Yoked Complexes of Human Choriogonadotropin and the Lutropin Receptor: Evidence that Monomeric Individual Subunits Are Inactive
Prema Narayan,
Judy Gray 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. Prema Narayan, Department of Biochemistry and Molecular Biology, Life Sciences Building, University of Georgia, Athens, Georgia 30602-7229. E-mail: narayan{at}bmb.uga.edu.
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ABSTRACT
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Human choriogonadotropin (hCG) contains an
-subunit, common to other members of the glycoprotein hormone family, and a unique ß-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGß were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-ß-
-LHR-C and N-
-ß-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human
-subunit and hCGß can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGß, but not
, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGß revealed that low levels of endogenous
-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGß to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.
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INTRODUCTION
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HUMAN CHORIOGONADOTROPIN (hCG) plays a central role in human reproduction by rescuing the corpus luteum and maintaining the relatively high level of progesterone production that is required for sustaining early pregnancy. All members of the glycoprotein hormone family, including CG, LH, FSH, and TSH, are structurally related and contain two distinct subunits,
and ß. Within a given species, the
subunit is common to each glycoprotein hormone, which has a unique ß-subunit that determines receptor binding specificity (1, 2, 3). hCG binds to its cell surface receptor, the LH receptor (LHR), a member of the glycoprotein hormone receptor family, that in turn belongs to the superfamily of G protein-coupled receptors (4). Binding of hCG to LHR leads to signal transduction primarily through the cAMP pathway, although activation of phospholipase Cß can also occur with high concentrations of hCG (5, 6). LHR contains a relatively large N-terminal ectodomain (7, 8) that is characterized by a motif of imperfect leucine-rich repeats and believed to be responsible for high affinity hormone binding (9, 10, 11, 12, 13). Several models of the receptor ectodomain have been developed using molecular modeling techniques (14, 15, 16, 17); however, a thorough understanding of the three-dimensional folding pattern of the receptor ectodomain and its mechanism of hormone binding is not available.
Previous studies have demonstrated that both subunits of heterodimeric hCG contain receptor contact sites (18, 19, 20, 21, 22, 23, 24), and it has been known for some time that significant hormonal activity occurs only after association of the two distinct subunits (1). Several reports have indicated that the individual subunits are capable of receptor binding, albeit with relatively low affinities (25, 26, 27, 28), but one caveat of these observations is the possibility of contamination of the subunit preparations with intact hormone. One study, for example, noted that the limited ability of purified subunits from bovine LH to compete with 125I-LH to a rat testicular membrane fraction was essentially abolished by additional purification with immunoaffinity columns resulting in IC50s of the subunits that are at least 104-fold greater than that of LH (29).
The observation that each of the bovine LH subunits is capable of forming homodimers (30), as is the ß-subunit of human LH (31) and CG (32), raises the intriguing possibility that free subunits, either monomeric or homodimeric, may exhibit biological activity through glycoprotein hormone receptors or other receptors, e.g. those for growth factors. Interestingly, the crystal structure of hydrogen fluoride-treated hCG showed that, despite no obvious amino acid sequence homology between the
- and ßsubunits, they exhibited similar three-dimensional polypeptide folding characterized by three extended loops and a central cystine-knot motif reminiscent of certain growth factors such as nerve growth factor and platelet-derived growth factor that mediate signaling through the formation of homodimers and heterodimers (33, 34). The possibility of activity in a homodimer was tested by Lobel et al. (32), who linked two hCGß-subunits in tandem and showed that this fusion protein formed a ß-ß homodimer. This molecule was able to bind LHR and elicit a cAMP response, albeit with an apparent 1000-fold reduction in affinity compared with hCG. However, they did not determine if the hCGß monomer was also functional. The free human
(h
)-subunit has also been reported to display biological activity (35). However, this activity is distinct from the steroidogenic activity of the heterodimer mediated by LHR.
