Cystine Knot of the Gonadotropin alpha  Subunit Is Critical for Intracellular Behavior but Not for in Vitro Biological Activity*

(Received for publication, February 3, 1997, and in revised form, May 4, 1997)

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

From the Dagger  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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The common alpha  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 alpha  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 alpha  subunit. The secretion rate for these monomers was comparable with wild type except for the alpha -10-60 mutant, which was 40% lower. The recovery of the alpha 7-31 and alpha 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 beta  subunit with double cysteine mutants did not enhance the recovery of alpha  mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the alpha 10-60 or alpha 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 alpha  and beta  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 alpha 10-60 and alpha 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.


INTRODUCTION

Human chorionic gonadotropin (CG),1 lutropin (LH), follitropin, and thyrotropin are members of the glycoprotein hormone family that share a common alpha  subunit but differ in their hormone-specific beta  subunits. The amino acid sequence of the alpha  subunit is identical within a species (1), suggesting that the tertiary structure of the alpha  subunit requires a degree of flexibility to adapt to a unique conformation relative to the beta  domain(s). The human alpha subunit has 10 highly conserved cysteine residues, which form five disulfide bonds. Based on the crystal structure of the alpha subunit in hCG, the proposed pairs are 10-60, 28-82, 32-84, 7-31, and 59-87 (Fig. 1) (2, 3). Bonds 28-32 and 32-84 comprise a ring structure penetrated by a bond bridging cysteine residues 10 and 60 resulting in a core that forms three hairpin loops. This structure is a common feature to the large growth factor family including tumor growth factor-beta , activins, nerve growth factor, and platelet-derived growth factor (4-8). Chemical reduction (1) or site-specific mutations of individual cysteine residues in either the common alpha  or CGbeta subunit, alters dimer assembly and secretion rate (9, 10). Mutants lacking either the 7-31 or 59-87 bond were secreted (10), and heterodimers containing the alpha 7-31 mutation bound to the LH/hCG receptor with an affinity comparable with the wild-type hCG, whereas the 59-87 mutant interacted with a lower binding affinity. However, mutants comprising the cystine knot (10-60, 28-82, and 32-84) were not secreted in sufficient quantities to allow examination of the biological activity. To address this issue, we used a single chain gonadotropin model where the CGbeta subunit was genetically fused to the alpha  subunit (11, 12) to promote more efficient secretion of proteins bearing the alpha  and beta  subunit domains in the same complex. Using this approach, the rate-limiting step of subunit assembly could be circumvented, and mutations that otherwise block dimer formation could be evaluated. Here we show that single chains containing mutations in the cystine knot (alpha 10-60, 28-82, and 32-84) are secreted and biologically active, implying that quaternary relationships between the subunits in the heterodimer primarily influence the intracellular behavior rather than receptor binding/signal transduction.


Fig. 1. Disulfide bond assignments of the common human alpha  subunit.
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MATERIALS AND METHODS

Enzymes for preparing vectors and for polymerase chain reaction were purchased from Promega (Madison, WI), Boehringer Mannheim, or Stratagene (La Jolla, CA). Oligonucleotides were prepared by the Washington University Sequencing Facility. Cell culture media and reagents were prepared by the Washington University Center for Basic Cancer Research (St. Louis, MO). Fetal bovine serum, dialyzed fetal bovine serum, and the neomycin analogue, G418, were purchased from Life Technologies, Inc. For metabolic labeling, [35S]cysteine-methionine (Promix) (Amersham Corp.) or [35S]cysteine (ICN, Irvine, CA) were used. Monoclonal antibodies A407 and B109 were gifts from Dr. Robert Canfield and Dr. Steve Birken (Columbia University, New York).

Construction of alpha  Cysteine Mutants

Previously, the intracellular behavior of single cysteine mutants was assessed (10, 13). Double mutants were only constructed that deleted the 7-31 and 59-87 disulfide bridges. Here, we designed all of the analogs to contain double substitutions for each disulfide bond that were based on the assignments from the recently published crystal structure (2, 3). Thus, both monomeric alpha  subunit and the single chain analogs containing such modifications were constructed.

