©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Roles of Exoloop 1 of the Human Follicle-stimulating Hormone Receptor in Hormone Binding and Receptor Activation (*)

Inhae Ji , Tae H. Ji (§)

From the (1)Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The follicle-stimulating hormone (FSH) receptor is a member of the glycoprotein hormone receptor subfamily of the seven-transmembrane receptor superfamily. These receptors have an extracellular N-terminal half of 350 amino acids and a membrane-associated C-terminal half of 350 amino acids. The N-terminal halves have the high affinity hormone binding site. On the other hand, the C-terminal half of the lutropin/choriogonadotropin receptor has the receptor activation site. However, little is known about the activation site and mechanism of the FSH receptor, although the existing evidence indicates crucial differences in the activation of the FSH receptor and the lutropin/choriogonadotropin receptor. As a first step to resolve this issue, we examined the upstream juxtamembrane five amino acids, Asp-Ile-His-Thr-Lys, of the exoloop 1. Ala scan and multi-substitutions show that the five amino acid sequence is important for both hormone binding and receptor activation to induce cAMP synthesis, despite its short length. Specifically, His is important for high affinity hormone binding, whereas Asp, Thr, and Lys are crucial for receptor activation. The data suggest that the five amino acids may form a turn of helix that is an extension of the transmembrane helix 2. In this helical arrangement, Asp, Thr, and Lys are grouped to form a hydrophilic face of the helix, suggesting a correlation between this arrangement and receptor activation. In addition, the diverse and differential roles of the five amino acids indicate that high affinity hormone binding and receptor activation are discernible functions. These novel observations will be helpful for understanding the activation mechanism of the FSH receptor.


INTRODUCTION

FSH()is a member of the glycoprotein hormone family of LH, CG, and TSH. They are heterodimeric glycoproteins composed of a common subunit and a hormone-specific subunit. For each mammalian species, the subunit is encoded by a single gene (1) and have identical amino acid sequences, whereas the subunits are encoded by distinct genes(2, 3) . These hormones bind to their complementary receptors with high specific affinities, and there is no cross-activity between them except for LH and hCG, which recognize the LH/CG receptor.

Glycoprotein hormone receptors have similar structures with sequence homologies: an extracellular N-terminal half and a membrane-associated C-terminal half(4, 5, 6, 7, 8, 9, 10, 11, 12) . This C-terminal half includes seven transmembrane domains, three cytoloops, three exoloops, and a C-terminal cytoplasmic tail. In addition to these structural similarities, the glycoprotein hormone receptors share a similar, if not identical, signaling pathway involving G-protein and adenylyl cyclase(13, 14) . Among these three glycoprotein hormone receptors the LH/CG receptor has been most extensively investigated. Studies using photoaffinity labeling (15) and synthetic receptor peptides (16) indicate that multiple contact points comprise the hormone contact site of the LH/CG receptor. The receptor has high and low affinity hormone contact sites. The high affinity site is in the extracellular N-terminal half(17, 18, 19) , and the low affinity site is in the membrane-associated C-terminal half(20) . The low affinity site alone is capable of activating the LH/CG receptor to induce hormone action (19-22). The TSH receptor also has the high affinity hormone contact site in the N-terminal half(23, 24) , which appears to consist of multiple hormone contact points(25, 26) .

The information on the sites for hormone contact and receptor activation of the FSH receptor is scarce. The existing evidence suggests that the N-terminal half of the FSH receptor is capable of high affinity hormone binding (27) and has a few hormone contact points (28, 29). Considering these similarities in the structures and the signaling pathways of the glycoprotein receptors, one would presume that the structure-function relationship of the FSH receptor is similar to that of the LH/CG receptor and the TSH receptor. However, the existing data indicate otherwise. There are notable differences in receptor activation by LH/CG and FSH. For instance, the C-terminal residues of FSH and hCG play crucial roles for receptor activation but in hormone-specific manners(30) . Furthermore, a synthetic peptide corresponding to the C-terminal amino acids 83-92 of the subunit is capable of binding to and activating the LH/CG receptor (21). However, it does not bind to the FSH receptor. Because of these reasons, we set out to define the receptor activation site of the FSH receptor.


MATERIALS AND METHODS

Mutagenesis

Full-length human cDNA (a kind gift from Chip Albanese and Dr. J. L. Jameson) was ligated in the antisense direction into EcoRI/KpnI site in pAlternate (Promega, Madison, WI). Single strand DNA was prepared and mutagenesis performed using mutagenic oligonucleotides and the Altered Sites Mutagenesis System (Promega) as described previously(31) .

Functional Expression of and Assays for Mutant FSH Receptors

hFSH-R/pcDNA3 was CsCl-purified and transfected into human kidney 293 cell line using the calcium phosphate method(31) . Cells were selected in the presence of 300 µg/ml G-418 for 2-3 weeks by replacing medium every 3 days, and stable cells expressing recombinant FSH receptors were established. These stable cells were used for I-FSH binding and intracellular cAMP assay as described previously(31) . Human FSH was provided by the National Hormone and Pituitary Program.


