COMMUNICATION:
The Biologic Action of Single-chain Choriogonadotropin Is Not Dependent on the Individual Disulfide Bonds of the beta  Subunit*

(Received for publication, November 7, 1996, and in revised form, January 10, 1997)

David Ben-Menahem Dagger , Masataka Kudo §, Mary R. Pixley , Asomi Sato , Nobuhiko Suganuma , Emerald Perlas §, Aaron J. W. Hsueh § and Irving Boime par

From the Department of Molecular Biology & 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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Disrupting disulfide loops in the human chorionic gonadotropin beta  subunit (CGbeta ) inhibits combination with the alpha  subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CGbeta subunits bearing paired cysteine mutations to a wild-type alpha  (WTalpha ) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CGbeta monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro. The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction. Moreover, these studies demonstrate that the single-chain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.


INTRODUCTION

Mutational and structural studies have revealed that small clusters of amino acids rather than large structural motifs often contribute to most of the energy involved in protein-protein interactions such as hormone binding to its receptor (for review, see Refs. 1-3). However, it is not clear how the conformation of a peptide ligand contributes to the coupling of binding to signal transduction. This is especially an issue for the function of hormone-receptor complexes involving multisubunit ligands. A convenient model for studying the tertiary and quaternary determinants in signaling is the glycoprotein hormone family which include human chorionic gonadotropin (hCG),1 lutropin (LH), follitropin (FSH), and thyrotropin. Each is a non-covalent heterodimer composed of a common alpha  and unique beta  subunit which allows recognition of the corresponding G-protein-coupled receptor (for review, see Ref. 4).

Recent crystallographic studies of hCG revealed a significant structural similarity to several growth factor families, e.g. transforming growth factor beta , which contain a cystine knot motif composed of three pairs of bridged cysteine residues (5-8). Each hCG subunit contains a cystine knot configuring three disulfide-bonded loops which are the major structural motifs (7, 8). Based on the crystallographic studies of hCG (7, 8), the disulfide bonds in the CGbeta subunit are at positions 9-57, 34-88, 38-90, 23-72, 93-100, and 26-110 (Fig. 1A). The folding intermediates associated with the ordered formation of these bridges to acquire an assembly-competent form is well documented (9, 10). Current models of hCG action presume that the conformation of the dimer and the highly interactive contacts between the two subunits are critical for function (11-14). One method to examine the functional role of the structural motifs in the glycoprotein hormones is to assess the biologic activity of variants containing mutated cysteine residues. However, breaking single disulfide bonds of the beta  subunit inhibits secretion and assembly with the alpha  subunit, and as a result dimer recovery is dramatically reduced (Ref. 15 and Table I). Since only the heterodimer binds to the receptor, it is virtually impossible to examine the bioactivity of these variants. Recently, a single gene encoding a protein containing CGbeta and alpha  subunit was constructed (16, 17). The tethered hormone exhibited secretion kinetics and bioactivity similar to that of the non-covalent heterodimer. Here we compile into a single protein subunits that cannot combine efficiently, namely the alpha  subunit and CGbeta with cysteine to alanine mutations. Because this tethered construction by-passes the assembly step, we can examine the role of the disulfide bonds on the biological activity of hCG. The data show that they are primarily required for assembly with the alpha  subunit and secretion of the heterodimer. The extensive native subunit interaction in the dimer which is altered by these mutations is not critical for receptor binding and signal transduction.


Fig. 1. A, proposed structure of CGbeta subunit and disulfide bond assignments. The bonds of the cystine knot are represented by solid lines. B, construction of single-chain variants. LTR denotes the sequence of the promoter contained in the Harvey murine sarcoma virus inserted in the expression vector pM2.
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Table I.