Monomeric Subunit

The alpha  cysteine mutants previously constructed in vector pM2 were used (10). Exon III of the alpha  subunit contains a unique XbaI restriction site within the sequence encoding amino acid residues 34 and 35 (Fig. 2A). Because the vector also contains a single XbaI site, fragments bearing alpha 10, alpha 28, or alpha 32 mutations were exchanged with XbaI-digested pM2 containing alpha 60, alpha 82, or alpha 84 substitutions. This resulted in double cysteine mutants alpha 10-60, alpha 28-82, and alpha 32-84, respectively. Construction of the pM2 alpha 7-31 and pM2 alpha 59-87 were described previously (10).


Fig. 2. A, construction of Cys mutations in alpha  subunit monomer. B, design of tethered variants.
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Tethered Analogs

Single chain variants were constructed with the carboxyl end of the beta  subunit fused to the amino end of the alpha  subunit using overlap polymerase chain reaction mutagenesis (11) (Fig. 2B). All constructs generated by polymerase chain reaction were sequenced to ensure that the final products contained no misincorporated residues.

Transfection, selection of stable CHO clones, metabolic labeling, and immunoprecipitation with subunit-specific antiserum were previously described (10, 14, 15). Analysis by Western blotting was performed using conditioned media that were concentrated with Centriprep-10 columns (Amicon, Beverly, MA). Samples were loaded on 12.5% SDS-polyacrylamide gels and electroblotted to nitrocellulose (Hybond ECL, Amersham International, UK), and the proteins were detected by the Western Light kit (Tropix, Bedford, MA).

To determine the biological activity, the variants were quantitated either by an hCG RIA (Diagnostic Products Corporation, Los Angeles, CA) containing CGbeta polyclonal antiserum or an hCG dimer-specific enzyme-linked immunosorbent assay kit (Organon, Oss, The Netherlands). Both assays gave comparable results. Conditioned media were incubated with human fetal kidney 293 cells stably transfected with human LH/CG receptor, and the binding affinities (10) and cAMP accumulation were determined (16). Iodination of hCG (CR-127; 14,900 IU/mg) was performed as described (17); the specific activity and maximum binding of 125I-labeled hCG, as determined by radioligand receptor assay (18), were 53,000 cpm/ng and 40%, respectively. Nonspecific binding was determined by adding a 1000-fold excess of unlabeled ligand (Pregnyl, Organon); specific binding routinely was 10-12% of total 125I-labeled hCG added. The cAMP levels were also determined in 293 cells expressing human LH receptors by radioimmune assay (16).


RESULTS

Intracellular Behavior of Cys Mutations in the Monomeric alpha  Subunit

The proposed disulfide bonds in the alpha  subunit are at positions 7-31, 10-60, 28-82, 32-84, and 59-87 (Fig. 1). Previously, we demonstrated that alpha  subunits containing single mutations at Cys-10, -28, -60, -82, and -84 were not secreted and in most cases were degraded intracellularly (10). These mutants also failed to assemble with hCGbeta subunit, whereas mutants with alterations at Cys-7, -31, -32, -59, or -87 were secreted and assembly-competent. Because a free thiol group could alter intracellular behavior (9, 10, 19, 20), double mutants in the alpha  subunit were constructed using the assignments based on the crystallographic analysis (2, 3). To assess secretion/stability of these mutants, transfected cells were labeled with [35S]cysteine and subjected to pulse-chase analysis. The intracellular (lysate) and extracellular (media) forms were immunoprecipitated and resolved on SDS gels (Fig. 3). Except for the 10-60 variant, the secretion rate of the mutants and wild-type subunit were comparable. As described previously, the alpha WT, 7-31, and 59-87 mutants were efficiently secreted because greater than 95% of the subunits were recovered from the media (10). In contrast, the recovery of the alpha 10-60, 28-82, and alpha 32-84 double mutants was 21, 41, and 20%, respectively (Fig. 3; Table I). The decreased recovery was not due to lower synthesis but rather to enhanced degradation, since at zero time of chase the intracellular accumulation of the mutants is comparable (Fig. 3). These data suggest the cystine knot structure is critical for maximum alpha  subunit secretion. Although the secreted non-combined alpha WT is heterogeneous, the media form of alpha 10-60 is more homogeneous, suggesting that a post-translational modification is affected by this mutation.


Fig. 3. Secretion kinetics of alpha  subunit cysteine mutants. Cells expressing alpha WT, alpha 10-60, alpha 28-82, or alpha 32-84 were pulse-labeled and chased for the indicated times (in hours). Lysates (L) and media (M) were immunoprecipitated with alpha  antiserum, followed by SDS-polyacrylamide gel electrophoresis.
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Table I. Secretion of alpha  subunit cysteine mutants

Values represent the mean ± S.E. of at least two experiments.

Variant t1/2a Recovery
Monomerb Dimerc

min %
 alpha WT 130  ± 12 92  ± 4 83  ± 1
7-31 135  ± 18d >95d 77  ± 6
10-60 195  ± 34 21  ± 6 <5
28-82 123  ± 30e 41  ± 6e 49  ± 5
32-84 114  ± 6e 20  ± 4e <5
59-87 142  ± 36d >95d 90  ± 5

a Time when half of the maximal signal is detected in the media with free alpha  subunit only.
b The amount of hormone retrieved from the media at steady state and expressed as a fraction of the total (lysate + medium).
c Clones co-expressing hCGbeta WT and alpha  subunits were pulse-labeled and chased for up to 24 h. The samples from each clone were divided into two aliquots and immunoprecipitated with alpha  or hCGbeta antisera. Recovery is determined by comparing the quantity of alpha  subunit in the secreted dimer (detected with beta  antiserum) with the total alpha  subunit pool (lysate + media) using polyclonal alpha  subunit antiserum.
d Data from previous study (10).
e Mean ± range of two experiments.

To evaluate the efficiency of dimer formation, the alpha  constructs were co-transfected with the hCGbeta gene (21), and clones synthesizing excess beta  subunit were selected to ensure that it would not limit assembly. The cells were labeled, and equal aliquots of lysate/medium were precipitated with subunit-specific polyclonal antisera (Fig. 4, Table I). Recovery of the 10-60 and 32-84 mutants in the secreted heterodimer was less than 5% although synthesis of all the mutants was at significant levels. In the case of the 28-82 mutant (50% recovery), the combination efficiency is relatively unaffected, since the amount of alpha  subunit precipitated by either alpha  or beta  subunit antiserum was comparable. Pulse-chase experiments demonstrated that the alpha 7-31, 28-82, and 59-87 mutants combined efficiently with the beta  subunit, and their recovery paralleled the uncombined alpha  subunit synthesized in the absence of co-transfected beta  subunit (Table I). The low amount of dimers containing the alpha 10-60 or alpha 32-84 mutant reflects a decrease in combination efficiency and/or an enhanced intracellular degradation of the mutated alpha subunit. The data show that the disulfide bonds 10-60 and 32-84 (and to a lesser extent 28-82) are critical for assembly.


Fig. 4. Clones expressing hCGbeta WT and either alpha WT, alpha 10-60, alpha 28-82, or alpha 32-84 were labeled for 1 h (Lysates) or 7 h (Media). The samples from each clone were divided into two aliquots and immunoprecipitated with alpha  or hCGbeta antisera, followed by SDS-polyacrylamide gel electrophoresis.
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Expression of Tethered Variants

Due to decreased dimer accumulation in the media, studies on the biologic action of the heterodimers containing the 10-60 or 32-84 mutation are difficult. However, if the beta  subunit is covalently linked to the alpha  subunit, the rate-limiting assembly step is bypassed, and sufficient material could be obtained to examine signal transduction. The disulfide bond mutants described above were linked to the wild-type beta  subunit to form single chain analogs (see "Materials and Methods"), and the intracellular behavior was examined by pulse-chase experiments. The secretion kinetics of the single chain variants (Fig. 5, Table II) paralleled the corresponding monomers. The variants including the nonmutated tether (CGbeta alpha ) were secreted at a comparable t1/2 (CGbeta alpha t1/2 = 106 min), while the single chain alpha 10-60 mutant is secreted much slower (t1/2 = 244 min; see Table II). Thus, even in the single chain construct, the 10-60 bond is critical for secretion. However, compared with the corresponding heterodimers (compare Tables I and II), recovery of the mutants 10-60 and 32-84 dramatically increases when incorporated in a single chain.


