Cis- and Trans-Activation of Hormone Receptors: the LH Receptor
Inhae Ji,
ChangWoo Lee,
YongSang Song,
P. Michael Conn and
Tae H. Ji
Department of Chemistry (I.J., C.L., Y.S., T.H.J.), University of Kentucky, Lexington, Kentucky 40506-0055; and Oregon Regional Primate Research Center and Department of Physiology (P.M.C.), Oregon Health Science University, Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. Tae H. Ji, Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055. E-mail: tji{at}uky.edu.
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ABSTRACT
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G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. Although initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases that these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the LH receptor, a GPCR that does not form stable dimers. The LH receptor consists of a 350-amino acid amino-terminal domain, which is responsible for high-affinity binding to human CG, followed by seven-transmembrane domains and connecting loops. This seven-transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.
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INTRODUCTION
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G PROTEIN-COUPLED receptors (GPCRs) constitute the major portion of the human genome, and nearly 2000 of them have been cloned (1). Among the receptors, rhodopsins and the adrenergic receptors have been most extensively investigated and defined. In this receptor subfamily, the ligands bind to the transmembrane bundle where hormone signals are generated (1, 2). Therefore, the ligand-binding site is generally thought to be the site for the signal generation, and a liganded receptor generates a hormone signal intramolecularly (1, 2). However, there are other GPCRs, which have distinct sites for hormone binding and signal generation (Fig. 1
). For example, the family of glycoprotein hormone receptors and the receptors for neurotransmitters, such as Ca2+, metabotropic glutamate, and
-aminobutyric acid, bind their hormones at the N-terminal extracellular exo-domain, whereas the hormone signals are generated in the membrane-associated endo-domain (1). In this case, the liganded exo-domain modulates the endo-domain to generate a hormone signal (Fig. 1B
). Therefore, intramolecular signal generation has been presumed. Furthermore, when some neurotransmitter receptors form a disulfide-linked dimer (3, 4), the dimeric receptor complex binds two ligand molecules. This full occupation of both ligand-binding sites in a receptor dimer is thought to generate a signal within the dimeric complex (3). It is unclear, however, whether only one or both of the two endo-domains in the dimeric complex need to be activated for signal generation. Of particular interest is the case in which only one of the two exo-domains is liganded. It is unclear whether the liganded exo-domain is capable of intramolecularly activating its cognate endo-domain (cis-activation) and/or intermolecularly activating the endo-domain of the unliganded receptor (trans-activation) (Fig. 1C
). These are fundamental questions of far reaching implications on the understanding of signal generation and human diseases and disorders.

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Figure 1. Hypothetical Models of Monomeric and Dimeric Cis-Activation and Trans-Activation
A, Domain structure of LHR showing the exo-domain where the ligand binds and the endo-domain where the hormone signal is generated. A putative disulfide linkage between exoloops 1 and 3 in the endo-domain is shown as a black bar. B, Cis- and trans-activation of monomeric LHRs. The ligand is shown in red. C, Cis- and trans-activation of putative dimeric LHRs. D1, Normal parts are shown in gray and defective parts are shown in blue. Trans-activation of a mutant LHR that is defective in ligand binding in the exo-domain shown in blue but is capable of generating a hormone signal in the endo-domain shown in gray (LHR-hCG/+cAMP) by a mutant LHR that is capable of binding ligand in the exo-domain shown in gray but incapable of generating a hormone signal in the endo-domain shown in blue (LHR+hCG/-cAMP). D2, Trans-activation of a mutant LHR that is defective in ligand binding in the exo-domain shown in blue but is capable of generating a hormone signal in the endo-domain shown in gray (LHR-hCG/+cAMP) by a chimera with a functional LHR exo-domain shown in gray linked to the blue nonfunctional CD 8 transmembrane and cytoplasmic domain (ExoCD).
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Regardless of monomeric or dimeric receptors, the central question is whether a liganded exo-domain of a receptor is capable of trans-activating the endo-domain of other unliganded receptors, in addition to cis-activation of its cognate endo-domain. The test of this challenging hypothesis requires a receptor system that has a clearly defined hormone-binding site, which is physically distinct from the signal generation site. LH/CG receptor (LHR) offers such a system. It is a GPCR with a unique structure and activation mechanism (1). It consists of two halves of approximately 350 amino acids each, an extracellular N-terminal exo-domain and a membrane-associated C-terminal endo-domain (5, 6) as shown in Fig. 1A
. The exo-domain, alone, is capable of high-affinity hormone binding (7, 8, 9) with hormone selectivity (10, 11, 12) but without hormone action (9, 13, 14). Hormone signal is generated in the endo-domain (15), which is structurally equivalent to the entire molecule of other GPCRs such as the rhodopsins and adrenergic receptors (1). Human CG (hCG) initially binds to the exo-domain, and the resulting hormone/exo-domain complex undergoes a conformational change and modulates the endo-domain to activate adenylyl cyclase (1, 15, 16). Therefore, mutations in the exo-domain tend to affect hormone binding (17, 18), whereas signal generation is usually impaired by mutations in the endo-domain (19, 20).
Among the hundreds of mutant LHRs generated in our laboratory during the past decade, some are defective in hormone binding (LHR-hCG), whereas others can bind the hormone but cannot induce cAMP production (LHR+hCG/-cAMP). A breakthrough has been made when some pairs of the two types of mutants were coexpressed in cells and, thus, rescued cAMP induction (Fig. 1D1
).
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RESULTS
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The bulk of the LHR exo-domain comprises the leucine-rich repeats (LRRs) (5, 21), which are involved in binding hCG (17, 22, 23). Some mutations in the LRRs, Ile55 to Ala (I55A) of LRR2, I80A of LRR3, and I105A of LRR4, impair hormone binding (24, 25). On the other hand, Lys583 at the boundary of exoloop 3 and transmembrane domain 7 in the endo-domain is essential for cAMP induction. The K583R mutation abrogates cAMP induction without impacting hormone binding and surface expression (26). The activities of these mutants are shown in Fig. 2
.

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Figure 2. Rescue of cAMP Production by Coexpression of LHR-hCG and LHR+hCG/-cAMP
HEK 293 cells were transfected with LHR-hCG plasmids, LHR+hCG/-cAMP plasmid, or both. Cells were assayed for 125I-hCG binding in the presence of increasing concentrations of nonradioactive hCG (A). The results were analyzed by Scatchard plot to determine the Kd values as described in Materials and Methods. In addition, intact cells were treated with increasing concentrations of unlabeled hCG and intracellular cAMP was measured (B). Experiments were repeated four to six times in duplicate with P values of <0.05.
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Rescue of cAMP Induction
When 293 cells were cotransfected with the LHRK583R and LHRI55A plasmids, the cells bound hCG with the same high affinity as the wild-type binding as shown by the similar dissociation constant (Kd) values (Fig. 2
). The cells also produced cAMP in an hCG dose dependent manner. The maximum cAMP level of LHRK583R and LHRI55A was approximately 43-fold higher than the basal cAMP level and 33% of the wild type maximum cAMP level. These are significant, but the EC50 value for cAMP was 4-fold higher than the wild-type value, indicating the higher hCG concentration needed to induce the maximum cAMP level for LHRK583R and LHRI55A as compared with the wild-type LHR. The cells cotransfected with the LHRK583R and LHRI80A plasmids bound hCG and responded to produce cAMP. However, the cells cotransfected with the LHRK583R and LHRI105A plasmids bound hCG but did not produce cAMP. These results suggest a specificity of the pairing. To test whether the rescue of cAMP induction was caused by changes in the G protein and/or adenylyl cyclase activity, the cells were treated with cholera toxin, which activates Gs
, leading to the stimulation of adenylyl cyclase and cAMP production. All of the cells expressing the wild-type LHR, LHRK583R, LHRI55A, LHRI80A, LHRI103A, LHRK583R/I55A, LHR K583R/I80A, or LHR K583R/I103A produced similar amounts of cAMP (data not shown). This result indicates no significant changes in G protein and adenylyl cyclase in the cells, regardless of cotransfection.
Surface Expression of Nonbinding Receptors
Before determining the pairing specificity, it was necessary to demonstrate that the nonbinding receptors, LHRI55A, LHRI80A, and LHRI105A, were indeed expressed on the surface of the cotransfected cells. To detect nonbinding receptors, the Flag epitope was attached to the N terminus of mature receptors and assayed for binding of 125I-anti-Flag monoclonal antibody as described previously (24, 27) and also in Materials and Methods. Cells were cotransfected with the LHRK583R plasmid and one of the Flag-LHRI55A, Flag-LHRI80A, and Flag-LHRI105A plasmids. The antibody showed significant specific binding to all of the cells, indicating the surface expression of Flag-LHRI55A, Flag-LHRI80A, and Flag-LHRI105A (Table 1
). These results are consistent with the previous report showing that Flag-LHRI55A, Flag-LHRI80A, and FlagLHRI105A are expressed on the cell surface without coexpression of LHRK583R (24). Next, we tested how these Flag-LHR mutants coexpressed with LHRK583R respond to hCG. The cells cotransfected with LHRK583R and either Flag-LHRI55A or Flag-LHRI80A produced cAMP in response to hCG (Fig. 3
) as did the corresponding non-Flag mutants (Fig. 2
). In contrast, the cells transfected without LHRK583R did not respond. Also, the cells cotransfected with Flag-LHRI105A and LHRK583R bound hCG but did not produce cAMP. These results indicate that LHRI55A and LHRI80A, with or without the Flag epitope, were expressed on the cell surface and capable of inducing cAMP production in the presence of coexpressed LHRK583R.

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Figure 3. cAMP Induction by Flag-LHR Mutants
HEK 293 cells expressing Flag-LHR mutants only or Flag-LHR mutants with LHRK593R were treated with increasing concentrations of hCG and assayed for intracellular cAMP induction as described in the legend of Fig. 2 . The maximum cAMP induction levels were presented as the percentage of the wild-type maximum cAMP level.
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In these experiments transiently transfected cells were used. However, stably coexpressed cell lines also responded to hCG and produced cAMP with the similar pairing specificity and cAMP induction levels (data not included). The results were similar when CHO cells and Cos 7 cells were cotransfected with the plasmid pair of LHRK583R and LHRI55A or LHRK583R and LHRI80A (data not shown). The data show that the cAMP rescue is a general phenomenon, independent of the cell types. To avoid variations in receptor concentrations, batches of transfected cells expressing 8,00015,000 of each receptor species per cell were used. Furthermore, cells expressing more than 100,000 receptors/cell showed the rescue of cAMP induction.
Specificity of Pairing LHR+hCG/-cAMP and LHR-hCG
A simple explanation for our observations is that hCG bound to the exo-domain of the LHR+hCG/-cAMP and the resulting hCG/exo-domain complex interacted with and activated the endo-domain of the LHR-hCG. To test this possibility we generated receptors with double mutations, K583R and I55A or K583R and I80A. The cells transfected with either of the double-mutant plasmids were incapable of binding hormone or producing cAMP in response to hCG (data not shown). Furthermore, cAMP was not induced from the following coexpressed pairs of the double mutants: LHRK583R with LHRK583R-I55A or LHRK583R-I80A, LHRI55A with LHRK583R-I55A or LHRK583R-I80A, and LHRI80A with LHRK583R-I55A or LHRK583-I80A. These results are consistent with the intermolecular interaction of the liganded exo-domain of LHR+hCG/-cAMP with the endo-domain of unliganded LHR-hCG and the subsequent activation of the unliganded LHR-hCG. In addition, cAMP rescue by coexpression of LHRK583R with LHRK583R/I55A or LHRK583R/I80A likely requires an LHR+hCG and an LHR-cAMP. Because the gonadotropin receptors act as monomers as shown by chemical cross-linking, gel permeation, and immunological and surface plasmon resonance studies (28, 29, 30), the results suggest transient interactions between the receptor pairs.
Activation by the Hybrid of the LHR Exodomain Attached to the CD 8 Endodomain
Next, we raised the intriguing question whether an LHR exo-domain+hCG not connected to the endo-domain could activate an LHR-hCG. We approached the problem using a hybrid of the LHR exo-domain attached to the transmembrane domain of CD 8 (ExoCD) that lacks the extracellular domain of CD 8 (Fig. 1D2
), which was generated by Hsueh and associates (14). ExoCD was expressed on the cell surface and bound hCG, but was incapable of inducing cAMP, as shown in Fig. 4
and reported previously (14). Coexpressed ExoCD with LHRI55A or LHRI80A bound hCG and induced cAMP production, but coexpressed ExoCD with LHRI105A, LHRK583R/I55A or LHRK583/I80A did not. All of these cells produced similar levels of cAMP in response to cholera toxin, indicating the integrity of adenylyl cyclase and the G proteins. It is clear that the exo-domain does not have to be attached to the endo-domain for hCG binding and activation of the endo-domain. In addition, the interaction of the seven-transmembrane domains is not necessary for rescue.

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Figure 4. Rescue of cAMP Induction by the ExoCD Chimeras
The LHR exo-domain was linked to the transmembrane and cytoplasmic domain of CD 8. The resulting chimera (ExoCD) was coexpressed with LHR-hCG mutants and assayed for hCG binding and cAMP induction as described in the legend of Fig. 2 .
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More Effective Intermolecular (Trans-) Activation than Intramolecular (Cis-) Activation in Some Mutants
In addition to the nonbinding mutant LHRs, there are mutants that marginally bind the hormone with low binding affinity and induce low cAMP production. For example, LHRL29A and LHRL53A bind hCG with an affinity nearly 10-fold lower than the wild-type affinity (Fig. 5
). They produce cAMP, but the amount is so small that it can be detected only when hCG is highly potent, the background is low, and transfected cells are healthy. The cells cotransfected with LHRK583R and either LHRL29A or LHRI53A produced significantly higher amounts of cAMP in an hCG dose-dependent manner than did the cells transfected only with LHRL29A or LHRI53A. Furthermore, cotransfected ExoCD also enhanced the hCG-dependent cAMP production by LHRL29A or LHRI53A. These results suggest that an active exo-domain of an LHR can more effectively and intermolecularly trans-activate the endo-domain of another LHR with low binding activity than the defective exo-domain of the mutant could intramolecularly cis-activate its cognate endo-domain. In addition, the results show that an exo-domain complexed with hCG can activate the endo-domain of another LHR independent of its hormone binding capacity.

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Figure 5. Rescue of Partially Active LHR Mutants by LHRK583R or ExoCD
L29A of LRR1, I53A and I55A of LRR2, I80A of LRR3, L103A and I105A of LRR4, L179A of LRR7, and L202A of LRR8 are partially or totally impaired in hCG binding (24 ). They were coexpressed with either LHRK583R (A and B) or with ExoCD (C and D), and assayed for maximum cAMP induction in response to hCG as described in the legend to Fig. 2 . Some of the results are presented in panels AD and the summary is in panel E.
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A Correlation of the Rescue and the Location of the Nonbinding Mutation
We have seen that some, but not all, nonbinding mutants could be rescued. To better define these two groups, LRR mutants with low or nonbinding activity were screened for rescue. Figure 5E
shows that the receptors with mutations of L29A, I53A, I55A, and I80A in LRRs 13 were effectively rescued but those with mutations of L103A, I105A, L179A, and L202A at the latter LRRs were not. A simple explanation for these results and the data from Fig. 5
, AD, is that the exo-domains with no mutation or mutations in LRRs 13 allow an active exo-domain of another receptor to intermolecularly interact and activate the endo-domain. In contrast, mutations in the LRRs 48 prevent intermolecular activation. LHRs are generally more effective, although slightly so, than the corresponding Flag-LHRs. The flag epitope might have caused the differences. It is also possible that our more extensive experience in handling cells expressing LHRs than those expressing Flag-LHRs led to better results for LHRs. Undoubtedly, we have not reached the technically mature stage in transfection and expression of molecules in eukaryotic cells.
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DISCUSSION
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Our results show that a liganded LHR exo-domain can trans-activate the endo-domain of other unliganded LHRs (Fig. 1
). Interestingly, trans-activation differs from cis-activation with respect to the maximum cAMP induction and EC50 values. They raise the inevitable question concerning the relationship of cis activation and trans-activation not only for LHR and other GPCRs but also for those hormone receptors with a single-transmembrane domain such as growth factor receptors.
It has been an enigma how a hormone receptor, e.g. LHR, can generate two or more signals (1) capable of activating two enzymes, adenylyl cyclase and PLC, and to generate two distinct signal pathways. Particularly, it is unclear whether one receptor molecule can generate only one signal at a time and thus, many receptor molecules are needed to generate multiple signals. This could be accomplished when a liganded receptor molecule activates one G protein molecule at a time, which is consistent with the current understanding on the interactions of GPCR. For example, these receptors interact with a variety of cytoplasmic signal molecules (31, 32), yet have limited contact points for G proteins (33). On the other hand, if a receptor molecule generates multiple signals at the same time or sequentially, it is puzzling how one receptor molecule could activate two or more G proteins at the same time or sequentially. The situation is more confusing if desensitization is considered. Trans-activation offers a mechanism for a liganded hormone receptor to cis-activate itself and generate a signal. Subsequently, it could trans-activate other receptor molecules for multiple signal generation, yet each receptor interacting with only one G protein to generate a signal at a time. This mechanism would allow one liganded receptor to generate multiple signals without a receptor simultaneously interacting with multiple G proteins. It could modulate receptor desensitization (32) and phosphorylation (34). It would be interesting to see whether activation of PLC is simultaneously and equally rescued along with activation of adenylyl cyclase. This question is of particular interest because activation of the two effectors is biphasic, requiring different hCG concentrations.
Our unpublished observations show the importance of the ratio of LHR+hCG/-cAMP and LHR-hCG for cAMP rescue. This is not surprising because trans-activation is likely dependent on the ratio of LHR+hCG/-cAMP and LHR-hCG, because too many LHR+hCG/-cAMP could jam LHR-hCG, thus becoming unproductive. This is reminiscent of an optimal ratio of antigen and antibody for successful antibody reaction. Secreted exo-domain complexed with hCG was unable to induce cAMP production by the truncated endo-domain lacking the exo-domain (14). It is unclear whether the failed cAMP rescue was caused by some folding problems with the secreted exo-domain and truncated endo-domain, disconnection between the two domains, undesirable concentration ratio of the domains, or other factors.
Trans-activation appears to occur regardless of the hormone-binding ability of the unliganded receptor, because the rescued, unliganded LHRs are either incapable or partially capable of hormone binding. For example, LHRL29A and LHRI53A are partially active and can more effectively induce cAMP production by trans-activation than by cis-activation. The efficiency of trans-activation varies depending on the nature of mutations in LHR-hCG: for example, the location of the mutation and the substituting amino acids. Receptors with the mutations in the upstream LRRs can be trans-activated, but those with mutations in the downstream LRRs could not be. In particular, the receptors with a mutation near the hinge region and therefore close to the endo-domain could not be trans-activated. A simple explanation is that the liganded exo-domain of a receptor may have difficulty in seeking out such mutated sites and replacing them. It is also possible that the mutations near the hinge region may be irreplaceable due to the crucial role of the hinge region in modulating the signal generation. Another possibility is that the mutations close to the hinge region constrain the flexibility of the junction between the exo-domain and endo-domain.
The hinge region contains a suppressor (35), P254S255, which interacts with the exo-domain 2 of LHR (36, 37) and constrains signal generation in the endo-domain (36, 38). Apparently, hormone binding relieves the constraint as part of signal generation (35). Therefore, mutations close to the suppressor may interfere with the relief of the constraint by the liganded exo-domain of LHR+hCG/-cAMP. In contrast to the suppressor, there is an activator (35) located around Gly109 in LRR4 (25). Again, the LHRI105A, an LHR-hCG with a mutation near this activator, could be rescued by LHR+hCG/-cAMP or ExoCD.
Regardless of whether the successful pair of coexpressed LHR+hCG/-cAMP and LHR-hCG was transiently interacted or stably associated as a dimer, this intermolecular trans-activation is novel and provides new insights into the mechanisms of receptor activation. For example, a hormone receptor on the cell surface is thought to be activated upon binding its cognate hormone, an implication of intramolecular activation. Similarly, a dimeric receptor complex is activated when the complex interacts with two hormone molecules as shown by the crystal structure of the metabotropic glutamate receptor (3). The underlying mechanisms for trans-activation of a monomeric receptor and a dimeric receptor could be different. For example, monomeric receptors are more likely to collide and trans-activate, whereas dimeric receptors need to make a specific interaction before trans-activation. Although LHRs have been the subject of extensive chemical cross-linking and photoaffinity labeling studies for the past 20 yr, a cross-linked LHR dimer has never been demonstrated (39, 40). Chemical cross-linking has been proven to be the most successful, classic method to capture dimers and oligomers of hormone receptors and other proteins including growth factor receptors and GPCR (4, 41). In addition, immunological and surface plasmon resonance studies failed to show the gonadotropin receptor dimer (29, 30). On the other hand, fluorescence energy transfer studies showed that a fraction of LHRs aggregate (42) during desensitization and internalization (43). Surface receptors cluster in clathrin-coated pits before endocytosis (44). Furthermore, a crucial difference in the two seemingly conflicting observations is that chemical cross-linking and immunological assays are capable of detecting stable dimers (45), whereas fluorescence energy transfer could detect transient complexes (46). Therefore, it is worthwhile to examine the stability of the interaction between the pair of coexpressed LHR mutants.