Secretion of hCG beta  Cys mutants

Results are expressed as mean ± S.E. of 3-6 experiments.
Hormone Secretion rate (t1/2)a
Recovery from media
Single-chain  beta Subunit Single-chainb Heterodimerc

min %
WT 86  ± 7 150d 76  ± 5 >80
34 -88 351  ± 38 320d 24  ± 3 4  ± 1
38 -90 158  ± 9 200 55  ± 4 <1 
9 -57 173  ± 24 220 39  ± 15 7  ± 1
23 -72 105  ± 5 180d 47  ± 4 22  ± 4
93 -100 149  ± 11 170d 71  ± 8 <1 
26 -110 59  ± 7 34d 89  ± 4 9  ± 4

a Time (min) when half of the maximal secreted hormone is detected in the media.
b The amount of hormone retrieved from the media at steady state is expressed as a fraction of the total (lysate + medium).
c Determined in cells expressing both WT-alpha and mutated monomeric CGbeta subunits. Cells were labeled for 16 h with [35S]Cys, and equal aliquots of lysate and media were immunoprecipitated with alpha - or CGbeta antisera. Heterodimer recovery corresponds to the assembled CGbeta in the media as a fraction of total beta  synthesized (lysate + medium).
d Taken from Ref. 15.


EXPERIMENTAL PROCEDURES

Vector Construction

A KpnI-XhoI fragment (2.7 kilobases; Fig. 1B) containing single-chain hCG (CGbeta alpha ) in vector pM2HA (16) was subcloned into pBluescript II KS (Stratagene). Construction of cysteine (Cys) to alanine (Ala) mutants in the CGbeta subunit gene (CGbeta Delta Cys) was described (15). The ApaI fragment containing the first 135 amino acids of CGbeta sequence in CGbeta alpha was exchanged for the ApaI fragment in CGbeta Delta Cys. The new KpnI-XhoI fragment was inserted into pM2AH and rechecked by restriction enzyme analysis. The mutations were confirmed using Taq DyeDeoxy Terminator Cycle Sequencing Kit and an ABI prism DNA Sequencer (Perkin Elmer).

Cell Culture and Metabolic Labeling

Clones of transfected Chinese hamster ovary (CHO) cells were maintained as described (15, 16). We used polyclonal alpha  antiserum for precipitation and Western blots since WTalpha subunit is tethered to the mutated CGbeta sequence, and the immunoreactivity of all variants to the antiserum should be similar. Pulse-chase experiments were performed in 12-well dishes (300,000 cells/well) (15). Precipitates were resolved on SDS gels and were quantitated with a PhosphorImager (Molecular Dynamics) or a laser densitometer (LKB).

Radioligand Assays

Condition medium obtained from cells grown in serum-free F12 was concentrated (using Amicon Centriprep 10 concentrators), the concentrate was diluted (1:15) in phosphate-buffered saline, and the volume reduced again. The mutants were quantitated using our polyclonal alpha  antiserum and the double antibody RIA (Diagnostic Products Corp.). A human kidney-derived cell line (293), stably transfected with the cDNA-encoding human LH/CG receptor, was used for receptor binding and cAMP production (18).


RESULTS

Secretion of hCG Single-chain Mutants

To assess the intracellular effect of the disulfide loops in the CGbeta domain, we examined the secretion of tethered hCG variants in which both cysteines of a proposed pair were mutated. Transfected CHO cells stably expressing the Cys mutants were metabolically labeled and subjected to SDS-polyacrylamide gel electrophoresis analysis under reduced conditions. The electrophoretic migration of the secreted proteins is slower than the corresponding intracellular species (Fig. 2A) which reflects both the processing of the N-linked and addition of the O-linked oligosaccharides to the CGbeta subunit just prior to secretion (15). The experiment reveals that the secretion kinetics and extent of recovery of the mutants from the media are variable. For example, the 34-88 mutant is not detected in the media after 4 h of labeling (lane 7). Pulse-chase analysis shows that the unmodified single-chain hCG (CGbeta alpha ) is secreted efficiently (t1/2 = 85 min; recovery = 75%; Table I) which is comparable to that of the heterodimer (15). As previously observed for the monomeric CGbeta subunit (15), release of the 26-110 mutant is accelerated (t1/2 = 60 min, recovery 90%). In contrast, mutations in the cystine knot (i.e. 9-57, 34-88, and 38-90) resulted in variants that were secreted slower and less efficiently. This is especially evident for the 34-88 mutant where the t1/2 is 350 min and 25% of the protein is recovered. The reduced recovery suggests that the mutants were altered resulting in degradation of a significant fraction of the pool. This is substantiated by pulse-chase analyses that show in contrast to CGbeta alpha , where most of the variant is secreted, 75% of the 34-88 mutant that accumulates in the lysate does not exit the cell and is no longer detected after 24 h (Fig. 2B). Thus, the disulfide bonds which comprise the core of the subunit, namely 9-57, 34-88, and to a lesser extent 38-90, are essential for efficient secretion of the CG single chain. The secretion kinetics generally parallel those seen for CGbeta monomer (Table I), which implies that these mutation-induced alterations in intracellular behavior are not the result of the single-chain construction.


Fig. 2. A, expression of single-chain hCG, WT, and CGbeta Cys mutants, in transfected CHO cells. Stable clones (300,000 cells/well) were labeled with 25 µCi/ml Pro-mix (Amersham) for 4 h. Lysate (L) and medium (M) samples were precipitated with alpha  antiserum. B, secretion kinetics of single-chain hCG variants. Cells were pulse-labeled and chased for the indicated times as described under "Experimental Procedures."
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Conformational Changes of the Cys Analogs

If the conformation is altered by breaking the disulfide bonds, we should detect differences in electrophoretic mobility and immunoreactivity on SDS gels. To test this prediction, Western blots were performed under non-reduced conditions using a monoclonal antibody directed against alpha  subunit epitopes that are exposed primarily in the heterodimer (designated A407; Ref. 19). The antibody recognized the CGbeta alpha , suggesting that dimer-like structure is preserved despite the tethering (Fig. 3A, lanes 1 versus 6). The mutants lacking the bonds that form late in the folding sequence of CGbeta monomer, i.e. 23-72, 26-110, and 93-100 (9, 10), were immunoreactive (Fig. 3A, lanes 2, 7, and 8). The recognition of the 23-72 and 26-110 Cys variants by the dimer-specific antibodies presumably reflects the limited assembly of such mutated CGbeta monomers with the alpha  subunit (Table I and Ref. 15). It is unclear why the 93-100 Cys mutant is detected because no discernible assembly is seen with monomer CGbeta subunit bearing this mutation (Table I and Ref. 15), but this single-chain mutant may be partially configured to a heterodimeric form. Of significance, analogs lacking bonds of the cystine knot which are the first to form in CGbeta (9, 10) display little or no signal (Fig. 3A, lanes 3-5). Comparable results were obtained with B109 (data not shown), a monoclonal antibody raised against a CGbeta epitope specific to the heterodimer (12). When the same blot was reprobed with polyclonal alpha  antiserum (Fig. 3B), all analogs and free alpha  monomer were detected. This indicates there was sufficient protein in the blot and illustrates the discrimination between the conformation of free and dimerized subunit by the monoclonal antibody (lane 1).


Fig. 3. Western blot analysis of the secreted heterodimer and single-chain forms of hCG. Samples were electrophoresed under non-reducing conditions without heating. Blotting was performed on nitrocellulose membrane and visualized by the Western light chemiluminescent detection system (Tropix Inc., Bedford, MA). A, probing with dimer-specific monoclonal anti-alpha antibody (A407, 1:5000 dilution). B, panel A was reprobed with polyclonal alpha  antiserum (1:8000 dilution). Different volumes of each sample were analyzed to give comparable intensities of the main band.
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As previously reported, the CGbeta alpha migrated slightly faster than the heterodimer under non-reduced conditions (Fig. 3, lanes 1 and 6, and Ref. 16). In addition, the electrophoretic migration of each analog differs compared with CGalpha further demonstrating conformational changes induced by disrupting the disulfide bridges. We also observed slower migrating species on the blots and that this heterogeneity for the CGbeta alpha and the Cys mutants is variable; it is most pronounced for mutants 38-90, 9-57 (panel B, lanes 3 and 4), and, for the 34-88 mutant, the higher molecular weight protein represents a considerable fraction of the total (lane 5). The high molecular weight forms of CGbeta alpha are non-covalent since heating (95 °C, 5 min) under non-reduced conditions converts them to the expected size of the single chain (data not shown). The larger forms of the Cys variants apparently consist mainly of covalent polymers since they are resistant to heat (95 °C, 5 min) but dissociate when boiled in the presence of beta -mercaptoethanol (data not shown). Aggregate formation, often associated with misfolded forms of a protein, was observed when Cys mutants of CGbeta subunit monomers were analyzed under non-reduced conditions (10) and during purification of native urinary hCG (20, 21). Taken together, the data imply that bonds of the cystine knot motif are the scaffold for the structure, and the cysteine mutations induce conformational changes in the single chains. In contrast to the inability of such CGbeta subunits to efficiently combine and thus interact with the alpha  subunit, the mutated beta  domains in the single chain are nevertheless tethered to an alpha  domain and secreted. The improved recovery (Table I) now permits analysis of the effects of disrupting the disulfide-bonded loops on biological activity.

Receptor Binding and Signal Transduction

Binding of single-chain variants to the human LH/CG receptor stably expressed in 293 cells was examined. We unexpectedly found that all CG variants bind to the receptor including those mutants that as subunits assemble poorly (i.e. 34-88, 9-57, 93-100, and 38-90). The single-chain mutants, except analogs 38-90 and 34-88, displayed similar dose-response curves to the purified heterodimer (CR129)2 and CGbeta alpha (Fig. 4A); the affinity of mutants 38-90 and 34-88 was apparently reduced by 7- and 10-fold, respectively (Table II). We cannot exclude the possibility that these changes are due to the observed aggregation (see above; Fig. 3B, lanes 3 and 5); purification is needed to address this point. That the binding affinities observed were not due to an altered specificity induced by the single-chain configuration, we tested the action of single-chain FSH (22). As expected, this protein did not displace the bound hCG (Fig. 4A). The signal-transducing responses of the hCG variants were also assessed by quantifying adenylate cyclase activation. All these mutants increased cAMP levels, and the signal transduction paralleled binding efficiency, resulting in similar coupling ratios (Fig. 4B and Table II). These data show that bioactivity was preserved and mutations in the CGbeta monomer which block assembly with the alpha  subunit are without effect on the efficiency of signal transduction when expressed as single chains.


Fig. 4. Determination of biological activity of the hCG variants. Binding (panel A) and signal transduction (panel B) were measured with human kidney 293 cells expressing human LH/CG receptor. The concentration of the variants in conditioned media was determined by RIA. Displacement curves of 125I-hCG are presented at each dose of unlabeled hormone as the percentage of maximal binding of the tracer. Adenylate cyclase activity was determined by RIA. In both panels, the data are presented as the mean ± S.E. of two cultures.
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Table II.

Receptor binding and cAMP stimulation of single-chain hCG analogs

IC50 and EC50 define the hormone concentration (ng/ml) at 50% displacement of the tracer and half-maximal adenylate cyclase stimulation, respectively. The results are from 2-4 experiments (mean ± S.E.) each conducted in duplicate.
Hormone Binding IC50 cAMP EC50 Coupling factor (IC50/EC50)

Heterodimer 9.5  ± 2.4 4.5  ± 0.6 2.1
CGbeta alpha 10.3  ± 2.1 4.6  ± 1.6 2.2
34-88 93  ± 24 32  ± 5 2.9
38-90 67  ± 11 51  ± 21 1.3
 9-57 20  ± 4 13  ± 1 1.5
23-72 8.0  ± 2 5.0  ± 2 1.6
93-100 15  ± 4 13  ± 2.4 1.2
26-110 23  ± 6 9.1  ± 4.5 2.5