Fig. 5. Secretion kinetics of alpha  cysteine mutants in CG single chain. The experimental procedure was similar to that described in the Fig. 3 legend except that hCGbeta antiserum was used for immunoprecipitation.
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Table II. Secretion of alpha  mutants in CG tether

See the legend of Table I for description of quantitation. CGbeta alpha corresponds to the nonmutated CG single chain.

Variant t1/2 Recovery

min %
CGbeta alpha 106  ± 4 74  ± 5
7-31 107  ± 5 81  ± 5
10-60 244  ± 36 36  ± 3
28-82 102  ± 15 55  ± 5
32-84 138  ± 32 45  ± 6
59-87 102  ± 13 87  ± 4

Because little heterodimer is formed with alpha  mutants 10-60 and 32-84, we suspected that the conformation of the single chain bearing these mutated alpha  domains would be changed. To address this issue, conditioned media were examined by Western blot and probed by two dimer-specific monoclonal antibodies: A407 and B109 (22-26) (Fig. 6). An A407 epitope resides in amino acid residues 5 and 6, 11-13, and 81 of the alpha  subunit (23). Under nonreducing conditions, A407 recognizes 7-31, 28-82, 59-87, nonmutated CGbeta alpha single chain, and CG heterodimer (Fig. 6A). However, 10-60 and 32-84 are barely detected (Fig. 6A). These low signals are not due to a difference in the recovery of the blotted protein, since reprobing with polyclonal beta  antiserum shows comparable signals for all mutants, including 10-60 and 32-84 (Fig. 6B). Moreover, it is apparent that the migration of the mutants under nonreduced conditions is altered by the mutations, implying that the conformation of the single chain mutants and CGbeta alpha are not the same.


Fig. 6. Conformational difference of alpha  Cys-mutated single chains. Conditioned media incubated with clones expressing either mutant (7-31, 10-60, 28-82, 32-84, 59-87), CG single chain (beta alpha ), or CG dimer were loaded on SDS gel under nonreducing conditions. They were probed with dimer-specific monoclonal antibodies. A, monoclonal antibody A407. The same membrane was reprobed with beta  polyclonal antiserum (B). C, monoclonal antibody B109. The same membrane was reprobed with CGbeta polyclonal antiserum (D).
[View Larger Version of this Image (42K GIF file)]

Similar data were obtained with B109, which is specific for the dimer form of the beta  subunit (24-26) (Fig. 6C). Tethers containing mutations 7-31, 28-82, and 59-87 and the nonmutated beta alpha are recognized as well as the heterodimer, but single chains containing either the 10-60 or 32-84 mutation show very weak signal compared with the same blotted membrane using CGbeta antiserum (Fig. 6D). Both A407 and B109 recognized higher molecular weight proteins in CGbeta alpha 28-82 and 59-87. They are likely noncovalently linked because when the samples are boiled under nonreducing conditions, the "aggregates" disappeared (data not shown). (It is not clear why the aggregates are detected by monoclonal antibodies but not by polyclonal antiserum; we are currently purifying these proteins for extensive characterization.) These results suggest that the conformation of single chain mutants 10-60 and 32-84 differ substantially from the nonmutated single chain and the heterodimeric forms.

Biologic Activity of Single Chain Variants

Because we could now obtain sufficient quantities of single chains bearing the cystine knot mutants, the influence of the disulfide bonds on receptor binding/signal transduction was examined using human kidney 293 cells expressing the LH/hCG receptor. For these bioassays, conditioned media were quantitated with a beta -specific polyclonal based radioimmune assay (see "Materials and Methods"). It is apparent that except for the 59-87 mutant, the binding affinity of all of the mutants was comparable with the nonmutated tether (Fig. 7A, Table III). The binding affinity of single chain 59-87 was reduced 10-fold, similar to that for the heterodimer containing this mutation in the monomeric alpha  subunit as previously reported (10).