The exo-domain of the metabotropic glutamate receptor forms a disulfide-linked dimer, and each monomeric unit binds the ligand (3) as the Ca2+ receptor (4) and m3 muscarinic receptor (47) do. It is unclear whether these neurotransmitter receptors are cis-activated and/or trans-activated. Interestingly, however, some isoforms of these receptors do not form dimers (48). The same question can be raised about dimers and oligomers of the hormone receptors with a single-transmembrane domain, which is involved in receptor Tyr phosphorylation and activation (49). It is not quite clear how the dimeric exo-domains activate the Tyr-kinase of the endo-domain.
LHR is a crucial component of human reproduction. Our observations explain how heterozygotes of two defective mutant LHRs could be reproductive and pass the genes onto the next generation (50, 51). This study opens new insights into understanding the mechanisms of mutant receptors and introduces different therapeutic approaches to those mutants such as complementation rather than replacement of a defective receptor.
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MATERIALS AND METHODS
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Mutagenesis and Functional Expression of Receptors
Mutant LHR cDNAs were prepared in a pSELECT vector using the non-PCR-based Altered Sites Mutagenesis System (Promega Corp., Madison, WI), sequenced, and subcloned into pcDNA3 (Invitrogen, San Diego, CA) as described previously (24, 52). After subcloning pcDNA3, the mutant cDNAs were sequenced again. ExCD8 was kindly provided by Dr. Hsueh (14). The exo-domain of LHR was fused to the CD8 TM region with the cytoplasmic C-terminal tail through a thrombin cleavage site present in the thrombin receptor exo-domain, producing LHR exo-domain (···NPCED (355)/thrombin receptor (A36TLDP·····NESGL (66)/CD8 TM (I162YIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV).
Varying concentrations of plasmids were transfected into HEK 293 cells by the calcium phosphate method (53). Transiently transfected cells were assayed 6072 h after transfection. Stable cell lines were established in MEM containing 8% horse serum and 500 µg/ml G-418. All assays were carried out in duplicate and repeated three to four times. Means and SDs were calculated.
125I-hCG Binding and Intracellular cAMP Assay
hCG was provided by the National Hormone and Pituitary Program and radioiodinated as described previously (39). Cells were assayed for 125I-hCG (150,000 cpm) binding in the presence of increasing concentrations of nonradioactive hormone. Kd values were determined by Scatchard plots. For intracellular cAMP assay, cells were washed twice with MEM and incubated in the medium containing isobutylmethylxanthine (0.1 mg/ml) for 15 min. Increasing concentrations of hCG was added, and the incubation was continued for 45 min at 37 C. After the medium was removed, the cells were rinsed once with fresh medium without isobutylmethylxanthine, lysed in 70% ethanol, freeze-thawed in liquid nitrogen, and scraped. After the cell debris was pelleted at 16,000 x g for 10 min at 4 C, the supernatant was collected, dried under vacuum, and resuspended in 10 µl of the cAMP assay buffer that was provided by the manufacturer. cAMP concentrations were determined with an 125I-cAMP assay kit (Amersham Pharmacia Biotech, Arlington Heights, IL) following the manufacturers instruction and validated for use in our laboratory.
RIA for Flag-LH Receptor
Flag-LHR expressed on intact cells was assayed as established and verified previously (27). Flag-LHR was prepared by inserting the FLAG epitope, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (5'-GAC TAC AAG GAC GAT GAC GAT AAG-3'), between the C terminus of the signal sequence and the N terminus of mature receptors. Mouse anti-Flag monoclonal M2 antibody (Sigma, St. Louis, MO) was iodinated with 125I according to the published procedure for radioiodination of hCG (39), and 125I-anti-Flag antibodies were purified on a Sephadex G-150 column. Binding of 125I-anti-Flag (150,000 cpm) to HEK 293 cells expressing Flag-LHR was carried out in the presence of increasing concentrations of nonradioactive anti-Flag antibody in MEM containing 0.3 mg/ml of BSA for 810 h at 4 C. 125I-anti-Flag antibody (27) was used to determine the concentrations of receptors incapable of binding the hormone, in comparison with 125I-hCG used to determine the concentration of receptors capable of binding the hormone.
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ACKNOWLEDGMENTS
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We appreciate the helpful suggestions of Hanlee P. Ji, M.D., and Brian Kobilka, M.D.
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FOOTNOTES
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This work was supported by NIH Grants HD-18702 and DK-51469.
Abbreviations: GPCR, G protein-coupled receptor; hCG, human CG; LHR, LH/CG receptor; LRR, leucine-rich repeat.
Received for publication December 14, 2001.
Accepted for publication February 13, 2002.
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