|
|
|
|||||||
|
|||||||||
(Received for publication, February 3, 1997, and in revised form, May 4, 1997)
From the The common
Volume 272, Number 29,
Issue of July 18, 1997
pp. 18098-18103
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Is Critical for
Intracellular Behavior but Not for in Vitro Biological
Activity
,
,
,
Department of Molecular Biology and
Pharmacology, Washington University School of Medicine, St. Louis,
Missouri 63110 and the Division of Reproductive Biology, Department of
Gynecology/Obstetrics, Stanford University Medical Center,
Stanford, California 94305-5317
subunit of
glycoprotein hormones contains five disulfide bonds. Based on the
published crystal structure, the assignments are 7-31, 59-87, 10-60,
28-82, and 32-84; the last three comprise the cystine knot, a
structure also seen in a variety of growth factors. Previously, we
demonstrated that the efficiency of secretion and the ability to form
heterodimers by subunits bearing single cysteine residue mutants in
the cystine knot were significantly reduced. These results suggested
that the cystine knot is critical for the intracellular integrity of
the subunit. To assess if the presence of the free thiol affected the
secretion kinetics, we constructed paired cysteine mutants of each
disulfide bond of the subunit. The secretion rate for these
monomers was comparable with wild type except for the -10-60
mutant, which was 40% lower. The recovery of the 7-31 and
59-87 mutants was greater than 95%, whereas for the cystine knot
mutants, it was 20-40%. Co-expression of the wild-type chorionic
gonadotropin 
subunit with double cysteine mutants did not enhance
the recovery of mutants in the media. Moreover, compared with
wild-type, the efficiency of heterodimer formation of the 10-60 or
32-84 mutants was less than 5%. Because subunit assembly is
required for biological activity, studies on the role of these
disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the and
subunits are genetically fused. The recovery of secreted tethered
gonadotropins bearing mutations in the cystine knot was increased
significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain 10-60 and 32-84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in
vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or
signal transduction.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. M. Smallwood, J. Williams, Q. Xu, D. J. Leahy, and J. Nathans Mutational Analysis of Norrin-Frizzled4 Recognition J. Biol. Chem., February 9, 2007; 282(6): 4057 - 4068. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Jablonka-Shariff, T. R. Kumar, J. Eklund, A. Comstock, and I. Boime Single-Chain, Triple-Domain Gonadotropin Analogs with Disulfide Bond Mutations in the {alpha}-Subunit Elicit Dual Follitropin and Lutropin Activities in Vivo Mol. Endocrinol., June 1, 2006; 20(6): 1437 - 1446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Garcia-Campayo, A. Jablonka-Shariff, and I. Boime A Single-chain Bifunctional Gonadotropin Analog Is Secreted from Chinese Hamster Ovary Cells as Two Distinct Bioactive Species J. Biol. Chem., October 22, 2004; 279(43): 44286 - 44293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Horn, G. B. Hurst, A. Mayasundari, N. A. Whittemore, E. H. Serpersu, and C. B. Peterson Assignment of the Four Disulfides in the N-terminal Somatomedin B Domain of Native Vitronectin Isolated from Human Plasma J. Biol. Chem., August 20, 2004; 279(34): 35867 - 35878. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mayasundari, N. A. Whittemore, E. H. Serpersu, and C. B. Peterson The Solution Structure of the N-terminal Domain of Human Vitronectin: PROXIMAL SITES THAT REGULATE FIBRINOLYSIS AND CELL MIGRATION J. Biol. Chem., July 9, 2004; 279(28): 29359 - 29366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. B. Fralish, P. Narayan, and D. Puett Consequences of Single-Chain Translation on the Structures of Two Chorionic Gonadotropin Yoked Analogs in {alpha}-{beta} and {beta}-{alpha} Configurations Mol. Endocrinol., April 1, 2003; 17(4): 757 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Narayan, J. Gray, and D. Puett Yoked Complexes of Human Choriogonadotropin and the Lutropin Receptor: Evidence that Monomeric Individual Subunits Are Inactive Mol. Endocrinol., December 1, 2002; 16(12): 2733 - 2745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Hsu, K. Nakabayashi, and A. Bhalla Evolution of Glycoprotein Hormone Subunit Genes in Bilateral Metazoa: Identification of Two Novel Human Glycoprotein Hormone Subunit Family Genes, GPA2 and GPB5 Mol. Endocrinol., July 1, 2002; 16(7): 1538 - 1551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Garcia-Campayo and I. Boime Independent Activities of FSH and LH Structurally Confined in a Single Polypeptide: Selective Modification of the Relative Potencies of the Hormones Endocrinology, December 1, 2001; 142(12): 5203 - 5211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hiro'oka, D. Maassen, P. Berger, and I. Boime Disulfide Bond Mutations in Follicle-Stimulating Hormone Result in Uncoupling of Biological Activity from Intracellular Behavior Endocrinology, December 1, 2000; 141(12): 4751 - 4756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Darling, R. W. Ruddon, F. Perini, and E. Bedows Cystine Knot Mutations Affect the Folding of the Glycoprotein Hormone alpha -Subunit. DIFFERENTIAL SECRETION AND ASSEMBLY OF PARTIALLY FOLDED INTERMEDIATES J. Biol. Chem., May 12, 2000; 275(20): 15413 - 15421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Rose, R. E. Gaines Das, and A. H. Balen Definition and Measurement of Follicle Stimulating Hormone Endocr. Rev., February 1, 2000; 21(1): 5 - 22. [Abstract] [Full Text]
|
|
|
|
|
|
P. Narayan, J. Gray, and D. Puett A Biologically Active Single Chain Human Chorionic Gonadotropin Analog with Altered Receptor Binding Properties Endocrinology, January 1, 2000; 141(1): 67 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Jackson, P. Berger, M. Pixley, C. Klein, A. J. W. Hsueh, and I. Boime The Biological Action of Choriogonadotropin Is Not Dependent on the Complete Native Quaternary Interactions between the Subunits Mol. Endocrinol., December 1, 1999; 13(12): 2175 - 2188. [Abstract] [Full Text]
|
|
|
|
|
|
M. Kanda, A. Jablonka-Shariff, A. Sato, M. R. Pixley, E. Bos, T. Hiro’oka, D. Ben-Menahem, and I. Boime Genetic Fusion of an {alpha}-Subunit Gene to the Follicle-Stimulating Hormone and Chorionic Gonadotropin-{beta} Subunit Genes: Production of a Bifunctional Protein Mol. Endocrinol., November 1, 1999; 13(11): 1873 - 1881. [Abstract] [Full Text]
|
|
|
|
|
|
R. J. Darling, J. A. Wilken, A. K. Miller-Lindholm, T. M. Urlacher, R. W. Ruddon, S. A. Sherman, and E. Bedows Functional Contributions of Noncysteine Residues within the Cystine Knots of Human Chorionic Gonadotropin Subunits J. Biol. Chem., March 30, 2001; 276(14): 10692 - 10699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Ben-Menahem, A. Jablonka-Shariff, R. K. Hyde, M. R. Pixley, S. Srivastava, P. Berger, and I. Boime The Position of the alpha and beta Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes J. Biol. Chem., August 3, 2001; 276(32): 29871 - 29879. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |