©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Exoloop 3 of the Luteinizing Hormone/Choriogonadotropin Receptor
LYS IS ESSENTIAL AND IRREPLACEABLE FOR HUMAN CHORIOGONADOTROPIN (hCG)-DEPENDENT RECEPTOR ACTIVATION BUT NOT FOR HIGH AFFINITY hCG BINDING (*)

(Received for publication, September 22, 1995; and in revised form, January 22, 1996)

Ki-Sung Ryu Roger L. Gilchrist Inhae Ji Seung-Jo Kim Tae H. Ji (§)

From the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 C-terminal 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 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 and Lys, that are located at the boundaries with the transmembrane columns 6 and 7, respectively. All substitutions including Arg for Lys 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 beta-like structure for exoloop 3.


INTRODUCTION

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 activation 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, 10, 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 to Lys (Fig. 1), whereas the other two exoloops are twice as long. In this communication, we report a novel observation that Lys of exoloop 3 is crucial and irreplaceable for receptor activation of the LH/CG receptor. Lys is located at the boundary between exoloop 3 and the transmembrane column 7. No other amino acid of exoloop 3 including Lys demonstrates this crucial role.


Figure 1: The arrangement of the LH/CG receptor and comparison of exoloop 3 sequences of glycoprotein hormone receptors. The LH/CG receptor was arranged according to the putative seven-transmembrane columns. The sequence of exoloop 3 is shown. The amino acids in gray circles are more important for cAMP induction than those in blank circles. The sequences of exoloop 3 of the receptors for LH/CG, FSH, and TSH are aligned. The sequences of cloned LH/CG receptors and FSH receptors of different species show complete homology whereas the sequences of cloned TSH receptors show some divergency among species (h, human; d, dog; r, rat).




MATERIALS AND METHODS

Mutagenesis and Functional Expression of LH/CG Receptors

Mutant 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 µg/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.

I-hCG Binding and Intracellular cAMP Assay

Stable cells were assayed for I-hCG binding in the presence of 150,000 cpm of 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 times g for 10 min at 4 °C, the supernatant was collected, dried under vacuum, and resuspended in 10 µl of the cAMP assay buffer, which was provided by the manufacturer (Amersham Corp.). cAMP concentrations were determined with a I-cAMP assay kit (Amersham Corp.) following the manufacturer's instruction validated for use in our laboratory.

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 mMN-ethylmaleimide, and 10 mM EDTA) and pelleted by centrifugation at 1300 times 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 times g for 60 min. The supernatant (500 µl) was mixed with 150,000 cpm of I-hCG and 6.5 µl 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 µl of buffer A containing bovine -globulin (5 mg/ml) and 750 µl of buffer A containing 20% polyethylene glycol 8000. After incubation at 4 °C for 10 min, samples were pelleted at 1300 times 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.


RESULTS

The 11 amino acids of exoloop 3, Lys, Val, Pro, Leu, Ile, Thr, Val, Thr, Asn, Ser, and Lys, were individually substituted with Ala to produce 11 substitution mutants. They are LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, LH/CG-R, and LH/CG-R (Fig. 2).


Figure 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 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 50 cpm, and nonspecific bindings were 100 cpm including the background. Nonspecific bindings were normally less than 2% (100 cpm) of specific binding. In contrast, specific bindings of samples with >70,000 receptors/cell were >10,000 cpm. Therefore, the noise/signal ratios were less than 2%. Usually, 10-30% of the total input I-hCG bound as suggested in the Scatchard plots. However, several mutants with <54,000 receptors/cell bound 1-10% of the total input 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 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 < 0.05 was considered to have a significant difference from the wild type.



K(d) values of all mutant receptors were in the range of 408-827 pM (p geq 0.05) except for LH/CG-R and LH/CG-R 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, LH/CG-R, LH/CG-R, LH/CG-R,LH/CG-R, LH/CG-R, and LH/ CG-R. On the other hand, LH/CG-R, LH/CG-R, LH/CG-R, and LH/CG-R 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. Therefore, the poor surface expression of LH/CG-R, LH/CG-R and LH/CG-R appears to be caused by low level production of the receptors, which are capable of hCG binding. In the case of LH/CG-R, 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 were trapped in the cells as are LH/CG-R, LH/ CG-R and LH/CG-R, probably due to defective cell surface expression.

EC values for the cAMP induction by some of the mutant receptors are significantly diverse (p leq 0.05), suggesting important roles for some of exoloop 3 amino acids. Particularly, LH/CG-R was not capable of inducing cAMP, indicating the importance of Lys in cAMP induction. Other mutant receptors showed considerably reduced affinities for cAMP induction in the order of LH/CG-R (p leq 0.002) >LH/CG-R(pleq0.026)>LH/CG-R (p leq 0.015) > LH/CG-R (p leq 0.003) > LH/CGR (p leq 0.006). Since the substituted residues are hydrophobic except for neutral Asn, the results underscore the importance of strongly hydrophobic residues at those positions.

To learn more about the importance of Lys, 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 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 in cAMP induction. Surprisingly, even the substitution of Arg for Lys 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) .


Figure 3: Multiple substitution of Lys. Lys 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.



Substitutions of Lys 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 (p leq 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 geq 0.1) except for LH/CG-R (p leq 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 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 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, 19, 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-Val-Pro-Leu-Ile-Thr-Val-Thr-Asn-Ser-Lys, 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, Pro, Ile, Val, Asn, and Lys than on Lys, Leu, Thr, Thr, and Ser ( Fig. 1and Fig. 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 three amino acids (Thr-Thr-Ser), which possess a CH(2)-OH group. Furthermore, these two groups of amino acids are arranged in an alternate sequence except for Val-Pro. A simple explanation of this arrangement is that these residues may form a beta-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-Ile-Val-Asn, shows an interesting trend for cAMP induction. The EC 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, is the most important for cAMP induction and the following residues play gradually less important roles. Overall, Lys 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 resulted in the loss of the cAMP inducibility. This complete irreplaceability indicates the strict structural requirement for Lys and appears to be specific. Also, our results indicate that the changes in the affinity (EC 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. 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 C-terminal amino acid of exoloop 3 of TSH receptors that corresponds to Lys 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 alpha 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, LH/CGR, and LH/CG-R 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 result 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 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 (where Del is deletion), LH/CG-R, LH/CG-R, LH/CG-R and LH/CG-R (data not shown). These results indicate that Lys, Pro, Ile, and Asn 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 of the LH/CG receptor in cAMP induction without the loss of the hormone binding affinity. Lys is also important for the receptor's surface expression as are Lys, Pro, Ile, and Asn of exoloop 3.

Note Added in Proof-While this manuscript was under review, Fernandez and Puett (Fernandez, L. M., and Puett, D.(1996) J. Biol. Chem.271, 925-930) reported the importance of [Abstract/Full Text] Lys.


FOOTNOTES

*
This work was supported by Grant HD-18702 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944. Tel.: 307-766-6272; Fax: 307-766-5098; Ji{at}uwyo.edu.

(^1)
The abbreviations used are: LH, luteinizing hormone; CG, choriogonadotropin; hCG, human choriogonadotropin; FSH, follicle-stimulating hormone.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.