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Molecular Endocrinology, doi:10.1210/me.2002-0349
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*CHORIONIC GONADOTROPIN
Molecular Endocrinology 17 (7): 1192-1202
Copyright © 2003 by The Endocrine Society

Structural Analysis of Yoked Chorionic Gonadotropin-Luteinizing Hormone Receptor Ectodomain Complexes by Circular Dichroic Spectroscopy

Gregory B. Fralish, Brian Dattilo and David Puett

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Life Sciences Building, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bmb.uga.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1–341 (encoded by exons 1–10 and a portion of 11), and two C-terminal ECD deletion fragments, 1–294 (encoded by exons 1–10) and 1–180 (encoded by exons 1–7). The fusion proteins, including yoked CG (N-ß-{alpha}-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating ß-strand/{alpha}-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8–10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE G PROTEIN-COUPLED LH receptor (LHR) plays a critical role in human reproductive physiology and male sexual differentiation, with important processes such as gonadal steroidogenesis and ovulation regulated by high affinity binding of LH or chorionic gonadotropin (CG) to LHR (1, 2). The two ligands, LH and CG, are members of the family of glycoprotein hormones, which also includes FSH and TSH (3). These proteins all share a heterodimeric structure, consisting of a common {alpha}-subunit and a hormone-specific ß-subunit. LH and CG exhibit a high degree of homology (>90% similarity), with most of their dissimilarity arising from a 30-amino acid residue C-terminal peptide (CTP) of human (h) CG-ß. Indeed, the two hormones display similar (albeit distinct) binding affinities for LHR (3). The crystal structures of hCG (4, 5) and hFSH (6) reveal a common fold for the growth factor-like cystine-knot motif that is probably shared by all of the family members.

The three glycoprotein hormone receptors, i.e. for LH/CG, FSH, and TSH, form a subfamily in the group of rhodopsin-like G protein-coupled receptors (GPCR) (1, 2). They are characterized by a large N-terminal ectodomain (ECD), comprising about half the mass of the receptor and composed of an N-terminal cysteine (Cys)-rich region, a Leu-rich-repeat (LRR) region, and a C-terminal hinge region, also rich in Cys residues, that binds ligand with high affinity in the absence of the transmembrane (TM) domain (Fig. 1AGo) (1, 2, 7). Homology modeling of a large portion of the LHR ECD has been done by our laboratory, in collaboration with Drs. Isaacs and Lapthorn (University of Glasgow, Scotland, UK), and by others (Fig. 1BGo) (8, 9, 10). The number and delineation of the LRRs in the different models vary, but all are in agreement that the repeats found in the LHR ECD sequence could accommodate the hallmark ß-strand/{alpha}-helix repeat fold. However, the intrastrand regions of different LRR proteins display diverse structural characters that are dependent upon the length of this region (11). Thus, the precise nature of the fold is unclear. None of the models included the conserved N-terminal Cys-rich region, and only one of the reports suggested a possible fold for a portion of the C- terminal Cys-rich cluster (10). From the modeling studies and subsequent mutagenesis mapping of the ECD, it has become apparent that charge-charge interactions play a critical role in the high affinity binding of hCG to LHR ECD (9, 10, 12, 13), as do hydrophobic interactions (13). Further, peptide competition studies (14, 15) suggest a noncontiguous hormone footprint indicative of an extended hormone-receptor interface, whereas independent LRR and exon truncation (16, 17) and deletion (18) studies associate the N-terminal region of the ECD with high affinity binding.



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  		Figure 1. ECD Model and Fusion Protein Schematics

A, Schematic representation of the tripartite delineation of the ECD. The residues purported to reside in each region are indicated. B, Homology model of the LHR ECD. [Reprinted with permission from N. Bhowmick et al.: Mol Endocrinol 10:1147–1159, 1996 (10 ). © The Endocrine Society.] C, Schematic representation of Y341, Y294, Y180, and YhCG. YhCG has been characterized previously (22 ). Y341, Y294, and Y180 represent the soluble hormone receptor fusions. Y341 and Y294 are predicted to contain eight or nine LRRs (9 10 ), whereas Y180 is predicted to contain six LRRs. The naturally occurring CTP in hCG-ß was used to link the subunits of the hormone and an additional CTP was used to link the hormone to the receptor. All fusion proteins contain identical arrangements of the hCG subunits and Factor Xa protease recognition sequence between hormone and receptor.

 
Direct structural data of the ECD for LHR and the other glycoprotein hormone receptors are extremely limited. This is due in large part to the incompatibility of the ECDs with most commonly employed overexpression systems. Recently, two groups have reported high levels of expression of the LHR ECD in bacteria (19) and Chinese hamster ovary cells (20). We have successfully expressed and characterized a novel yoked hCG-LHR ECD (1–341) fusion protein (YECD) in insect cells (21). The feasibility of this approach was supported by studies on the previously described yoked hormone (YhCG) (22), and the yoked hormone-receptor complex (YHR) (23, 24). From studies on YHR, it was apparent that hCG fused to the N terminus of LHR was capable of fully stimulating the receptor and blocking binding of exogenous hCG. YECD also appears to be a very stable complex, with an apparent high affinity interaction between hCG and the LHR ECD and is expressed at levels sufficient for structural studies (21).

The major goal of this study was to obtain good estimates of the secondary structure, {alpha}-helix, ß-strand, and ß-turn, of the LHR ECD and two C-terminal deletion fragments for comparison with a proposed model (10). We report herein the structural characterization, using circular dichroism (CD) spectroscopy, of yoked hCG-LHR ECD (1–341) (Y341, i.e. the number of ECD amino acid residues in the rat construct) and expression, purification and structural characterization of two C-terminally truncated hormone-receptor fusion proteins, yoked hCG-LHR ECD (1–294) (Y294) and yoked hCG-LHR ECD (1–180) (Y180). The CD spectrum for the fused hormone, YhCG, was also obtained and when subtracted from the YECD spectrum yielded a difference spectrum consistent with that of a ß-strand/{alpha}-helix protein. Estimates of percent helix from the CD spectra of Y294 and Y180 were found to be similar to each other but less than Y341. These results suggest that: 1) the C-terminal 47 amino acid residues of the ECD stabilize the helicity of the entire ECD or, less likely, are themselves helical in nature, and that 2) the ECD has a repetitive folding pattern that may extend further into the C-terminal Cys-rich cluster than expected (10). The approach of fusing ligand and receptor has enabled high expression levels of complexes to be achieved and has provided a new approach for obtaining direct structural data for determining the molecular characteristics of GPCR ECDs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The structural nature of the ECD has been elusive primarily because of difficulties encountered when trying to overexpress the protein in heterologous systems. We expressed the ECD in insect cells in the absence of hCG, but the level of secretion was too low for structural studies. Recently, we reported the purification and characterization of a functional single chain hCG-ECD protein expressed in insect cells. It was observed that when the ligand was fused to the ECD expression levels near 1.5 mg/liter were achieved (21). The boost in expression was proposed to be ligand-associated stabilization of the ECD, which may be flexible and unstable. This suggestion was supported by a subsequent report where the porcine LHR ECD was produced in Chinese hamster ovary cells at high levels through coexpression with hCG (20). We also coexpressed the ECD with hCG in insect cells and separated the product using low pH, but the yield was insufficient for the spectral studies. Thus, we have used the yoked hCG-LHR ECD system to obtain sufficient quantities of proteins for CD spectroscopy.

The C-terminal region of the ECD (amino acid residues 205–341 of rat LHR ECD) has been shown to contribute little to the high affinity binding of hCG (16, 17, 18). Thus, we have generated truncated versions of YECD consisting of the first 180 and 294 amino acid residues of the ECD (denoted Y180 and Y294, respectively) with the identical yoked hormone-receptor arrangement of YECD, while retaining the Factor Xa recognition sequence (Fig. 1CGo). Y294 is of interest given its existence as an alternative splicing product (residues 1–297 in porcine LHR and 1–294 rat LHR), whose mRNA is detectable in vivo (25, 26). Y180 was chosen because it represents the smallest portion of the ECD that can still bind hormone with significant affinity, albeit some 10-fold reduced compared with intact LHR (17). The cDNAs were cloned, recombinant baculoviruses were generated, and expression of the proteins in insect cells was optimized. Interestingly, expression levels for the C-terminally truncated proteins were greater than that of the full-length product, e.g. Y294 and Y180 expressed at respective levels of 1.5- to 2-fold and 4- to 5-fold higher than that of Y341 (data not shown).

Purification of the various proteins was achieved using ammonium sulfate precipitation and antibody affinity chromatography, and typical protein yields were in the range of 40–60%. Proteins eluted from the M2-Flag column were homogeneous as determined by SDS-PAGE under reducing conditions and silver staining (Fig. 2Go). The broader bands of YhCG and Y180 are believed to result from their greater mobilities, relative to Y294 and Y341, in the 10% polyacrylamide gels with the resulting isoforms presumably yielding increased apparent heterogeneity. Factor Xa-treated and untreated samples were analyzed by Western blot analysis (Fig. 3Go). Using antibodies to the Flag and CTP, the receptor and hormone components of the protein complex can be discerned. Western blotting with these antibodies indicates intact receptor species resolving at molecular weights greater than would be predicted from just the amino acid sequence consistent with the addition of carbohydrate. For Y341, the receptor is more diffuse in the gel, suggesting a greater amount of glycosylation heterogeneity compared with Y180 and Y294. Y341 contains three more putative N-glycosylation sites than Y180, but the same number as Y294, suggesting there may be a requirement for the C-terminal region of the ECD for complete maturation. Also, when the Factor Xa-treated samples were probed with the CTP antibody, the hormone regions of the protein were resolved to equivalent positions on the gel. The efficiency and specificity of the cleavage is excellent with little undigested protein present.



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  		Figure 2. SDS-PAGE of Y341, Y294, Y180, and YhCG

The purity of the individual proteins was assessed using 10% SDS-PAGE under reducing conditions and silver staining. All lanes were loaded with 50–100 ng of protein and silver stained. The approximate molecular mass of the proteins estimated from the molecular mass standards shown are 83, 77, 60, and 39 kDa for Y341, Y294, Y180, and YhCG, respectively.

 


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  		Figure 3. Factor Xa Digestion and Western Blot Analysis of YECDs

Y341, Y294, and Y180 were treated with Factor Xa protease for 4 h at room temperature. The samples (5–10 ng protein) were resolved by 10% SDS-PAGE, then transferred to PVDF membrane and probed for the Flag or CTP epitope. Intact controls were probed for the Flag epitope. The sizes of the molecular mass standards are indicated.

 
Proteins were tested for binding activity using a method previously described (21). The data indicate that Y294 can bind exogenous 125I-hCG at a level similar to that of the Y341 (Fig. 4Go). Binding to Y341 and Y294 is only detectable if the proteins are first treated with Factor Xa (Fig. 4Go), suggesting that the fused hormone occupies the binding site for hCG. This is not surprising in these fusion proteins because the ligand is constrained by the CTP-Factor Xa cleavage site linker from diffusing further away from the ECD binding site than the length of the linker, ensuring that the single chain gonadotropin is always either bound to the ECD or in close proximity.



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  		Figure 4. Total Specific Binding of Factor Xa-Treated and Untreated Samples of Y341, Y294, and Y180

Soluble binding assays were performed as previously described (21 ). The ordinate represents total specific counts (cpm) bound to PVDF membrane squares blotted with 10 ng purified protein that was either treated or not treated with Factor Xa. After incubation with 1 nM 125I-hCG, specific counts were calculated by subtracting counts obtained in the presence of excess unlabeled hCG from total bound 125I-hCG. Stoichiometric amounts of YhCG and ECD were present in the incubations with 125I-hCG, and no corrections were made for competition of YhCG and hCG binding.

 
Numerous studies using detergent-solubilized cells have shown that the intact ECD of rat and human LHR binds hCG with the same affinity as the full-length receptor, if not somewhat slightly higher affinity (2). Several C-terminal deletion fragments of the ECD have also been characterized with respect to ligand binding (17), as have a number of deletions within the ECD in full-length LHR in intact and in detergent-solubilized cells (18). Pertinent to the ECD N-terminal fragments investigated herein are several reports on the N-terminal ECD fragments (1–293, 1–294, and 1–295), of rat and human LHR (17, 18, 26, 27, 28, 29, 30). Expressing the binding results as (Kd)wt/(Kd)mut, i.e. the ratio of the Kd of the full-length receptor to that of the deletion mutant, the mean from these six reports is 1.1 with a range of 0.6–1.6. Thus, the 1–294 fragment we studied binds ligand with essentially the same affinity as does the full-length ectodomain of LHR. This is confirmed with the reduced binding without pretreatment with Factor Xa.

Y180 did not show binding activity under these conditions (Fig. 4Go). There is only one report on hCG binding to the 1–180 fragment, in which a binding ratio, (Kd)wt/(Kd)mut, of 0.1 was found (17), i.e. the N-terminal fragment of the LHR ECD binds hCG with an affinity some 10-fold less than that of full-length receptor. Thus, we believe that the reduction in the affinity of this length of ECD for hormone prevents the detection of specific binding in our system under the conditions used (1 nM 125I-hCG), where there are stoichiometric amounts of single chain hCG and ECD 1–180 present after enzymatic digestion. However, because Y294 and Y341 retain high-affinity binding, low levels of specific binding are detectable, even with equivalent amounts of single chain hormone and ECD present.