DISCUSSION

Structural analyses of hCG revealed that the overall shape of the glycoprotein hormones is elongated rather than globular with the disulfide-bonded loops primarily surface-exposed (7, 8). However, despite their relatively large structures, conformational changes induced by deleting each of the disulfide bonds and altering any of the loops in the CGbeta domain does not abolish binding/signal transduction nor does it significantly affect coupling. These results were not anticipated since it is well accepted that the intracellular/extracellular behavior of the glycoprotein hormones is conformationally sensitive (11-14). Mutations inhibiting heterodimer formation and eliminating dimer-specific epitopes in the tethered analogs did not significantly affect the bioactivity of the single-chain mutants. The glycoprotein hormone-specific quaternary structure, i.e. the heterodimer, signals key functional intracellular events such as efficient secretion and hormone-specific processing of the subunit N-linked oligosaccharides (23). Our data imply that analogs with different conformations can bind to the receptor and activate adenylate cyclase. Alternatively, the hormone could exist in a series of resonant conformers, and the receptor favors only the binding-competent species. We consider this less likely because for Cys knot mutants no signal indicative of the native form was seen with monoclonal antibody. Therefore, we would conclude that the tertiary and quaternary features created by single disulfide loops in the beta  subunit and the tight interaction between subunits are essential for the normal intracellular behavior of the gonadotropins, but not for the coupling of receptor binding/signal transduction in vitro.

The bioactivity of Cys analogs can be explained by several models. (a) As previously proposed for numerous protein-protein interactions (reviewed in Refs. 1-3) including the gonadotropins and their receptors (24-28), the contact sites at the hormone receptor interface are likely established by small clusters of amino acid residues. These determinants for bioactivity could not be disrupted by the conformational changes induced by the Cys mutations; (b) hCG may contain redundant binding/signaling determinants, eliminating one is compensated by others in the intact loops; (c) binding of the complex to the receptor results in conformational changes of the CGbeta subunit that restores activity of the Cys mutated analogs.

The 91-110-amino acid stretch of CGbeta envelops the alpha  subunit and includes domains for bioactivity such as the "determinant loop" (i.e. residues 93-100; Ref. 29). Because the single-chain 26-110 or 93-100 mutants were as active as CGbeta alpha , a major role for this sequence is apparently to stabilize the noncovalent heterodimer (7, 8). Thus, the primary and/or secondary structure of loci within each of these individual loops rather than their tertiary structure are likely determinants for functional receptor recognition.

By-passing the assembly step with the single-chain approach enabled us to expand the spectrum of analogs for structure-function analysis. Similarly, mutations in other multisubunit hormones and growth factors of the cystine knot superfamily which result in inefficient assembly, secretion, and loss of bioactivity (6 and references therein) can be examined.


FOOTNOTES

*   This work was supported by grants from the Organon Company and National Institutes of Health Grant N01-HD67922.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Supported by a grant from the Lalor Foundation.
   Present address: Dept. of Obstetrics/Gynecology, Nagoya University School of Medicine, Nagoya, Japan.
par    To whom correspondence should be addressed. Tel.: 314-362-2556; Fax: 314-361-3560.
1   The abbreviations used are: hCG, human chorionic gonadotropin; LH, lutropin; FSH, follitropin; WT, wild type; RIA, radioimmunoassay; CGbeta alpha , unmodified single-chain hCG.
2   National Hormone and Pituitary Agency.

Acknowledgments

We thank Dr. Steven Birken for providing monoclonal antibodies and his helpful suggestions and Susan Carnes for excellent assistance in preparing the manuscript. We thank Drs. M. Muyan, D. Ornitz, and D. Towler for reading the manuscript and Takashi Hiro'oka and Burkhard Hirsch for FSH control.