Fig. 7. A, displacement of 125I-hCG binding to human LH receptors by single chain mutants. Stably transfected 293 cells expressing human CG/LH receptors were incubated with 125I-hCG in the presence of varying concentrations of single chain mutants. Displacement curves are presented as the percentage of maximal binding of 125I-hCG in the absence of mutants (mean ± S.E. of three independent experiments) performed in duplicate. B, dose-dependent cAMP stimulation by single chain mutants. Stably transfected 293 cells expressing human CG/LH receptors were incubated in the presence of varying concentrations of single chains for 2 h. cAMP concentration was measured by radioimmune assay. Data are the means ± S.E. of three independent experiments in duplicate. bullet , CGbeta alpha ; open circle . 7-31; X, 10-60; black-square, 28-82; square , 32-84; black-triangle, 59-87.
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Table III. Biological activity of alpha  cysteine mutants in CG single chain


Binding IC50a cAMP ED50b Coupling factor (IC50/ED50)c

ng/ml ng/ml
CGbeta alpha d 23.3  ± 7.9 4.3  ± 0.8 3.4  ± 1.4
7-31 6.7  ± 1.7 7.4  ± 1.8 1.0  ± 0.3
10-60 26.9  ± 8.4 11.9  ± 3.5e 2.9  ± 1.2e
28-82 6.5  ± 2.1 6.9  ± 0.5 1.0  ± 0.2
32-84 14.1  ± 3.1 12.9  ± 3.6 1.6  ± 0.6
59-87 254.3  ± 55.4 43.2  ± 15.5 8.1  ± 2.7

a Concentrations required to displace 50% of the maximal 125I-hCG bound. Values are presented as mean ± S.E. of at least three independent experiments.
b Concentration inducing half-maximal cAMP levels. Data are the mean ± S.E. of at least two independent experiments.
c The ratio of IC50 versus the ED50. Numbers are shown as mean ± S.E. of at least two independent experiment sets using same samples.
d CGbeta alpha corresponds to the nonmutated CG single chain.
e Mean ± range of two experiments.

Adenylate cyclase activation by the single chain was also determined (Fig. 7B). The data demonstrate that signal transduction parallels receptor binding affinity, suggesting that the determinant(s) necessary for bioactivity is conserved between CG heterodimer and single chain (Fig. 7B, Table III). Thus, despite differences in the intracellular behavior and immunoreactivity to conformationally sensitive monoclonal antibodies, the mutants are nevertheless biologically active in vitro, with coupling of receptor binding to signal transduction unaffected by these structural modifications.


DISCUSSION

Within a species, the amino acid sequence of the alpha  subunit is identical (1), and it is the unique structure of the beta  subunit that ensures binding of each dimer to its cognate receptor. The alpha  subunit, therefore, must be very flexible, since it combines with each of the four glycoprotein beta  subunits and beta  subunits from different species (27). Further evidence for this adaptability is the conformational changes occurring during dimer formation (for review, see Refs. 1 and 28-34). Presumably, the configuration of the disulfide bonds of the alpha  subunit is critical for its interaction with the beta  subunit. Previous studies on these bonds in the monomeric alpha  subunit demonstrated that single mutations in the cystine knot blocked secretion and/or inhibited heterodimer formation (10). Although the results implicated a critical role for these bonds in the intracellular behavior of the subunit, we could not exclude the possibility that the free thiol, rather than the disrupted bond per se, led to the observed changes (9, 10, 19, 20). To address this issue, double cysteine mutations of all the proposed pairs were constructed in the alpha  subunit. It was apparent that mutations in the knot altered the recovery of the subunit from the media. Recovery of variants containing mutations outside the cystine knot, alpha 7-31 and alpha 59-87, was comparable with the wild type alpha  subunit. These changes in the secretion patterns presumably reflect alterations in the folding of the subunit. Consistent with this hypothesis are the experiments showing that, compared with the wild-type alpha  subunit, the cystine knot variants 10-60 and 32-84 combined much less efficiently to the beta  subunit, with less than 5% of these mutants recovered as dimers. Thus, assessing the role of the alpha 10-60 and alpha 32-84 bonds in the biological action of hCG was precluded. Because tethers containing either mutation at 10-60 or 32-84 were recovered in the media, we could determine their biologic activity. Unexpectedly, it was observed that all of the cystine knot and the alpha 7-31 mutants exhibited high affinity binding for the receptor. When corrected for binding, signal transduction was unchanged regardless of the mutation. These results imply that disulfide bonds of the cystine knot are required for maximal secretion/assembly but that for receptor recognition, not all of the bonds in the core are needed. The single chain bearing the alpha 59-87 mutant exhibited a 10-fold decrease in receptor binding. Several studies have demonstrated the importance of the C-terminal residues 88-92 in the alpha  subunit for maximum binding affinity (1, 35-38). It is likely that disrupting the 59-87 bond perturbed the configuration of the carboxyl-terminal region, leading to the observed decrease in receptor binding. That the altered intracellular behavior of these tethers is not accompanied by a significant change in biologic activity, especially the 10-60 and 32-84 mutants, suggests that the receptor could recognize different conformations of the hormone. While this conclusion is supported by the absence of recognition by the dimer-specific monoclonal antibodies, we cannot exclude a greater liability of the epitopes in the 10-60 and 32-84 mutants to the SDS-polyacrylamide gel electrophoresis conditions used.