CD spectra for all of the proteins were measured between 250 and 190 nm. This 60-nm wavelength interval is in agreement with recommended ranges for the algorithms used for secondary structure analysis: 200–240 nm for CONTINLL, based upon the original CONTIN program (31, 32) with ridge regression but utilizing a locally linearized model to select reference proteins, and 200–260 nm for SELCON (32, 33, 34, 35). The CD spectra of Y341 and YhCG were measured, and that for Y341 shows a more negative mean residue ellipticity ([{theta}]) compared with YhCG in the 200–240 nm range, as well as a marked increase in [{theta}] at wavelengths below 200 nm (Fig. 5Go). These differences indicate the presence of more helicity in the Y341 protein compared with YhCG. Secondary structure estimates, calculated using two commonly employed methods (31, 33, 34, 35), revealed values that did not vary significantly based on the two algorithms used (Table 1Go). Because helicity estimates from CD spectra are by far the most reliable of the secondary structural elements (32), the helical content of the free ECD was estimated by analyzing the difference spectrum using the same methods. As shown in Table 1Go, the helicity of the ECD is estimated to be 18–20%. The estimate of helicity in YhCG from the CD spectrum is slightly higher but in reasonable agreement with the expected value from the crystal structure (4, 5); however, the ß-strand estimate from the recorded spectrum was significantly lower than expected. Similar observations were made with single chain hFSH (36).



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  		Figure 5. CD Spectra of Y341 and YhCG

Representative CD spectra of the purified Y341 and YhCG proteins were recorded and are presented as [{theta}], mean residue ellipticity. The spectrum of YhCG was subtracted from that of Y341 (with {theta} in millidegrees) and the calculated difference spectrum (Y341-YhCG) was then converted to the appropriate units for comparison. Estimates of secondary structure are given in Table 1Go. deg, Degrees.

 

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  		Table 1. Secondary Structure Analysis of YhCG, Y341, Y294 and Y180 from CD Spectra and of LHR ECD 1-341, 1-294, and 1-180 from CD Difference Spectra

 
The CD spectra of Y294 and Y180 were determined and found to be very similar, when shown as mean residue ellipticity (Fig. 6Go); however, both displayed reduced (less negative) ellipticity compared with Y341 in the 200–240 nm range. Because the spectra were highly reproducible between protein preparations and measurements, we performed an analysis of helical content in each construct. The secondary structural elements of the spectra were extracted as described above and are compared in Table 1Go. Because the percent helix for Y294 and Y180 are about the same, this implies that the larger protein, Y294, contains more helical residues than does Y180, and both yield lower helical values than Y341. The self-consistent method of extracting secondary structural information from CD spectra was able to reliably deconvolute just the Y341 spectrum. Thus, only the results obtained from the CONTINLL program are tabulated for Y294 and Y180. However, for comparison, helical estimates were calculated for Y294 and Y180 using another method (37) that yielded values consistent with those from the CONTINLL method. In addition, estimates of secondary structure were obtained by subtracting the CD spectrum of YhCG from that of Y294 and of Y180, e.g. as described above for Y341, followed by standard analysis. The results are given in Table 1Go and indicate a significant reduction in the amount of secondary structure present in the C-terminal truncated ECDs.



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  		Figure 6. CD Spectra of Y341, Y294, and Y180

The CD spectra of Y341, Y294, and Y180 were measured and are given as mean residue ellipticity. The spectra are shown as mean ± SEM to demonstrate the reproducibility of the results between protein preparations. The same subtractive analyses were done with Y294 and Y180 as for Y341 in Fig. 5Go to obtain the secondary structure estimates provided in Table 1Go.

 
There are no ab initio or homology models of the N-terminal and C-terminal Cys-rich regions; however, a suggestion was made that a portion of the C-terminal hinge region may have a cytokine-like fold (10). The ß-strand/{alpha}-helix motif of a typical LRR consists of approximately 6 amino acid residues forming a ß-strand, some 11 or 12 in a turn, and about 7 in an {alpha}-helix (2, 10). Thus, excluding contributions from the two Cys-rich regions of the LHR ECD, the LRR region is expected to contribute about 56–63 and 48–54 amino acid residues in {alpha}-helix and ß-strand, respectively, depending upon whether one considers 8 or 9 LRRs (9, 10). Realizing the imperfect nature of the LHR ECD LRRs and the variable lengths of the individual LRRs, the number of amino acid residues expected in secondary structure can be relaxed somewhat to consider an average of 7 ± 1 in a typical {alpha}-helix and 6 ± 1 in a typical ß-strand. It then follows that the 8 or 9 LRRs could contribute roughly 48–72 (or 60 ± 12) and 40–63 (or 52 ± 12) residues in {alpha}-helix and ß-strand, respectively.