REFERENCES

  1. Wells, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1-6 [Abstract/Free Full Text]
  2. Davies, D. R., and Cohen, G. H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7-12 [Abstract/Free Full Text]
  3. Jones, S., and Thornton, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13-20 [Abstract/Free Full Text]
  4. Segaloff, D. L., and Ascoli, M. (1993) Endocr. Rev. 14, 324-347 [Abstract]
  5. McDonald, N. Q., and Hendrickson, W. A. (1993) Cell 73, 421-424 [Medline] [Order article via Infotrieve]
  6. Sun, P. D., and Davies, D. R. (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 269-291 [CrossRef][Medline] [Order article via Infotrieve]
  7. Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J., and Isaacs, N. W. (1994) Nature 369, 455-461 [CrossRef][Medline] [Order article via Infotrieve]
  8. Wu, H., Lustbader, A. J., Liu, Y., Canfield, R. E., and Hendrickson, W. A. (1994) Structure 2, 545-558 [Medline] [Order article via Infotrieve]
  9. Huth, J. R., Mountjoy, K., Perini, F., and Ruddon, R. W. (1992) J. Biol. Chem. 267, 8870-8879 [Abstract/Free Full Text]
  10. Bedows, E., Huth, J. R., Suganuma, N., Bartels, C. F., Boime, I., and Ruddon, R. W. (1993) J. Biol. Chem. 268, 11655-11662 [Abstract/Free Full Text]
  11. Combarnous, Y. (1992) Endocr. Rev. 13, 670-691 [Medline] [Order article via Infotrieve]
  12. Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y., and Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031 [Abstract/Free Full Text]
  13. Jiang, X., Dreano, M., Buckler, D. R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W. A., and ElTayar, N. (1996) Structure 3, 1341-1353
  14. Liu, C., and Dias, J. A. (1996) Arch. Biochem. Biophys. 329, 127-135 [CrossRef][Medline] [Order article via Infotrieve]
  15. Suganuma, N., Matzuk, M. M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307 [Abstract/Free Full Text]
  16. Sugahara, T., Pixley, M. R., Minami, S., Perlas, E., Ben-Menahem, D., Hsueh, A. J. W., and Boime, I. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2041-2045 [Abstract]
  17. Narayan, P., Wu, C., and Puett, D. (1995) Mol. Endocrinol. 9, 1720-1726 [Abstract]
  18. Jia, X.-C., Oikawa, M., Bo, M., Tanaka, T., Ny, T., Boime, I., and Hsueh, A. J. (1991) Mol Endocrinol. 5, 759-768 [Abstract]
  19. Krichevsky, A., Birken, S., O'Connor, J. F., Bikel, K., Schlatterer, J. P., and Canfield, R. E. (1994) Endocrine 2, 511-520
  20. Lustbader, J., Birken, S., Pollak, S., Levinson, L., Bernstine, E., Hsiung, N., and Canfield, R. (1987) J. Biol. Chem. 262, 14204-14212 [Abstract/Free Full Text]
  21. Birken, S., Maydelman, Y., Gawinowicz, M. A., Pound, A., Liu, Y., and Stockell Hartree, A. (1996) Endocrinology 137, 1402-1411 [Abstract]
  22. Sugahara, T., Sato, A., Kudo, M., Ben-Menahem, D., Pixley, M. R., Hsueh, A. J. W., and Boime, I. (1996) J. Biol. Chem. 271, 10445-10448 [Abstract/Free Full Text]
  23. Corless, C. L., Matzuk, M. M., Ramabhadran, T. V., Krichevsky, A., and Boime, I. (1987) J. Cell Biol. 104, 1173-1181 [Abstract]
  24. Ryan, R. J., Charlesworth, M. C., McCormick, D. J., Milius, R. P., and Keutmann, H. T. (1988) FASEB J. 2, 2661-2669 [Abstract/Free Full Text]
  25. Campbell, R. K., Dean-Emig, D. M., and Moyle, W. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 760-764 [Abstract]
  26. Yoo, J., Ji, I., and Ji, T. H. (1991) J. Biol. Chem. 266, 17741-17743 [Abstract/Free Full Text]
  27. Chen, F., Wang, Y., and Puett, D. (1991) J. Biol. Chem. 266, 19357-19361 [Abstract/Free Full Text]
  28. Weiss, J., Axelrod, L., Whitcomb, R. W., Harris, P. E., Crowley, W. F., and Jameson, J. L. (1992) N. Engl. J. Med. 326, 179-183 [Medline] [Order article via Infotrieve]
  29. Ward, D. N., and Moore, W. T., Jr. (1979) in Animal Models for Research on Contraception and Fertility (Alexander, N. A., ed), pp. 151-164, Harper & Row, New York

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