Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Proc Natl Acad Sci U S A. 1991 May 15; 88(10): 4498–4502.
doi: 10.1073/pnas.88.10.4498
PMCID: PMC51688
PMID: 2034689

A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor.

S H Bass, M G Mulkerrin, and J A Wells
Author information Copyright and License information PMC Disclaimer

Abstract

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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

 
icon of scanned page 4498
4498
icon of scanned page 4499
4499
icon of scanned page 4500
4500
icon of scanned page 4501
4501
icon of scanned page 4502
4502
 

Images in this article

Image

Image
on p.4501

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • Nicoll CS, Mayer GL, Russell SM. Structural features of prolactins and growth hormones that can be related to their biological properties. Endocr Rev. 1986 May;7(2):169–203. [PubMed] [Google Scholar]
  • Chawla RK, Parks JS, Rudman D. Structural variants of human growth hormone: biochemical, genetic, and clinical aspects. Annu Rev Med. 1983;34:519–547. [PubMed] [Google Scholar]
  • Isaksson OG, Edén S, Jansson JO. Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol. 1985;47:483–499. [PubMed] [Google Scholar]
  • Cunningham BC, Jhurani P, Ng P, Wells JA. Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis. Science. 1989 Mar 10;243(4896):1330–1336. [PubMed] [Google Scholar]
  • Cunningham BC, Wells JA. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. [PubMed] [Google Scholar]
  • Cunningham BC, Henner DJ, Wells JA. Engineering human prolactin to bind to the human growth hormone receptor. Science. 1990 Mar 23;247(4949 Pt 1):1461–1465. [PubMed] [Google Scholar]
  • Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature. 1987 Dec 10;330(6148):537–543. [PubMed] [Google Scholar]
  • Baumann G, Stolar MW, Amburn K, Barsano CP, DeVries BC. A specific growth hormone-binding protein in human plasma: initial characterization. J Clin Endocrinol Metab. 1986 Jan;62(1):134–141. [PubMed] [Google Scholar]
  • Cosman D, Lyman SD, Idzerda RL, Beckmann MP, Park LS, Goodwin RG, March CJ. A new cytokine receptor superfamily. Trends Biochem Sci. 1990 Jul;15(7):265–270. [PubMed] [Google Scholar]
  • Bazan JF. Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6934–6938. [PMC free article] [PubMed] [Google Scholar]
  • Patthy L. Homology of a domain of the growth hormone/prolactin receptor family with type III modules of fibronectin. Cell. 1990 Apr 6;61(1):13–14. [PubMed] [Google Scholar]
  • Fuh G, Mulkerrin MG, Bass S, McFarland N, Brochier M, Bourell JH, Light DR, Wells JA. The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain. J Biol Chem. 1990 Feb 25;265(6):3111–3115. [PubMed] [Google Scholar]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed] [Google Scholar]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed] [Google Scholar]
  • Strauch KL, Beckwith J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1576–1580. [PMC free article] [PubMed] [Google Scholar]
  • Spencer SA, Hammonds RG, Henzel WJ, Rodriguez H, Waters MJ, Wood WI. Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence. J Biol Chem. 1988 Jun 5;263(16):7862–7867. [PubMed] [Google Scholar]
  • Barnard R, Bundesen PG, Rylatt DB, Waters MJ. Evidence from the use of monoclonal antibody probes for structural heterogeneity of the growth hormone receptor. Biochem J. 1985 Oct 15;231(2):459–468. [PMC free article] [PubMed] [Google Scholar]
  • Godowski PJ, Leung DW, Meacham LR, Galgani JP, Hellmiss R, Keret R, Rotwein PS, Parks JS, Laron Z, Wood WI. Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8083–8087. [PMC free article] [PubMed] [Google Scholar]
  • Amselem S, Duquesnoy P, Attree O, Novelli G, Bousnina S, Postel-Vinay MC, Goossens M. Laron dwarfism and mutations of the growth hormone-receptor gene. N Engl J Med. 1989 Oct 12;321(15):989–995. [PubMed] [Google Scholar]
  • Klapper MH. The independent distribution of amino acid near neighbor pairs into polypeptides. Biochem Biophys Res Commun. 1977 Oct 10;78(3):1018–1024. [PubMed] [Google Scholar]
  • Chothia C. The nature of the accessible and buried surfaces in proteins. J Mol Biol. 1976 Jul 25;105(1):1–12. [PubMed] [Google Scholar]
  • Rose GD, Geselowitz AR, Lesser GJ, Lee RH, Zehfus MH. Hydrophobicity of amino acid residues in globular proteins. Science. 1985 Aug 30;229(4716):834–838. [PubMed] [Google Scholar]
  • Argos P. An investigation of protein subunit and domain interfaces. Protein Eng. 1988 Jul;2(2):101–113. [PubMed] [Google Scholar]
  • Janin J, Miller S, Chothia C. Surface, subunit interfaces and interior of oligomeric proteins. J Mol Biol. 1988 Nov 5;204(1):155–164. [PubMed] [Google Scholar]
  • Barnard R, Bundesen PG, Rylatt DB, Waters MJ. Monoclonal antibodies to the rabbit liver growth hormone receptor: production and characterization. Endocrinology. 1984 Nov;115(5):1805–1813. [PubMed] [Google Scholar]
  • Cleary S, Mulkerrin MG, Kelley RF. Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli. Biochemistry. 1989 Feb 21;28(4):1884–1891. [PubMed] [Google Scholar]
  • Chamow SM, Peers DH, Byrn RA, Mulkerrin MG, Harris RJ, Wang WC, Bjorkman PJ, Capon DJ, Ashkenazi A. Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments. Biochemistry. 1990 Oct 23;29(42):9885–9891. [PubMed] [Google Scholar]
  • Wang JH, Yan YW, Garrett TP, Liu JH, Rodgers DW, Garlick RL, Tarr GE, Husain Y, Reinherz EL, Harrison SC. Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains. Nature. 1990 Nov 29;348(6300):411–418. [PubMed] [Google Scholar]
  • Ryu SE, Kwong PD, Truneh A, Porter TG, Arthos J, Rosenberg M, Dai XP, Xuong NH, Axel R, Sweet RW, et al. Crystal structure of an HIV-binding recombinant fragment of human CD4. Nature. 1990 Nov 29;348(6300):419–426. [PMC free article] [PubMed] [Google Scholar]
  • Pakula AA, Young VB, Sauer RT. Bacteriophage lambda cro mutations: effects on activity and intracellular degradation. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8829–8833. [PMC free article] [PubMed] [Google Scholar]
  • Davies DR, Padlan EA, Sheriff S. Antibody-antigen complexes. Annu Rev Biochem. 1990;59:439–473. [PubMed] [Google Scholar]
Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
-