The fraction of {alpha}-helicity of Y341 estimated from the CD spectrum converts to about 68–87 (or 77 ± 10) amino acid residues including the Flag tag, YhCG, the CTP plus Factor Xa cleavage site, and the complete 341 amino acid residue ECD; subtraction of the YhCG contribution (estimated from the CD spectrum) results in a remainder of about 51–70 (or 60 ± 10) helical residues in the ECD (Table 2Go). This range is in excellent agreement with that expected after subtracting the CD spectrum of YhCG from that of Y341 and analyzing the difference spectrum, 61–68 (or 65 ± 4) residues. Subtraction of the number of helical residues in crystallized hCG (4, 5) from the analyzed CD spectrum of Y341 gives a somewhat higher range, 72 ± 12, but nonetheless one that is similar to that predicted by the model. These results, along with those obtained based upon identical analyses of the Y294 and Y180 spectra, are summarized in Table 2Go.


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  		Table 2. Estimates of the Number of Amino Acids in {alpha}-Helices (and ß-Strands) in Full-Length and C-Terminal Deletion Fragments of the LHR ECD Based upon the Measured CD Spectra

 
The assumptions made in these calculations from the model must be emphasized, namely that the Flag tag, Factor Xa cleavage site, and the two Cys-rich regions do not contain {alpha}-helical residues and that there are no major changes in {alpha}-helicity of YhCG or the ECD concomitant with association. Confirmation of these assumptions must await additional studies. It should be pointed out, however, that earlier CD studies on hCG indicated negligible helicity (38), consistent with the low percentage noted in the crystal structure (4, 5). CD spectra of the hCG subunits (39, 40), including disulfide-intact subunits and reduced, S-carboxymethylated subunits, tryptic fragments of the reduced, S-carboxymethylated subunits, and the CTP (41) revealed that only limited {alpha}-helicity could be induced in the helix-promoting solvent, trifluoroethanol, but only with reduced, S-carboxymethylated subunits and certain tryptic fragments. Thus, it seems unlikely that YhCG would undergo a large increase in helicity upon binding to the ECD. Furthermore, numerous secondary structure prediction programs suggest that the amino acid residues in the N- and C-terminal Cys-rich regions have no significant propensity to form secondary structure (DeMars, G. J., G. B. Fralish, and D. Puett, unpublished results).

The same approach to estimate the number of residues in ß-strands was employed (Table 2Go, values in parentheses). We emphasize, as discussed by others (32), that estimates of ß-structure from CD spectra tend to be low in proteins with a high content of ß-strands, e.g. hCG (4, 5). Furthermore, the accuracy of ß-strand predictions is significantly lower than that obtained for helix (32). Thus, we have placed less confidence in these values and less emphasis on these results here and in the subsequent discussion.

The results in Table 2Go were used to calculate the number of LRRS in the ECD and two fragments assuming 7 ± 1 {alpha}-helical residues per LRR (Table 3Go). These results show that the CD spectra are consistent with the eight to nine LRRs predicted for the full-length ECD. In contrast, the 1–294 fragment is estimated to contain only three to four LRRs instead of the eight to nine predicted from the model (9, 10), leading to a net loss of 5 ± 1 LRRs. Likewise, the 1–180 fragment is predicted to contain five LRRs, but only two to three were estimated by CD spectroscopy, leading to a net loss of two to three LRRs. The difference between the predicted number of LRRs from the model and that estimated from the CD spectra is attributed to the destabilization of the N-terminal and central portions of the ECD by the C-terminal or hinge region of the ECD.


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  		Table 3. Comparison of the Number of LRRs in the LHR ECD and N-Terminal Fragments Estimated from CD Spectroscopy and Expected from the Model

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study reports, for the first time, the CD spectrum of the full-length LHR ECD yoked to hCG. The spectrum of YhCG was distinct from that of native hCG (42), perhaps indicating some constraints imposed by the fusion of {alpha} and ß. This observation on hCG is in contrast to a report where the CD spectrum for single chain hFSH was stated to be identical to that of native FSH (36). The spectra were analyzed extensively with multiple algorithms and yielded consistent values for {alpha}-helix and ß-strand, with nearly 90% accuracy expected for the helical estimate (32). Subtraction of the spectral contribution of YhCG from that of the complex permitted an estimate of the type and amount of secondary structure present in the ECD alone, which should correspond to that of the ligand-occupied state. In view of the highly reproducible nature of the individual CD spectra, the difference spectrum is of high accuracy. The results of this analysis were compared with values that would be expected based upon the proposed homology model of the ECD with either 8 or 9 LRRs (9, 10, 13). The findings suggest that the ECD contains 18–20% helix, some 6–7% lower than was suggested based upon a CD spectrum of a shorter porcine LHR ECD (residues 1–277)-hCG complex (20).