Given that perturbing the cystine knot affects the intracellular behavior of both the monomeric alpha  subunit and the single chain molecule, the conformation of this region is likely to be relatively conserved regardless of the associated hormone-specific beta  subunit. This fixed core determinant may be necessary for the "escort" (42, 43) function of the alpha  subunit, which is critical for rescuing the pituitary beta  subunits from the endoplasmic reticulum because few, if any, uncombined LH, follitropin, and thyrotropin beta  subunits are secreted in the absence of co-expressed alpha  subunit (34, 39, 40). Thus, we would propose that the major conformational changes of the alpha  subunit resulting from assembly with the beta  subunit occurs in regions outside of the cystine knot, i.e. the three hairpin loops (2, 3, 23, 41). Consistent with this interpretation, the recovery of the variants with the 7-31 and 59-87 mutations, which lie outside the knot, was comparable with the wild type.

That the intracellular fate of single chains with mutations of paired cysteines and corresponding monomers parallel each other suggests that a misfolded epitope is not recognized by an intracellular transport component and/or that the mutants are trapped by a chaperone, which leads to enhanced degradation. Although the beta  domain increases the recovery of the mutated single chains, e.g. in CGbeta alpha 10-60 and CGbeta alpha 32-84, they are still less than that of the wild type. Thus, even the presence of the beta  subunit domain is not sufficient to completely override the alpha  Cys mutations.

Other members of the cystine knot growth factor superfamily display similar characteristics (6-8). Mutations of the paired cysteine residues within the knot of tumor growth factor-beta 1 or activin dramatically reduced secretion (6, 7). Thus, together with our data presented here, the cystine knot is apparently the basic frame of the glycoprotein hormone subunits and several growth factors and represents a critical determinant for secretion and assembly of functional ligand. Receptor recognition is apparently less dependent on the configuration of this structure. The data also show the importance of the single chain approach to study structure-function of multisubunit proteins where dependence on the assembly step is essential for biologic action.


FOOTNOTES

*   This work was supported by a grant from the Organon Company, National Institutes of Health Grant N01-HD67922, and DRTC Molecular Biology Core Grant NIH 5 P60 DK20579.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.
§   Present address: Dept. of Obstetrics/Gynecology, Handa City Hospital, 2-29 Toyo-cho, Handa, Aichi 475, Japan.
   To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8103, St. Louis, MO 63110.
1   The abbreviations used are: CG, chorionic gonadotropin; hCG, human CG; LH, lutropin; WT, wild type.

ACKNOWLEDGEMENTS

We thank Drs. David Ornitz, Mesut Muyan, and Edward Grotjan for critical review of the manuscript and Dr. Steven Birken for helpful discussions. Also, we are grateful to Susan Carnes for invaluable assistance in preparing this manuscript.


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