To address the question of whether the individual subunits of hCG can bind to and activate LHR, we have used two complementary approaches designed to maximize any potential interaction of the subunits with the receptor: 1) protein engineering to prepare and functionally characterize fusion proteins of yoked h
-LHR (Y
R) and yoked hCGß-LHR (YßR), obtained by linking each subunit to LHR via the hCGß carboxy-terminal peptide (CTP) and a factor Xa protease cleavage site (Fig. 1
); and 2) cotransfection of LHR with individual h
and hCGß subunits. We have previously demonstrated the feasibility of this fusion protein approach by covalently linking two single chain (yoked) hCG molecules, N-ß-
-C (36) and N-
-ß-C (37), to LHR to produce yoked hormone-receptor (YHR) complexes, N-ß-
-LHR-C (38) and N-
-ß-LHR-C (37). Human embryonic kidney (HEK) 293 and COS-7 cells expressing these single chain hormone-receptor complexes exhibited a high basal level of intracellular cAMP, indicating a stable interaction between the receptor and the covalently linked yoked hormone that resulted in constitutively active hormone-receptor complexes.
Herein we report that expression of YßR or cotransfection of LHR with hCGß in HEK 293 cells results in a high basal level of intracellular cAMP similar to that obtained with heterodimer. In contrast, expression of Y
R or cotransfection of LHR with h
did not increase basal cAMP levels. However, YßR or hCGß cotransfected with LHR into Chinese hamster ovary (CHO) or COS-7 cells demonstrated no agonist activity. Further investigation into this anomaly revealed, not previously reported and much to our surprise, that HEK 293 cells synthesize very low levels of
-subunit. Although, not readily detectable, the levels of
-subunit appear to be sufficient to form heterodimers with the transfected hCGß and activate LHR. The data obtained with CHO and COS-7 cells unequivocally demonstrate that individual subunits of hCG do not possess bioactivity even under conditions where subunit-receptor interactions are maximized and there is no possibility of contamination with intact hormone.
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RESULTS
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Apparent Activity of Subunits in HEK 293 Cells
To determine whether individual subunits of hCG can activate LHR, single chain subunit-receptor yoked constructs, Y
R and YßR, were prepared (Fig. 1
). The
-subunit was ligated to rat LHR via the 30-amino-acid residue CTP of hCGß and a Factor Xa recognition sequence. YßR was linked to LHR via its own CTP and the Factor Xa recognition sequence. The construction and characterization of YHR has been previously reported (38) and was included in these studies as a positive control and for comparative purposes. A myc-epitope tag was added between the signal sequence and the subunit sequence or yoked hormone, to place the tag at the N terminus of the mature protein. The epitope tag was used to determine cell surface expression of the transfected constructs by anti-myc antibody in cases where binding to hCG was inhibited by the presence of the covalently linked subunit or hormone and as an independent measure of receptor expression.
Myc-tagged Y
R, YßR, YHR, and LHR were transiently transfected into HEK 293 cells along with pcDNA3 as an empty vector control (mock transfected, MT). The addition of the myc tag has no effect on the bioactivity of LHR (39) or the yoked constructs (data not shown). Specific binding of 125I-hCG was detected in cells expressing LHR and Y
R, but not in cells expressing YßR, YHR, or pcDNA3. Competitive binding studies with increasing concentrations of unlabeled hCG showed that the IC50 values for LHR and Y
R were similar (Table 1
). To ensure that YßR and YHR were expressed on the cell surface, binding was performed with anti-myc antibody followed by detection with 125I-labeled secondary antibody. Compared with LHR, YßR and YHR were expressed at 76% and 63%, respectively (Table 1
). Interestingly, Y
R could not be detected by this method, suggesting that the myc-tag may not be accessible to the antibody in this construct. Therefore, although this method is useful to determine the presence of a protein, it may not be ideal for quantitative measurements of the different yoked proteins.
Consistent with the binding results, expression of YßR and YHR, but not Y
R, resulted in a dramatic stimulation (16- to 20-fold) of basal cAMP synthesis over LHR without further increase in cAMP levels after stimulation with hCG (Fig. 2A
and Table 1
). Interestingly, the basal cAMP level of YßR-expressing cells is similar to that of YHR-expressing cells, and the maximum cAMP levels achieved by these yoked receptors is approximately half that obtained by ligand stimulation of LHR. Basal cAMP levels in LHR- and Y
R-expressing cells were similar to that in MT cells and they responded well to exogenous hCG with EC50 values of 0.09 and 0.12 nM, respectively.

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Figure 2. Activity of Subunits in HEK 293 Cells
Dose-response curves of hCG-mediated increases in intracellular cAMP in HEK 293 cells transfected with yoked constructs (A) or cotransfected with LHR and subunits (B). The data shown represent means ± SEM of three independent experiments.
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Because the fusion protein of hCGß with LHR exhibited constitutive receptor activation, we then asked if the free subunits could also function as agonists. To address this question, h
and hCGß cDNAs were individually and together cotransfected with LHR. The same amount of 125I-hCG bound to cells transfected with LHR or cotransfected with LHR and free
-subunit (LHR/
), and IC50 values from competition binding were similar (Table 1
). In contrast, no binding was observed in cells transfected with LHR and free ß-subunit (LHR/ß), with LHR and free
- and ßsubunits (LHR/
/ß) or pcDNA3 (MT). Cell surface expression of LHR, determined by anti-myc antibody experiments, showed that LHR levels in LHR/
cells were similar to those in LHR but were 2- to 3-fold higher in LHR/ß and LHR/
/ß cells, indicating that the LHR was expressed on the cell surface even though hormone binding to receptor could not be detected (Table 1
). Basal cAMP levels in LHR/ß and LHR/
/ß cells were 28-fold higher than in LHR cells, and addition of hCG did not lead to further stimulation (Fig. 2B
and Table 1
). Again, the maximal cAMP levels were less than those obtained by ligand-mediated stimulation of LHR. It is interesting that the yoked constructs and LHR cotransfected with both subunits consistently and reproducibly produce lower levels of maximum cAMP compared with that obtained with LHR in the presence of a saturating dose of hCG, similar to that observed in our original report on the characterization of YHR (38). One possible explanation is that the active conformation of receptor in the yoked or cotransfected conditions is different and less responsive to increasing amounts of ligand. Basal and maximal cAMP levels, as well as EC50 values, were similar in LHR/
and LHR cells. The results from the fusion protein and the cotransfection experiments suggest that the ß-subunit by itself can activate LHR. However, as described below, further studies showed that this was not the case.
To ensure the specificity of activation of LHR by the covalently linked hCG or ß-subunit, two control constructs were constructed (Fig. 1
) and analyzed. First, an unrelated protein of similar size, bovine prolactin (PRL; 199 amino acid residues), was linked to LHR via the CTP and Factor Xa sequence (PRL-LHR). Second, the CTP/Factor Xa recognition sequence was yoked to LHR (CTP-LHR) to confirm that these sequences, which are common to all the yoked constructs, did not have any effect on LHR activation. Binding and signaling analyses of these constructs transfected into HEK 293 showed that they could not activate LHR (Fig. 3
). The yoked noncognate ligand/CTP-LHR complexes bound hCG with the same affinity as LHR and showed no increase in basal cAMP (Table 1
). The maximal levels of cAMP stimulation and EC50 values for PRL-LHR and CTP-LHR were similar to those obtained for myc-LHR.

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Figure 3. Bioactivity of LHR and the Control Constructs, YPR, and CTP-LHR, in HEK 293 Cells
A, Competitive binding assay wherein specific binding of 125I-hCG in the absence of hormone was normalized to 100%. B, Dose response of hormone-mediated increases in intracellular levels of cAMP. The data shown are means ± ranges of two independent experiments.
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Activity of Subunits in CHO and COS-7 Cells
To further confirm that hCGß by itself was an agonist, the activity of the subunits was determined by transfection of the yoked constructs and cotransfection of subunit and receptor into CHO cells. Competitive binding experiments showed that the binding affinity of hCG to cells expressing Y
R and YßR was the same as in cells expressing LHR (Fig. 4A
and Table 2
). 125I-hCG binding could not be detected with cells expressing YHR. Y
R and YßR did not show any increase in basal cAMP and responded to hCG with EC50 values similar to those exhibited by LHR (Fig. 4B
and Table 2
). However, YHR-expressing cells showed a significant increase in basal levels of cAMP without further stimulation by exogenous hCG. Cotransfection experiments showed that neither the
-subunit nor CGß by itself was capable of activating LHR. There was no significant difference in the binding affinity of hCG to LHR, LHR cotransfected with h
or hCGß (Fig. 4C
and Table 2
). No increase in basal levels of cAMP was observed, and the maximal levels obtained with a saturating dose of hCG were similar for LHR and LHR cotransfected with the individual subunits (Fig. 4D
). However, cotransfection of LHR with both hormone subunits abolished binding of 125I-hCG and resulted in elevated basal levels of cAMP indicating that the two subunits together can activate LHR. These results were surprising because they indicated that CGß by itself was not an agonist in CHO cells.

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Figure 4. Activity of Subunits in CHO Cells
Competitive binding assay and dose response of cAMP stimulation with yoked constructs are shown in panels A and B, respectively, and those with subunits cotransfected with LHR are given in panels C and D. The data presented represent means ± ranges of two independent experiments for the yoked constructs and means ± SEM of three independent experiments for the cotransfection experiments.
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To further confirm the requirement for both subunits in CHO cells, cotransfection experiments were performed with Y
R/ß and YßR/
. 125I-hCG binding in these cells was greatly reduced compared with cells expressing Y
R or YßR alone and similar to that obtained with YHR-expressing cells (Fig. 5A
). Basal levels of cAMP were elevated in the cotransfected cells compared with cells transfected with Y
R or YßR alone, and further stimulation of cAMP was not observed upon hCG addition (Fig. 5B
). The increase in basal cAMP levels was lower than that observed with YHR-expressing cells. These results suggested that the yoked subunit could form a heterodimer with the cotransfected free subunit, resulting in a functional hormone-receptor complex, and provide further evidence for the requirement of both subunits in the activation of LHR. Additionally, these results demonstrated that Y
R and YßR are not in a conformation that is inherently inactive. Interestingly, cotransfection of Y
R and YßR resulted in a 5-fold increase in basal cAMP compared with LHR, and these cells could be further stimulated with exogenous hCG, suggesting that some of the yoked receptors form homodimers or heterodimers to give fully active complexes.

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Figure 5. Cotransfection of Yoked Subunit-Receptor with Individual Subunits in CHO Cells
A, Total specific binding obtained with the different transfected constructs is expressed as a percent of LHR binding that was normalized to 100%. B, Intracellular levels of cAMP measured in the absence and presence of a saturating dose (200 ng/ml) of hCG. The data presented represent means ± ranges of two independent experiments.
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COS-7 cells were also transfected with the yoked constructs or cotransfected with LHR and the subunits. Similar to observations in CHO cells, 125I-hCG binding was readily detectable in COS-7 cells expressing Y
R and YßR compared with barely detectable levels in cells expressing YHR (Fig. 6A
). Y
R and YßR did not produce an increase in the basal levels of cAMP and responded to exogenous hCG to the same extent as LHR (Fig. 6B
). On the other hand, YHR produced a significant increase in basal cAMP and was unresponsive to further stimulation by exogenous hCG. Cotransfection of LHR with
only or CGß only did not show agonist activity of the subunits, whereas cotransfection of h
and hCGß subunits with LHR resulted in a 22-fold increase in basal cAMP.

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Figure 6. Activity of Subunits in COS-7 Cells
A, Total specific binding obtained with the different transfected constructs is expressed as a percent of LHR binding that was normalized to 100%. B, Intracellular levels of cAMP measured in the absence and presence of a saturating dose (200 ng/ml) of hCG. The data presented represent means ± ranges of two independent experiments.
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Further Characterization of Subunit Activity in HEK 293 Cells
To determine the reason for the selective activity of CGß in HEK 293 cells, additional experiments were performed in these cells. One possibility considered was that the HEK 293 cells expressed the
-subunit endogenously and this could associate with the transfected CGß to produce a heterodimer. To test this hypothesis, media from cells transfected with the yoked constructs were concentrated and equal amounts were subjected to Western blot analysis with an
-subunit-specific antiserum. The
-subunit was readily detected in media from cells transfected with YßR and YHR but not from cells transfected with vector alone (MT), LHR, or Y
R (data not shown). Media from cells transfected with only LHR or the subunits, or cotransfected with LHR and subunits, were analyzed on gels under reducing and nonreducing conditions. As expected, media from cells transfected with the
-subunit, cotransfected with
-subunit and LHR (LHR/
), or cotransfected with LHR and both subunits (LHR/
/CGß) but not with empty vector or LHR alone showed the presence of a band of the correct molecular size of the
-subunit (Fig. 7A
). Media from cells expressing CGß did not show the presence of the
-subunit. However, media from cells cotransfected with LHR and CGß (LHR/CGß) contained the
-subunit. Therefore, it appeared that LHR and CGß were both required for endogenous
-subunit expression in HEK 293 cells, and either alone did not generate easily detectable levels of
-subunit in the media. Based on the intensity of the
-subunit-specific bands, the amount of endogenous h
produced is considerably greater than that from the expression of the transfected h
-subunit cDNA. When media samples were probed with a hCGß-specific antiserum, specific bands were only detected in media from cells transfected with CGß, LHR/CGß, or LHR/
/ß cDNAs (data not shown).

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Figure 7. Western Blot Analysis of Media Samples from HEK 293 Cells Cotransfected with LHR and Subunits
Equal volumes of concentrated media samples were subjected to denaturing PAGE under reducing (A) or nonreducing conditions (B). The blots were probed with a polyclonal antiserum specific for the h -subunit. The sizes of the molecular mass standards are indicated. Highly purified urinary hCG was included as a control.
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To determine whether the secreted
-subunit could form a heterodimer with CGß, the media samples were analyzed under nonreducing conditions (Fig. 7B
). Bands corresponding to the size of the expected heterodimer (compared with hCG control) were detected only in media from LHR/ß and LHR/
/ß cells. The presence of free
-subunit in these samples suggests that the endogenous
-subunit is in great excess over the transfected CGß-subunit or that only a fraction is capable of associating with CGß.
Because both LHR and CGß, accompanied by an increase in intracellular cAMP, appeared to be required for endogenous
-subunit expression, we asked if cAMP was sufficient for endogenous
subunit synthesis. For this purpose, HEK 293 cells were transfected with a constitutively active LHR mutant, D556H, that stimulates cAMP synthesis in the absence of ligand. Additionally, MT cells were treated with 100 µM forskolin, a stimulator of adenylyl cyclase. When media from these cells were analyzed by Western blot analysis, the
-subunit was readily detected in the media, and intracellular levels of cAMP were greatly elevated compared with MT cells (Fig. 8
, A and B). Furthermore, in contrast to cotransfection of hCG with LHR, cotransfection of human FSHß and human TSHß with LHR did not result in an increase in basal levels of intracellular cAMP (Fig. 8D
) and, therefore, no production of
-subunit in the media (Fig. 8C
). However, addition of hCG activated LHR in these cells as judged by an increase in cAMP. These data suggest that cAMP is sufficient and required for synthesis of high levels of endogenous
-subunit.
To test the possibility that low levels of the
-subunit were constitutively synthesized in HEK 293 cells, equal numbers of HEK 293 cells were transfected with empty vector (MT),
-subunit, CGß, or the two subunits (
/ß) together. Media from these cells were concentrated approximately 200-fold and then analyzed by RIAs specific for the free
-subunit, CGß (free and heterodimer), or only heterodimer. The
-subunit-specific RIA measured very low levels of
-subunit in media from MT, CGß, and
/ß transfected cells, ranging from approximately 0.81 ng/ml of media before concentration, compared with 30 ng/ml in cells transfected with the
-subunit. In contrast,
-subunit could not be detected in MT media from CHO cells. Western blot analysis of concentrated media containing equivalent amounts of
-subunit (5 ng as determined by RIA) from each of these transfections further confirmed the presence of the
-subunit (data not shown).
To determine whether the low levels of
-subunit present in HEK 293 cells could form an active heterodimer with the transfected CGß, HEK 293 cell media containing high concentrations (1 µg/ml) of CGß were used in bioassays with CHO cells expressing LHR. A 3-fold increase in intracellular levels of cAMP compared with basal level was noted. In comparison, media from cells expressing the
-subunit (1 µg/ml) did not cause any increase in cAMP, whereas media from
/ß cotransfection containing 5 ng/ml of heterodimer led to a 5-fold increase in cAMP levels over basal values (data not shown). Furthermore, media from transfected CHO cells containing CGß at 1 µg/ml did not result in any increase in LHR-mediated intracellular cAMP levels. Taken together, these results suggest that the low levels of endogenous
-subunit present in HEK 293 cells can form an active heterodimer with transfected CGß in sufficient concentrations to activate LHR.