The LHR ECD, like that of the other glycoprotein hormone receptors, is delineated by three distinct regions: an N-terminal Cys-rich region (exon 1, residues 1–28 in rat LHR), which is important for binding (15, 18), a central LRR-containing region (exons 2–8, most of exon 9, residues 29–245 in rat LHR) also important in binding (1, 2), and a C-terminal Cys-rich region (exons 9, 10 and part of exon 11, residues 245–341 in rat LHR), which is not considered to be important for binding (17, 43) but is critical in signal transduction (30, 44, 45). The results obtained for Y341 suggest that most of the secondary structure ({alpha}-helicity) is expected to lie in the purported 8 (13) or 9 (10) typical LRRs, which are predicted to fold with a repeating motif of: ß-strand (6 residues), turn (11–12 residues), and {alpha}-helix (6–8 amino acids). Thus, the C-terminal Cys-rich region, which has been suggested to fold in a chemokine-like manner (10), is not predicted to contain significant amounts of helix or strand. This inference is based upon: 1) previous secondary structure predictions (PHD program), where sequences within the LRR region were estimated (with some 74% accuracy, in a comparative analysis with controls) to contain considerable fractions of {alpha}-helix and ß-sheet (9); 2) the analysis (albeit limited) of the CD spectrum of the porcine LHR ECD (1–277)-hCG noncovalent complex, which lacked most of the C-terminal Cys-rich domain, contained 25% helix (20); and 3) the N- and C-terminal Cys-rich regions contain no significant propensity to form secondary structure based upon numerous predictive methods (DeMars, G. J., G. B. Fralish, and D. Puett, unpublished results). Thus, the spectral analyses of Y341 and YhCG provide direct structural evidence in favor of the proposed homology model, accommodating eight or nine LRRs.

Upon truncating the receptor’s C terminus to 294 amino acids, there was a marked reduction in the mean residue ellipticity. The drop in [{theta}] represents a 50% reduction in the number of amino acids involved in helical secondary structure or a loss of some 5 ± 1 LRRs. This observation suggests that residues encoded by exon 11 of the ECD either contain a significant portion of helicity or are important for the stabilization of the structure of the entire ECD. For the former to be the case, a strong propensity for helix in this region would be required, as most of the truncated 47 residues would be helical. As discussed above, this seems unlikely.

Alternatively, these data suggest stabilization of the secondary structure of the ECD via amino acid residues present in the C-terminal hinge region. This intriguing observation suggests a mechanism by which the more N-terminal ECD region containing the LRRs is important for binding and hormone recognition, communicates structurally with the C-terminal region of the ECD, which is critical in the transfer of signal to the TM domain. An emerging model of receptor activation suggests that hormone binding results in the disruption of an interaction between regions of the hinge-domain in exon 9 (residues 250–268) and exoloop 2 of the TM domain (44). When this interaction is disrupted by mutation (46), by peptide competition (30), or through the generation of chimeras (44), the receptor is activated. As such, interactions between the ECD and TM domain are suggested to restrict the receptor in a conformation not productive for coupling to G protein, and when hormone binding occurs the receptor is relaxed by disrupting this interaction. This model may extend to other members of this class of GPCRs, as the TSH receptor is constitutively active when its N-terminal ECD is removed (47). The critical nature of the amino acid residues in exon 11 for secondary structural integrity in the entire ECD suggests that interactions exist between the receptor’s C-terminal hinge region and the central LRR region. Indeed, sequences truncated in Y294 have been shown to be critical in the transduction of signal, where mutation of the conserved charged residues Glu-332 and Asp-333 in rLHR resulted in a dramatic reduction of the ability of the hormone to activate receptor (45). Therefore, these data represent direct, structural support for the hinge region’s potential role in signal transduction as originally suggested by Hsueh et al. (44), although it is unclear how this structural transition may directly affect this process.

It should be noted that the secondary structure present in the full-length Y341 is not required for binding as Y294 displayed similar binding activity as Y341. This observation is consistent with numerous studies comparing soluble LHR ECDs of similar length to those studied here (1, 2, 7, 16, 17, 18, 26, 27, 28, 29). The receptor’s tolerance to structural variability in the hormone has been demonstrated (24, 48, 49); however, this study raises the possibility that the receptor itself can be structurally altered while still retaining the ability to recognize hormone. Thus, the interactions responsible for high affinity recognition by either the hormone or receptor are not necessarily determined by a strict three-dimensional (lock and key) configuration, but perhaps more of an induced fit.

