From the
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
FSH
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
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).
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
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
The helical wheel analysis of the sequence from
Asp
To determine the role of the side chain of
Asp
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
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
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
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.
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
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.
(
)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.
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.
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.
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) .
,
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.
, 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.
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.
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
to
Lys
.
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) .
, 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.
Table: Hormone binding affinity of truncated N-terminal
extension of the FSH receptor
or
transfected with pcDNA3 lacking the FSH receptor cDNA.
I-FSH binding was performed with intact cells or cells
solubilized in Triton X-100. K
values
were determined by Scatchard plots. NS, not significant.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.