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DISCUSSION
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It has been the long-held dogma that association of the two subunits of the heterodimeric glycoprotein hormones are required for hormonal activity (1). Using site-directed mutagenesis, several laboratories have identified amino acid residues on both subunits to be important in receptor binding (18, 19, 20, 21, 22, 23, 24). However, there are no structural data on the hCG-LHR complex, nor is there an understanding by which ligand binding leads to LHR activation. More recently, single chain proteins generated by covalently linking the
- and ß-subunits of the glycoprotein hormones have been reported to be biologically active (36, 40, 41, 42, 43). Variants of these single chain analogs in which the disulfide bonds of the cystine knot motif have been disrupted or additional intersubunit disulfide bonds introduced are still functional (44, 45, 46, 47). Furthermore, gonadotropins in which hCGß and FSHß are covalently linked to a single
-subunit show dual activity by binding and activating both LHR and FSHR (48, 49). The most significant conclusion to emerge from the studies of the single chain and heterodimeric analogs (50) is that the nonnative interactions between the
- and ß-subunits can produce hormones with different conformations that can recognize and activate the receptor. Further support for this conclusion is provided by the observation that single chain hCG ß-ß homodimers can bind and activate LHR, albeit with an approximately 1000-fold lower affinity than the heterodimer (32).
Based on these recent changes in our understanding of gonadotropin hormone-receptor interactions, it seemed pertinent to reexamine the question of whether both subunits were obligatory for hormone activity. Our study addressed the ability of h
- and hCGß-subunits to activate LHR. Because the affinity of each subunit to LHR would be predicted to be low, the two experimental approaches that we employed to test subunit activity were intended to maximize subunit-receptor interactions. Fusion proteins such as YHR, YßR, and Y
R presumably facilitate this process by shifting the equilibrium of binding, subunit + LHR
subunit-LHR complex, to the right. Cotransfection of subunit and receptor would potentially allow for subunit and receptor to interact as they are being synthesized in the endoplasmic reticulum and then trafficked to the plasma membrane as a subunit-receptor complex.
Our initial finding that hCGß, but not h
, could activate LHR in HEK 293 cells but not in CHO or COS-7 cells was surprising. HEK 293 cells are widely used in structure-function studies of gonadotropins and their receptors, as are CHO and COS-7 cells, and the conclusions rarely, if at all, change with cell type. In fact, YHR shows the same activity in HEK 293 and COS-7 cells (38). Therefore, we did not expect to obtain such disparate results in the different cell types. Our finding that low levels of
-subunit are constitutively synthesized in HEK 293 cells and are capable of forming an active heterodimer (albeit at low concentration) with transfected CGß provided an explanation for this discrepancy. If LHR is also expressed in these cells, as in LHR/ß or YßR transfections, then the low levels of heterodimer can presumably activate the receptor and produce sufficient amounts of cAMP to activate the endogenous
-subunit promoter leading to the synthesis of large amounts of
-subunit. This would, in turn, lead to sufficiently high levels of heterodimer to fully activate the receptor. To our knowledge, there are no reports in the literature describing the presence of the
-subunit in HEK 293 cells. When these studies were mostly completed, however, we became aware that
-subunit mRNA had been detected in untransfected HEK 293 cells by PCR (Keutmann, H. T., Massachusetts General Hospital, Boston, MA; personal communication).
This study addressing the ability of individual subunits of the hCG heterodimer to activate LHR has demonstrated that both subunits are required for functional activity of the hormone. Because a single chain hCG ß-ß homodimer displays low but demonstrable receptor activity (32) and our studies only examined the activity of monomeric form of the subunits, it is possible that some combination of homodimeric structure (either
-
or ß-ß) may mimic an
-ß heterodimer and lead to receptor binding and activation. The fact that the
and CGß-subunits have similar three-dimensional folding in spite of little sequence similarity (33, 34), coupled with the flexibility in the quartenary structure of the hormone as discussed above, suggests that two identical subunits could associate to form active homodimers. Bovine and human LHß, as well as hCGß, can form homodimers in solution (30, 31, 32); however, their receptor activity has not been directly tested. It is known that some tumor cells, e.g. choriocarcinoma, secrete high levels of hCGß, and it is believed that these cells depend of CGß for growth suggesting that a homodimeric form of hCGß may act as an autocrine growth factor in these tumor cells.