The CD spectrum (as mean residue ellipticity) of the Y180 protein was nearly identical to that of Y294, suggesting a repeating structural unit. Thus, these data provide direct structural evidence for a repeating motif in the LHR ECD, greatly bolstering the argument for al LRR fold. Importantly, after correcting for the different sizes of the two ECD C-terminal deletion fragments, it is estimated that two to three LRRs are destabilized as a result of the loss of interaction.

In summary, we have used the yoked hormone-receptor system to produce significant quantities of functional hormone-receptor ectodomain complexes for CD spectroscopy. Various analyses of the spectra for the full-length Y341 and the free YhCG suggest that the receptor contains 18–20% helix, supporting the current model with eight or nine LRRs. Surprisingly, C-terminal truncation of the ECD by 47 amino acid residues resulted in the destabilization of considerable secondary structure of the ECD with no loss in binding activity. The destabilization observed provides direct structural evidence for the interaction of this region with other parts of the receptor and offers intriguing possibilities for the role of the hinge region as a signal modulator.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cloning and Expression of Constructs
YhCG was cloned as described previously (22). A Flag tag (N–DYKDDDDK–C) was added to the 3' end of the YhCG sequence using PCR with primers YhCG1–5' (5'-ATGGAGATATTCCAGGGGCTGCTG-3') and YhCG1–3' (5'-TTACTTATCGTCATCGTCCTTGTAGTCAGATTTGTGATAATAAAC–3'). The PCR product was purified and cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA). Colonies were analyzed by restriction analysis and sequencing and then subcloned into the baculovirus transfer vector pVL1393 (BD Biosciences, PharMingen, San Diego, CA). Low-titer baculovirus stocks were generated through cotransfection of the pVL1393/YhCG and Baculogold, linearized baculoviral DNA (BD Biosciences, PharMingen). Medium from the transfected cells was used in serial amplifications to generate high-titer recombinant baculovirus (~108 plaque forming units/ml). YECD, containing a histidine tag, was cloned as previously described (21) and used as a template for PCR to generate Flag-tagged Y294 and Y180 cDNAs using methods described above with only the 3' primer sets differing: Y294–3' primer (5'-TTACTTATCGTCATCGTCCTTGTAGTCAAGCGTCTCGTTATCTGC-3') and Y180–3' (5'-TTACTTATCGTCATCGTCCTTGTAGTCCAGCGAGATTAGAGTC 3'). Thus, the truncated constructs only varied in the length of the ECD, retaining the exact Flag tag, subunit, linker arrangement, and Factor Xa recognition sequence. The PCR products were confirmed by restriction enzyme analysis and sequencing, and subcloned from the TOPO TA vector into the pVL1393 transfer vector.

The high-titer recombinant baculovirus stocks for these constructs were produced as described previously (21) and were used to induce the heterologous expression of the proteins in SF9 insect cells. The SF9 cells were grown in suspension in orbital shaker cultures at a density of 8 x 105 cells/ml. Expression of the proteins at low multiplicities of infection (MOI = 0.1) was sufficient for their characterization. Expression was monitored using RIAs specific to the ß-subunit (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and heterodimeric hCG (Diagnostic Products Corp., Los Angeles, CA).

Purification of Recombinant Proteins
Y341, Y294, Y180, and YhCG-medium from cells infected with the recombinant baculovirus was harvested and clarified with two sequential centrifugations at 1,400 x g and 17,700 x g. Ammonium sulfate was slowly added to the clarified medium to 60% saturation (80% saturation for YhCG) at 4 C and stirred vigorously overnight. The precipitated protein was pelleted by two centrifugations at 17,700 x g. The pellet was resuspended in TBS [50 mM Tris-HCl (pH 7.5), and 150 mM NaCl] and dialyzed extensively at 4 C against TBS. The resuspended and dialyzed protein sample was cycled at low flow rates (0.5 ml/min) over M2-Flag Ab affinity resin (Sigma-Aldrich Corp., St. Louis, MO) overnight. Binding of protein to the column was followed using RIA of the eluent fractions. The loaded column was washed extensively with TBS, and protein was eluted with 0.1 M glycine (pH 3.5). The column fractions were collected in tubes containing 1 M Tris-HCl (pH 8.0) to immediately neutralize the solutions. Fractions containing the protein were pooled and concentrated for subsequent analyses. Purity of the proteins was assessed using SDS-PAGE and silver staining, and molecular weight estimates were based upon the migration of protein standards (Invitrogen).

