Exoloop 3 of the Luteinizing Hormone/Choriogonadotropin Receptor

,

The luteinizing hormone/choriogonadotropin (CG) receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. It is comprised of an extracellular N-terminal half of 341 amino acids and a membrane-associated Cterminal half of 303 amino acids. The N-terminal half is capable of high affinity hormone binding whereas the C-terminal half is capable of low affinity hormone binding and receptor activation. However, the precise location of the receptor activation site is currently unknown. We present evidence for the first time that Lys 583 of exoloop 3 is crucial and irreplaceable for receptor activation to induce cAMP synthesis. Exoloop 3 is comprised of 11 amino acids and flanked by two Lys residues, Lys 573 and Lys 583 , that are located at the boundaries with the transmembrane columns 6 and 7, respectively. All substitutions including Arg for Lys 583 did not affect the high affinity human CG binding, but they resulted in the complete loss of cAMP synthesis induced by human CG. Ala substitutions of the other amino acids in exoloop 3 did not make such a dramatic impact on cAMP induction. The Ala scan revealed two distinct groups of amino acids in terms of their importance in cAMP induction, one group being more important than the other. Interestingly, these two groups of amino acids are arranged in an alternate sequence. This result suggests a specific structure similar to a ␤-like structure for exoloop 3.
Hormone binding to receptors on the cell surface activates the receptors and generates intracellular signals (1). These events are difficult to study due to a lack of appropriate assays and, perhaps, due to their possible transient existence. The LH/CG 1 receptor offers a unique model to study receptor acti-vation and signal generation, independent of high affinity hormone binding.
The LH/CG receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. It is comprised of an extracellular N-terminal half of 341 amino acids and a membrane-associated C-terminal half of 303 amino acids (2,3). Both halves have hormone contact points according to studies using photoaffinity labeling (4,5) and receptor peptides (6). The N-terminal half alone is capable of high affinity hormone binding (7)(8)(9) with no hormone action (9,10). In contrast, the C-terminal half is capable of low affinity hormone contact with cAMP induction (9 -11). These results suggest the relationship of the low affinity hormone contact with receptor activation. Such low affinity hormone contact and receptor activation are likely to occur at the extracellular domains of the C-terminal half of the receptor. The C-terminal half has three exoloops, which connect transmembrane columns. As a first step, we have examined exoloop 3. It is the shortest with 11 amino acids, from Lys 573 to Lys 583 ( Fig. 1), whereas the other two exoloops are twice as long. In this communication, we report a novel observation that Lys 583 of exoloop 3 is crucial and irreplaceable for receptor activation of the LH/CG receptor. Lys 583 is located at the boundary between exoloop 3 and the transmembrane column 7. No other amino acid of exoloop 3 including Lys 573 demonstrates this crucial role.

Mutagenesis and Functional Expression of LH/CG Receptors-Mu-
tant LH/CG receptor cDNAs were prepared in pSELECT vector using the Altered Sites Mutagenesis System (Promega) and subcloned into pcDNA3 (Invitrogen) as described (12). Mutant LH/CG receptor constructs were transfected into human embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines were established in the presence of 500 g/ml G418 and used for hormone binding and cAMP assays. All assays were carried out in duplicate and repeated 4 -6 times. The mean and standard deviation for repeats of each mutant was calculated and analyzed by Student's t test. In addition, values for different mutants were compared with the corresponding values of the wild type receptor using analysis of variance with 95% confidence. Untransfected cells do not express the LH/CG receptor and, therefore, have been used as a control. 125 I-hCG Binding and Intracellular cAMP Assay-Stable cells were assayed for 125 I-hCG binding in the presence of 150,000 cpm of 125 I-hCG and increasing concentrations of un-iodinated hCG. The K d values were determined by Scatchard plots. hCG, batch CR 127, was supplied by the National Hormone and Pituitary Program. For intracellular cAMP assay, cells were washed twice with Dulbecco's modified Eagle's medium and incubated in the medium containing isobutylmethylxanthine (0.1 mg/ml) for 15 min. Increasing concentrations of hCG were then added, and the incubation was continued for 45 min at 37°C. After removing the medium, the cells were rinsed once with fresh medium without isobutylmethylxanthine, lysed in 70% ethanol, freeze-thawed in liquid nitrogen, and scraped. After pelleting cell debris at 16,000 ϫ g for 10 min at 4°C, the supernatant was collected, dried under vacuum, and resuspended in 10 l of the cAMP assay buffer, which was provided by the manufacturer (Amersham Corp.). cAMP concentrations were determined with a 125 I-cAMP assay kit (Amersham Corp.) following the manufacturer's instruction validated for use in our laboratory.
125 I-hCG Binding to LH/CG Receptor in Solution-Transfected cells were washed two times on ice with ice-cold 150 mM NaCl, 20 mM HEPES, pH 7.4 (buffer A). Cells were scraped on ice in the buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 10 mM EDTA) and pelleted by centrifugation at 1300 ϫ g for 10 min. Cells collected from a 10-cm plate were resuspended in 0.6 ml of the buffer containing 1% Nonidet P-40, 20% glycerol, and the protease inhibitors (buffer B), incubated on ice for 15 min, and diluted with 5.4 ml of buffer A containing 20% glycerol and the protease inhibitors (buffer C). The mixture was centrifuged at 100,000 ϫ g for 60 min. The supernatant (500 l) was mixed with 150,000 cpm of 125 I-hCG and 6.5 l of increasing concentrations of cold hCG. Varying concentrations of cold hCG were dissolved in 0.9% NaCl and 10 mM Na 2 HPO 4 at pH 7.4. This mixture was incubated at 4°C for 12 h. The solution was then thoroughly mixed with 250 l of buffer A containing bovine ␥-globulin (5 mg/ml) and 750 l of buffer A containing 20% polyethylene glycol 8000. After incubation at 4°C for 10 min, samples were pelleted at 1300 ϫ g for 30 min and the supernatant removed. The pellets were resuspended in 1.5 ml of buffer A containing 10% polyethylene glycol 8000, centrifuged, and counted for radioactivity.
K d values of all mutant receptors were in the range of 408 -827 pM (p Ն 0.05) except for LH/CG-R Asn-5813 Ala and LH/CG-R Lys-5833 Ala that showed an improved K d value of 174 and 346 pM, respectively. These results indicate that the Ala substitution of the amino acid residues did not significantly reduce the hormone binding affinity. The number of receptors detected on intact cells was more than 120,000 for LH/CG-R Val-5743 Ala , LH/CG-R Leu-5763 Ala , LH/CG-R Thr-5783 Ala , LH/CG-R Val-5793 Ala , LH/CG-R Thr-5803 Ala , LH/CG-R Ser-5823 Ala , and LH/ CG-R Lys-5833 Ala . On the other hand, LH/CG-R Lys-5733 Ala , LH/ CG-R Pro-5753 Ala , LH/CG-R Ile-5773 Ala , and LH/CG-R Asn-5813 Ala showed less than 100,000 receptors/cell. To determine whether any of the latter mutant receptors were trapped inside the cells, cells were solubilized in Nonidet P-40 and assayed for hormone binding. The numbers of these receptors in Nonidet P-40 solution were not significantly different from their numbers detected on intact cells, except for LH/CG-R Pro-5753 Ala . Therefore, the poor surface expression of LH/CG-R Lys-5733 Ala , LH/CG-R Ile-5773 Ala and LH/CG-R Asn-5813 Ala appears to be caused by low level production of the receptors, which are   2. Ala scan of exoloop 3. The 11 amino acids of exoloop 3 were individually substituted with Ala, and the resulting mutant receptors were stably expressed on human 293 cells. The cells were assayed for hormone binding and hCG-dependent cAMP induction. For hormone binding, cells were incubated with a constant amount of 125 I-hCG and increasing concentrations of un-iodinated hCG as described. For cAMP assay, cells were incubated with increasing concentrations of cold hCG, and intracellular cAMP was determined. Counts of empty tubes (background) were ϳ50 cpm, and nonspecific bindings were ϳ100 cpm including the background. Nonspecific bindings were normally less than 2% (ϳ100 cpm) of specific binding. In contrast, specific bindings of samples with Ͼ70,000 receptors/cell were Ͼ10,000 cpm. Therefore, the noise/signal ratios were less than 2%. Usually, 10 -30% of the total input 125 I-hCG bound as suggested in the Scatchard plots. However, several mutants with Ͻ54,000 receptors/cell bound 1-10% of the total input 125 I-hCG with the noise/signal ratios of 2-6%. Each experiment was repeated 4 -6 times in duplicate. The mean and standard deviations of repeats for individual mutant data are presented. Their statistical significance was analyzed to determine p value by Student' t test. They are shown in the table section of the figure. NS, not significant. It was used when bindings were less than 2-fold of the noise level, and, therefore, K d values and numbers of receptors/cell could not be determined. In addition, values for different mutants were compared with the corresponding values of the wild type receptor using analysis of variance with 95% confidence. The resulting p values were discussed in the text, and a sample with p Ͻ 0.05 was considered to have a significant difference from the wild type. capable of hCG binding. In the case of LH/CG-R Pro-5753 Ala , receptors in Nonidet P-40 solution were approximately 9 times higher than that found on intact cells (data not shown). This result suggests that nearly 90% of LH/CG-R Pro-5753 Ala were trapped in the cells as are LH/CG-R Lys-5733 Ala , LH/ CG-R Ile-5773 Ala and LH/CG-R Asn-5813 Ala , probably due to defective cell surface expression.
EC 50 values for the cAMP induction by some of the mutant receptors are significantly diverse (p Յ 0.05), suggesting important roles for some of exoloop 3 amino acids. Particularly, LH/CG-R Lys-5833 Ala was not capable of inducing cAMP, indicating the importance of Lys 583 in cAMP induction. Other mutant receptors showed considerably reduced affinities for cAMP induction in the order of LH/CG-R Pro-5753 Ala (p Յ 0.002) ϾLH/CG-R Ile-5773 Ala (pՅ0.026)ϾLH/CG-R Val-5743 Ala (p Յ 0.015) Ͼ LH/CG-R Val-5793 Ala (p Յ 0.003) Ͼ LH/CGR Asn-5813 Ala (p Յ 0.006). Since the substituted residues are hydrophobic except for neutral Asn 581 , the results underscore the importance of strongly hydrophobic residues at those positions.
To learn more about the importance of Lys 583 , it was substituted with a panel of amino acids consisting of basic (Arg), acidic (Asp and Glu), neutral (Gln), hydrophilic (Tyr), and hydrophobic (Leu and Ala) residues (Fig. 3). In addition, Lys 583 was deleted to generate a deletion mutant receptor. A deletion mutant is useful to complement substitution mutations and to understand the effect of the original amino acid without introducing the effect of a new amino acid. None of these mutant receptors were capable of inducing cAMP, consistent with the essential and irreplaceable role of Lys 583 in cAMP induction. Surprisingly, even the substitution of Arg for Lys 583 resulted in the loss of the cAMP inducibility. Numerous substitutional studies on hCG and the LH/CG receptor showed that Lys and Arg could substitute each other without the complete loss of receptor activation as did Asp and Glu (12)(13)(14)(15)(16).
Substitutions of Lys 583 slightly affected the high affinity hormone binding and substantially the surface expression of the receptor. The substitution with Arg, Asp, or Glu resulted in the reduction of the K d values as did the deletion of Lys 583 (p Յ 0.01). The substitution with Gln, Tyr, or Leu resulted in little hormone binding to intact cells. However, when the cells transfected with these mutant constructs were solubilized in Nonidet P-40 and the solutions were assayed for hormone binding, all the mutant receptors were capable of binding hCG. The K d values of the mutant receptors in solution were not significantly different from those of the wild type receptor (p Ն 0.1) except for LH/CG-R Lys-5833 Ala (p Յ 0.002). The receptor concentrations after solubilization were similar in the range of 187,000 -541,000/cell. These partial and nearly complete losses of the receptor's surface expression indicate the importance of Lys 583 in surface expression. In addition, the mechanism to transport the receptor to the cell surface does not appear to be all or none, allowing for partial surface expression of the receptor.

DISCUSSION
Exoloop 3 is the shortest among three exoloops, consisting of 11 amino acids (Fig. 1). It links the transmembrane columns 6 and 7. These two transmembrane columns are likely to be constrained due to the short connecting loop. Therefore, it is possible that structural changes of exoloop 3 could have an impact on the orientation, geometry, and interaction of the two transmembrane columns as well as receptor activation (1). In fact, the mutation of Asp 556 3 Gly of the transmembrane column 6 resulted in a constitutively activated LH/CG receptor (17). Furthermore, the transmembrane column 6 is connected to cytoloop 3, which is considered to make contact with G s protein for the regulation of receptor activation (18 -20). Therefore, a physical linkage exists from exoloop 3 to cytoloop 3 and to G s protein. These observations prompted us to examine exoloop 3 as a first step to define the site for receptor activation on the exosurface domain of the receptor.
Our data indicate that exoloop 3 of the LH/CG receptor is important for the induction of cAMP synthesis and for the surface expression of the receptor but is not crucial for the high affinity hormone binding. Exoloop 3 consists of 11 amino acids, with two terminal Lys residues. The sequence, Lys 573 -Val 574 -Pro 575 -Leu 576 -Ile 577 -Thr 578 -Val 579 -Thr 580 -Asn 581 -Ser 582 -Lys 583 , is highly conserved among various species (21), suggesting its importance.
Receptor Activation to Induce cAMP Synthesis-The impacts of Ala substitutions were more pronounced on Val 574 , Pro 575 , Ile 577 , Val 579 , Asn 581 , and Lys 583 than on Lys 573 , Leu 576 , Thr 578 , Thr 580 , and Ser 582 (Figs. 1 and 2). A careful examination of these two groups reveals that the first group consists of primarily hydrophobic amino acids, in particular the four upstream residues. In contrast, the second group is comprised of primarily hydrophilic residues, including a unique group of FIG. 3. Multiple substitution of Lys 583 . Lys 583 was substituted with a panel of amino acids, and the mutant receptors were individually expressed on 293 cells. They were assayed and the data analyzed as described in the legend to Fig. 2. None of the mutant receptors was capable of inducing cAMP synthesis. three amino acids (Thr 578 -Thr 580 -Ser 582 ), which possess a CH 2 -OH group. Furthermore, these two groups of amino acids are arranged in an alternate sequence except for Val 574 -Pro 575 . A simple explanation of this arrangement is that these residues may form a ␤-like structure (Fig. 1). The amino acids on one side of the structure may play a more important role in cAMP induction than do the residues on the other side of the structure. The group of four residues, Pro 575 -Ile 577 -Val 579 -Asn 581 , shows an interesting trend for cAMP induction. The EC 50 values for cAMP induction by the corresponding Ala substitution mutants are in decreasing order (Fig. 2). This result indicates that in this group of four amino acids the first residue, Pro 575 , is the most important for cAMP induction and the following residues play gradually less important roles. Overall, Lys 583 is the most crucial and irreplaceable for cAMP induction. Therefore, the alternate sequence of the two groups of amino acids and their orderly importance for cAMP induction suggest a specific secondary structure of exoloop 3. In addition, they implicate the interaction of exoloop 3 with hCG and/or other domains of the LH/CG receptor for receptor activation to induce cAMP synthesis.
The substitution of Arg, Asp, Glu, Gln, Tyr, Leu, or Ala for Lys 583 resulted in the loss of the cAMP inducibility. This complete irreplaceability indicates the strict structural requirement for Lys 583 and appears to be specific. Also, our results indicate that the changes in the affinity (EC 50 value) and efficacy (the maximum level of synthesis) for cAMP induction by the mutant receptors are not necessarily in parallel. This further suggests the existence of distinct mechanisms to dictate the affinity and efficacy for cAMP induction.
The sequence of exoloop 3 of LH/CG receptors is conserved among species as is the exoloop 3 sequence of FSH receptors (Fig. 1). However, the sequences of the LH/CG receptor and the FSH receptor are not identical near the C-terminal end except for Lys 583 . Furthermore, the exoloop 3 sequences of TSH receptors are diverse among species and divergent from those of the LH/CG receptor and the FSH receptor. Specifically, the Cterminal amino acid of exoloop 3 of TSH receptors that corresponds to Lys 583 of the LH/CG receptor varies among species. Therefore, the importance of Lys at the exoloop 3 C-terminal of the TSH receptor is open to question. Although exoloop 3 of the TSH receptor appears to be important for cAMP induction (22), the role of exoloop 3 in cAMP induction may not be identical among the three glycoprotein hormone receptors. This conclusion is consistent with the recent observation that the three C-terminal amino residues of the ␣ subunits of the glycoprotein hormones play important roles in receptor activation. The roles are different in each hormone (13,15,16).
Surface Expression-LH/CG-R Lys-5833 Gln , LH/CG-R Lys-5833 Tyr , and LH/CG-R Lys-5833 Leu were not detected at all on intact cells whereas they showed the full hormone binding activity when cells were solubilized in Nonidet P-40. This re-sult suggests that the mutant receptors were trapped in the cells and the mechanism for surface expression was blocked. It is not clear whether this futile surface expression occurred at the endoplasmic reticulum during the membrane insertion of nascent receptors or during their transport to the plasma membrane. In addition to the three substituent receptors, the substitution of Arg and Asp for Lys 583 resulted in partial surface expression, leaving the majority of the mutant receptors trapped within the cells. The same result was seen with LH/ CG-R Lys-5833 Del (where Del is deletion), LH/CG-R Lys-5733 Ala , LH/CG-R Pro-5753 Ala , LH/CG-R Ile-5773 Ala and LH/CG-R Asn-5813 Ala (data not shown). These results indicate that Lys 573 , Pro 575 , Ile 577 , and Asn 581 are also important for successful surface expression of the receptor.
In conclusion, our data clearly demonstrate for the first time the essential and irreplaceable role of Lys 583 of the LH/CG receptor in cAMP induction without the loss of the hormone binding affinity. Lys 583 is also important for the receptor's surface expression as are Lys 573 , Pro 575 , Ile 577 , and Asn 581 of exoloop 3.