The observation that cotransfection of Y
R and YßR in CHO cells can result in an increase in the basal levels of cAMP suggests that at least a fraction of the yoked receptors are capable of forming an active dimer. Although our data so far suggest that this dimer most likely consists of Y
R and YßR, possibly formed during biosynthesis in the endoplasmic reticulum, we cannot formally rule out the possibility that two Y
R or YßR molecules associate to form a functional dimer. It also remains to be determined if both receptors can transduce the signal or if only one in a pair is active. Recent studies have shown that hCG bound to one LHR molecule can transactivate an adjacent receptor that is defective in hormone binding (51, 52).
Lastly, the specificity of the hormone-receptor interaction in the yoked constructs has been demonstrated by our control constructs, YPR and CTP-LHR. PRL, an unrelated protein of similar size to hCG, neither binds to nor activates LHR in spite of being covalently linked. CTP-LHR is also inactive, demonstrating that this linker, used extensively in our yoked constructs, does not contribute to the bioactivity of YHR.
In summary, our detailed study has established that individual subunits of hCG, at least in their monomeric form, are unable to bind to and activate LHR. Although we cannot rule out the possibility that homodimer formation occurs between the subunits when yoked to LHR, our results indicate that if it does occur, it does not lead to receptor activation.
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MATERIALS AND METHODS
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Materials
Highly purified urinary hCG was kindly provided by Dr. A. Parlow and NIDDKs National Hormone and Pituitary Program. 125I-hCG and the cAMP kit were purchased from Perkin-Elmer Life Sciences(Boston, MA). DMEM was obtained from Mediatech, Inc. (Herndon, VA). Serum, Waymouths MB752/1 medium, antibiotics, Lipofectamine, and anti-myc antibody were purchased from Invitrogen Life Technologies(Carlsbad, CA). Free
-subunit and hCG RIA kits, and hCG IRMA kits were procured from Biomerica (Newport Beach, CA), ICN Biomedicals, Inc.(Costa Mesa, CA), and Diagnostic Products Corp.(Los Angeles, CA), respectively. Biomax-10 ultrafiltration membranes and Immobolin P were products of Millipore Corp.(Bedford, MA). The ECL detection system was purchased from Amersham Pharmacia Biotech(Piscataway, NJ). The anti-
-subunit and anti-CTP antisera were kindly provided by Drs. Irving Boime and Vernon Stevens, respectively.
Construction of Expression Vectors
Myc-tagged Y
R, YßR, and YHR were generated by standard PCR amplification and subcloning methods. The CTP sequence [amino acid residues 116145 of hCGß and the Factor Xa protease recognition sequence (IEGR) were used to link each subunit to rat LHR and the myc-tag (EQKLISEEDL)] was inserted between the signal sequence and the beginning of the mature protein. Two control constructs were also prepared. Bovine PRL cDNA, including its signal sequence, was ligated to rat LHR via the CTP sequence to generate YPR, and the CTP sequence, with an N-terminal hCGß signal sequence, was also ligated to LHR (CTP-LHR). The clones were verified by sequencing and their schematic representations are shown in Fig. 1
. Myc-LHR cDNA was kindly provided by Dr. Mario Ascoli. All receptor and h
- and hCGß-subunit cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen Life Technologies).
Cells and Transient Transfections
HEK 293 cells were maintained at 5% CO2 in high-glucose DMEM supplemented with 10% (vol/vol) newborn calf serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.125 µg/ml amphotericin. CHO K1 and COS-7 cells were maintained in high-glucose DMEM supplemented with 10% FBS and antibiotics as described above. Cells were transfected with empty vector, yoked constructs, or cotransfected with individual
- or CGß-subunits and LHR using Lipofectamine as the transfection agent. For cotransfection experiments, empty vector DNA was used when necessary to keep the total amount of DNA being transfected constant. After 24 h, transfected cells were plated onto 12-well plates and used for experiments 48 h after transfection. HEK 293 cells were plated onto wells that had been precoated for 1 h with 0.1% gelatin in calcium- and magnesium-free PBS, pH 7.4.