Factor Xa Digestion and Western Blot Analysis of Y341, Y294, and Y180
Purified proteins were treated with Factor Xa protease (New England Biolabs, Inc., Beverly, MA) as previously described (21). Briefly, the proteins were dialyzed overnight against a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM CaCl2, then treated with protease (1:25 enzyme:substrate ratio) for 4 h at room temperature. The digestion was analyzed by SDS-PAGE (reducing conditions) and Western blot analysis using the M2-flag mouse monoclonal antibody (Sigma-Aldrich), which recognizes the C-terminal flag sequence. An antimouse IgG horseradish peroxidase-conjugated secondary antibody was used for detection of the protein using standard ECL reagents (Amersham Pharmacia Biotech). A polyclonal rabbit CTP antibody (1:1000 dilution from original stock), kindly provided by Dr. Vernon Stevens (Ohio State University, Columbus, OH) was employed to probe for hormone portions of the complexes (23).

Soluble Binding Assays
The ability of the yoked hCG-ECD proteins to bind exogenously added 125I-hCG was assessed using a method previously detailed (21). Purified, Factor Xa-treated (and untreated) protein (10 ng) was added to polyvinylidene difluoride (PVDF) membrane using the Dot Blot apparatus from Bio-Rad Laboratories, Inc. (Hercules, CA). The membrane was cut into squares and then incubated with 1 nM 125I-hCG, with and without 5 µg/ml unlabeled hCG (kindly provided by Dr. A. F. Parlow at the NIDDK, Torrance, CA) in TBS containing 0.2% (vol/vol) Tween 20 and 3% (wt/vol) BSA. The tubes containing the membranes were shaken overnight at room temperature, washed five times with TBS containing 1% Nonidet P-40, dried, and counted for {gamma} radiation. Specific counts bound (cpm) were calculated by subtracting counts obtained in the presence of excess unlabeled hCG. The data presented represent the mean ± SEM of three independent experiments.

CD
The CD spectra of the purified proteins were recorded using a Jasco 710 CD spectrometer. Before collection of the spectra, the samples were dialyzed against 5 mM phosphate buffer (pH 6.8) overnight. After dialysis, the sample was centrifuged through a 0.1 µm filter (Millipore Corp., Bedford, MA). The protein concentrations were determined by UV absorption spectroscopy from 220–350 nm with the extinction coefficients for the various proteins being estimated at 280 nm from the primary sequences assuming completely oxidized cystines (50). CD spectra of the proteins were recorded at room temperature using a 1 mm pathlength cell and protein concentrations of 5–10 µM. The settings for collection of the spectra were as follows: band width, 1 nM; sensitivity, 50 millidegrees; response, 2 sec; scan speed, 20 nm/min; step resolution, 0.2 nm; starting wavelength, 250 nm; lowest wavelength, 190 nm; and 5 scans/sample. The data presented represent the average of at least three separate spectral recordings with two or more different protein preparations. The Jasco standard analysis program was used to convert units and smooth data from replicate scans. Secondary structural analysis of YhCG and Y341 were performed using the SELCON3 (33, 34, 35) and the CONTINLL (31) programs based upon reference protein sets of 37 and 42 proteins, respectively. With the Y294 and Y180 spectra, estimates were based upon CONTINLL and the method described by Hennessey and Johnson (37).


   ACKNOWLEDGMENTS
 
We thank Dr. Prema Narayan for her interest and critical reading of the manuscript and Ron Seidel for helpful instruction on the CD spectrometer.


   FOOTNOTES
 
This work was supported by NIH Grant DK-33973. G.B.F. is the recipient of an ARCS (Academic Rewards for College Scientists) scholarship.

Present address for G.B.F.: Department of Cell Biology and Medicine, Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.

Present address for B.D.: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.

Abbreviations: CD, Circular dichroism; CG, chorionic gonadotropin (choriogonadotropin); CTP, C-terminal peptide (of hCG-ß); Cys, cysteine; ECD, ectodomain; GPCR, G protein-coupled receptor; h, human; LHR, LH (lutropin) receptor; LRR, Leu-rich repeat; PVDF, polyvinylidene difluoride; TM, transmembrane; YECD, yoked (single chain) hCG-LHR ECD complex (N-hCGß-{alpha}-CTP-factor Xa site-LHR ECD-Flag-C); YhCG, yoked (single chain) hCG (N-hCGß-{alpha}-Flag-C); Y341, same as YECD (complete ECD, residues 1–341); Y294, YECD with C-terminal deletion (residues 1–294); Y180, YECD with a C-terminal deletion (residues 1–180).

Received for publication October 9, 2002. Accepted for publication March 7, 2003.


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