RESULTS AND DISCUSSION

Human embryonic 293 cells were transfected with pcDNA3 containing no cDNA, the wild type FSH receptor cDNA, or the cDNA with a premature stop codon. The cDNA with a premature stop codon encodes a truncated N-terminal extracellular fragment (1-314). I-FSH did not bind to mock-transfected cells or to the cell lysate solubilized in Triton X-100 (). The hormone bound to cells transfected with pcDNA3 containing the FSH receptor cDNA () and induced cAMP production(30) . I-FSH bound to the Triton X-100 lysate of cells, which were transfected with the cDNA construct with a premature stop codon. The K value was comparable to that of the wild type receptor. However, solubilized truncated receptors in the lysate did not induce cAMP synthesis (data not shown). Furthermore, the hormone did not bind to the intact cells. These results indicate that the truncated N-terminal fragment was capable of high affinity hormone binding but was not expressed on the cell surface. In addition, the data suggest that the activation of the FSH receptor to induce cAMP synthesis requires intact receptor containing the membrane-associated C-terminal half of the receptor. This is consistent with previous observations indicating the presence of a FSH binding site in the N-terminal half of the receptor(27, 28) . Similarly truncated N-terminal halves of the LH/CG receptor and the TSH receptor are capable of high affinity hormone binding(17, 18, 19, 24) . This high affinity site is, however, incapable of activating the receptors(19) . There is a low affinity site in the membrane-associated C-terminal half of the LH/CG receptor, and it alone is capable of activating the receptor to induce hormone action(19, 20, 21, 22) .

To identify a potential site for receptor activation in the exodomains of the C-terminal half of the FSH receptor, the sequences of glycoprotein hormone receptors were compared. It shows that the upstream region of the exoloop 1 is the only divergent sequence among the highly conserved exodomains of the C-terminal halves of glycoprotein hormone receptors (Fig. 1). At the same time, this region is conserved among the same hormone receptors of different species. Therefore, this uniquely diverse region may play a role in the receptor function. To determine the roles of individual residues in this region, Asp, Ile, His, Thr, and Lys were individually substituted with Ala to produce FSH-R


Figure 1: A schematic presentation of the FSH receptor. The transmembrane organization of the FSH receptor is shown in which the upstream juxtamembrane five amino acids of the exoloop 1 are exaggerated. The sequences of the exoloop 1 of the glycoprotein hormone receptors are aligned. The upstream five amino acids of the exoloop 1 of the FSH receptor are arranged according to the helical wheel analysis.




Figure 2: Ala scan of the juxtamembrane upstream five amino acids of the exoloop 1. Asp, Ile, His, Thr, and Lys of the exoloop 1 were individually substituted with Ala to produce mutant receptors, FSH-R



Clearly, each amino acid was differently impacted by Ala substitution. In addition, the Ala substitution for an amino acid has differential effects on hormone binding and cAMP induction. These disparate and differential effects underscore the specific and differential roles of the individual amino acids on hormone binding and receptor activation. Furthermore, the data show that hormone binding and receptor activation are discernible functions. Particularly, the effects on hormone binding and receptor activation of the Ala substitution for Asp, Thr, or Lys were opposite. This indicates their differential roles in hormone binding and receptor activation. On the basis of these results, the five amino acids can be classified into three functionally different groups. In the first group, Asp, Thr, and Lys gained their affinities for hormone binding upon Ala substitution whereas the affinities for receptor activation were reduced or lost. In the second group, Ile was not noticeably affected. His belongs to the third group with an improved affinity for receptor activation and reduced affinity for hormone binding.

Despite the clear functional differences of the three groups, they are not physically separated into three groups. In fact, their arrangement is puzzling. For example, the three members of the first group are not physically grouped in a contiguous sequence. Instead, Asp, Thr, and Lys are interspersed between the other two groups. This is a particular concern because of the short length of the five contiguous amino acids. Therefore, one wonders how the five amino acids function distinctly while they are interspersed in a short linear sequence. A simple explanation is that they may not be linearly arranged. For example, they form a helix. Such a helix can be an extension of the putative transmembrane helix 2 (Fig. 1). In this case the one-turn helix extension can be stabilized by the ion pair between the carboxyl group of Asp and the amine of Lys. Although the transmembrane helix has not been unequivocally proven, the sequence upstream from Asp is comprised of excellent helix formers, Leu-Leu-Leu-Leu-Ile-Ala (32). Interestingly, Ser immediate downstream from Lys is a very poor helix former and thus may terminate the helix extension.

The helical wheel analysis of the sequence from Asp to Lys (Fig. 1) shows that Thr, Asp, and Lys are grouped to form a polar face of the helix. Asp is at the center of this polar face flanked by Thr and Lys. In this spatial arrangement the distance from each amino acid to Asp is in the order of Asp < Lys < Thr < Ile < His. In contrast, the extent of the loss in the cAMP inducibility upon the Ala substitutions is in the order of Asp > Lys > Thr > Ile > His. This inverse relationship between the distance of an amino acid to Asp and the extent of the mutation-dependent loss in the cAMP inducibility indicates that the farther an amino acid is from Asp, the lower the mutation-dependent loss in the cAMP inducibility of the mutant receptor is. The cAMP inducibility, therefore, appears to be related with the spatial arrangement of the amino acids, Asp being the most important residue.

To determine the role of the side chain of Asp on the cAMP inducibility, the amino acid was substituted with a series of amino acids with various side chains possessing acidic (Glu), hydrophobic (Ala and Val), hydrophilic (Ser and Tyr), and basic (Lys and Arg) groups. All of the resulting mutant receptors were capable of binding to the receptor with the K values not significantly different from that of the wild type receptor (Fig. 3). The substitutions with acidic and hydrophilic amino acids slightly improved the binding affinity, whereas the substitution with basic amino acids did not improve it. These results indicate that Asp is not crucial for the high affinity hormone binding. Nonetheless, it may play a minor role in hormone binding as the data in Fig. 2show. Similar conclusions were drawn from the mutational analysis of the LH/CG receptor(31, 33) .


Figure 3: Multi-substitutions of Asp of the FSH receptor. Asp was substituted with a series of amino acids, and the resulting mutant FSH receptors were expressed in 293 cells and assayed for hormone binding and cAMP induction as described above. The mutant receptors were reverted to the wild type receptor and assayed for hormone binding and cAMP induction. NS, not significant.



In contrast to the marginal impact on hormone binding, all of the substitution mutants except FSH-R

Whatever the substitutes at the amino acid position at 405 did in mature receptors, they resulted in unsuccessful coupling of the receptors to G-protein without the substituted amino acid's contacting G-protein. Therefore, the inability of some of the mutant FSH receptors to induce cAMP synthesis is likely to be caused by a defect in steps prior to activation of G-protein such as receptor activation, signal generation, or signal transfer(34) . Such a defect can be caused by substitution-dependent changes in the size, flexibility and polarity of the side chain, the spatial organization, and orientation of the Asp to Lys.

There are several reasons to believe that the effects of the mutations in the exoloop 1 are specific. The effects of the Ala substitution of individual amino acids from Asp to Lys are dramatically diverse. Furthermore, the effects of the mutation of an amino acid on hormone binding and receptor activation are also distinct. On the other hand, the substitution for Asp with different amino acids are identical. These strikingly contrasting results support the specificity of mutational effects, and they could not be explained by nonspecific effects. In addition, numerous reports indicates that mutations at the exo-domains of seven transmembrane receptors resulted in the defective coupling of the receptors to G-protein(34) .

Since a single mutant oligonucleotide was used to generate a mutant cDNA, unintended changes in other positions of the FSH receptor cDNA construct were not likely to occur. This is particularly true when compared with PCR derived mutagenesis. However, we cannot completely rule out the possibility of unintended mutations during mutagenesis and subcloning and resulting effects on receptor function. To exclude this possibility, the mutant constructs were reverted to wild type by converting Ala, Ala, Ala, Ala, or Ala of the mutants to Asp, Ile, His, Thr, or Lys, respectively. Similarly, multi-substitution mutants of Asp were reverted. These revertants were capable of binding FSH and producing cAMP with normal affinities, as were the cells transfected with the wild type receptor construct ( Fig. 2and Fig. 3). These results demonstrate that the substitutions themselves were responsible for the changes in hormone binding and cAMP induction of the mutant receptors.

In conclusion, our data not only demonstrate that receptor activation is distinct from high affinity hormone binding in the FSH receptor, but identify several amino acids that differentially affect hormone binding and receptor activation. They comprise a contiguous five-amino acid sequence and are present in the upstream juxtamembrane region of the exoloop 1 of the FSH receptor. They may form one turn helix extension from the transmembrane helix 2. Their unique spatial organization appears to be important for hormone binding and activation of the FSH receptor.

  
Table: Hormone binding affinity of truncated N-terminal extension of the FSH receptor

Human embryonic kidney 293 cells were transiently transfected with pcDNA3 containing the full-length wild type human FSH receptor cDNA transfected with the cDNA with a premature stop codon that encodes amino acids or transfected with pcDNA3 lacking the FSH receptor cDNA. I-FSH binding was performed with intact cells or cells solubilized in Triton X-100. Kvalues were determined by Scatchard plots. NS, not significant.



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. Tel.: 307-766-6272; Fax: 307-766-5098; E-mail, ji@uwyo.edu.

The abbreviations used are: FSH, follicle-stimulating hormone; LH, luteinizing hormone; CG, choriogonadotropin; hCG, human CG; TSH, thyroid-stimulating hormone.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.