Hormone Binding Assays
Binding assays were performed essentially as described previously (37). Cells plated in 12-well plates were incubated with 50100 pM of 125I-hCG and various concentrations of unlabeled hCG overnight at room temperature. Nonspecific binding was measured in the presence of 1 µg/ml hCG, and the resulting specific binding obtained in the absence of competitor was normalized to 100%. The competition curves were plotted as a percentage of the specific binding vs. increasing concentrations of urinary hCG on a log scale. The data were fitted to a sigmoidal equation to calculate IC50 values using the Prism software (GraphPad Software, Inc., San Diego, CA).
cAMP Assays
Cells in 12-well plates were washed twice with Waymouths MB752/1 medium containing 0.1% BSA and then incubated in the same medium containing 0.8 mM isobutylmethylxanthine for 15 min at 37 C. Increasing concentrations of hCG were added, and incubation was continued for 30 min at 37 C. The incubation medium was removed and cells were lysed at -20 C overnight. The ethanol supernatants were collected, dried, and dissolved in the assay buffer of the 125I-cAMP RIA kit that was used to determine intracellular cAMP concentrations. Cells treated with empty vector were treated with 100 µM forskolin for 18 h at 37 C.
Detection of Hormone Subunits in Transfected Cell Media
At 24 h post transfection, serum-containing medium was replaced with serum-free medium, and media were collected the following day. The media from the different transfections were concentrated to the same extent using Biomax-10 ultrafiltration membranes, and the concentration of free
- or CGß-subunits were measured, respectively, by a free
-subunit-specific RIA and an hCGß RIA that detects free subunit and heterodimer. Heterodimer concentrations were measured by the hCGß RIA or the hCG IRMA. All RIAs were performed according to the manufacturers protocols. Subunits were also detected by Western blot analysis. Media samples were electrophoresed on 16.5% sodium dodecyl sulfate-polyacrylamide gels under reducing (with 0.25 M ß-mercaptoethanol and heating) or nonreducing (without either ß-mercaptoethanol or heating) conditions and transferred to Immobilon P. The blots were probed with rabbit anti-
-subunit antiserum or rabbit anti-hCGß antiserum raised against amino acid residues 109145 of hCGß (anti-CTP) followed by horseradish peroxidase-labeled donkey antirabbit IgG. The blots were developed using the ECL detection system according to the manufacturers directions.
Detection of Receptor on the Cell Surface
Cells transfected with the myc-tagged constructs were transferred onto 12-well plates as described above. Cells were washed twice with Waymouths MB752/1 medium containing 0.1% BSA and incubated in duplicate with anti-myc antibody (1 µg/ml) in the same buffer for 4 h at room temperature. Nonspecific binding was determined by adding an excess of myc-peptide at a concentration of 100 µg/ml. Cells were washed twice and then incubated with 125I-rabbit antimouse IgG (
4 x 105 cpm/well) for 2 h at room temperature. The secondary antibody was removed, the cells were washed three times with PBS and then lysed with 1 N NaOH. The cell lysate was transferred to tubes, the wells rinsed with PBS and pooled, and tubes were counted in a
-counter.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Irving Boime for his generous gift of human
-subunit antiserum and for helpful suggestions, Dr. Vernon Stevens for the anti-CTP antiserum, Dr. Fritz Rottman for providing the bovine PRL cDNA, and Dr. Mario Ascoli for the myc-LHR cDNA. The contributions of Dr. Chengbin Wu during the early stages of this work are gratefully acknowledged. We thank Dr. Krassimira Angelova for comments on the manuscript.
 |
FOOTNOTES
|
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This work was supported by NIH Research Grant DK-33973.
Abbreviations: CHO, Chinese hamster ovary; CTP, carboxy-terminal peptide; h
, free human
; hCG, human choriogonadotropin; HEK, human embryonic kidney; LHR, LH receptor; MT, mock transfected; PRL, prolactin; Y
R, yoked h
-LHR; YßR, yoked hCGß-LHR; YHR, yoked hormone-receptor.
Received for publication June 7, 2002.
Accepted for publication September 3, 2002.
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