saenko, L. W. Hoyer, D. Scandella, and P. Lollar (1995)
J. Biol. Chem. 270, 14505-14509
   Abstract »    Full Text »    PDF »
Zone Mapping of the Binding Domain of the Rat Low Affinity Nerve Growth Factor Receptor by the Introduction of Novel N-Glycosylation Sites.
A. N. Baldwin and E. M. Shooter (1995)
J. Biol. Chem. 270, 4594-4602
   Abstract »    Full Text »    PDF »
A hot spot of binding energy in a hormone-receptor interface.
T Clackson and J. Wells (1995)
Science 267, 383-386
   Abstract »    PDF »
Human growth hormone and extracellular domain of its receptor: crystal structure of the complex.
A. de Vos, M Ultsch, and A. Kossiakoff (1992)
Science 255, 306-312
   Abstract »    PDF »
Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule.
B. Cunningham, M Ultsch, A. De Vos, M. Mulkerrin, K. Clauser, and J. Wells (1991)
Science 254, 821-825
   Abstract »    PDF »
Zinc mediation of the binding of human growth hormone to the human prolactin receptor.
B. Cunningham, S Bass, G Fuh, and J. Wells (1990)
Science 250, 1709-1712
   Abstract »    PDF »
Engineering human prolactin to bind to the human growth hormone receptor.
B. Cunningham, D. Henner, and J. Wells (1990)
Science 247, 1461-1465
   Abstract »    PDF »
Deciphering the message in protein sequences: tolerance to amino acid substitutions.
J. Bowie, J. Reidhaar-Olson, W. Lim, and R. Sauer (1990)
Science 247, 1306-1310
   Abstract »    PDF »
The MHC-binding and gp120-binding functions of CD4 are separable.
D Lamarre, A Ashkenazi, S Fleury, D. Smith, R. Sekaly, and D. Capon (1989)
Science 245, 743-746
   Abstract »    PDF »
High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.
B. Cunningham and J. Wells (1989)
Science 244, 1081-1085
   Abstract »    PDF »
Structure-Function of the Putative I-domain within the Integrin beta 2 Subunit.
Y.-M. Xiong and L. Zhang (2001)
J. Biol. Chem. 276, 19340-19349
   Abstract »    Full Text »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)