(Received for publication, October 10, 1995)
From the
The lutropin/choriogonadotropin receptor (LH/CG-R) contains a
relatively large extracellular domain, in addition to the seven
transmembrane helices (TMH), three extracellular loops (ECL), and three
intracellular loops typical of G protein-coupled receptors. While high
affinity ligand binding has been attributed to the N-terminal
extracellular domain, there is evidence that portions of the three ECLs
may function in ligand binding and transmembrane signaling. We have
investigated the role of several ionizable amino acid residues of rat
LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP
production. COS-7 cells were transfected with the pSVL expression
vector containing cDNAs of either wild-type or mutant rat LH/CG-R.
Several point mutants of Lys, located at the interface of
ECL III and TMH VII, bound hCG like wild-type receptor but exhibited
greatly diminished ligand-mediated signaling. Neither the point mutant,
Lys
Asp (ECL I), nor the double mutant,
Asp
Lys/Lys
Asp (ECLs I and
III, respectively), showed significant hCG binding to intact cells; in
detergent-solubilized cells, only the double mutant bound hCG. The
mutants Arg
Glu (interface of the extracellular
domain and TMH I) and Lys
Glu (ECL II) proved to
be similar to wild-type receptor in binding and signaling. Our results
establish that Lys
is important in signaling but not
ligand binding. Its location on the opposite side of the membrane from
G
precludes a direct interaction, thus emphasizing the
importance of a conformational change in the receptor and suggesting
that ligand binding to receptor and ligand-mediated receptor activation
are dissociable phenomena.
The LH/CG-R ()(1) is expressed on several
types of gonadal cells (2) and has a crucial role in
reproductive processes. Upon binding to their common receptor, LH and
CG increase adenylate cyclase activity. Although cAMP appears to be the
principal mediator of the actions of gonadotropins on most gonadal
cells, there is evidence to support activation of the phospholipase C
pathway as well, which results in the formation of inositol
1,4,5-trisphosphate and increased [Ca
]
levels (3, 4, 5) .
The LH/CG-R, FSH-R, and TSH-R are members of the glycoprotein hormone receptor family, characterized by a relatively large extracellular N-terminal region and a membrane-embedded C-terminal region containing seven TMHs. This C-terminal region is homologous to the small ligand binding members of the G protein-coupled receptor superfamily. However, unlike the small ligand binding receptors, where binding occurs in a cleft formed by the TMHs, the glycoprotein hormone receptors utilize their extracellular domain as the high affinity binding site for the heterodimeric glycoprotein hormones with molecular masses of 30-37 kDa (6, 7, 8, 9, 10) . This structural difference classifies these receptors as a distinct subfamily of the G protein-coupled receptor superfamily (11) .
The N- and C-terminal domains of the rat LH/CG-R each contain over 300 amino acid residues, the latter distributed over three ECLs, seven TMHs, three intracellular loops, and a cytoplasmic tail(1) . If the LH/CG-R spans the membrane similar to the small ligand binding G protein-coupled receptors, one would expect the seven putative TMHs to form a pocket like that of the bacteriorhodopsin and rhodopsin receptors(12, 13) ; the six hydrophilic connecting loops are essential in maintaining this conformation. Additionally, although the high affinity binding site of the LH/CG-R is located in the ECD, there is evidence to support the presence of a lower affinity binding site in the C-terminal domain of the receptor(14, 15) . Therefore, the ECLs represent potential hormone contact sites.
In an attempt to define the role of
the LH/CG-R ECLs in hormone binding and signaling, several Arg and Lys
residues were replaced: Arg (at the boundary between the
ECD and TMH I), Lys
(ECL I), Lys
(ECL II)
and Lys
(ECL III). Additionally, a reciprocal mutation of
Asp
and Lys
was characterized. The relative
positions of these amino acid residues are shown in Fig. 1.
These particular residues are invariant at the homologous positions in
the LH/CG-R and the FSH-R of all known species; in the TSH-R, the
positions equivalent to residues 401 and 583 are His and Gly,
respectively, and the other residues are invariant. Our results
revealed that Lys
is not involved in hormone binding but
is essential for full receptor activation.
Figure 1:
Schematic representation of the major
portion of the C-terminal region of LH/CG-R, including the seven
putative transmembrane helices, the three exoplasmic loops, and the
three cytoplasmic loops. The single mutations of the full-length
LH/CG-R prepared and characterized in this study are indicated:
Arg
Glu (R341E), Lys
Asp (K401D), Lys
Glu (K488E),
Lys
Glu(K583E), Lys
Gln (K583Q), Lys
Arg (K583R), and
Lys
Pro (K583P) LH/CG-R. In addition, a
reciprocal mutation was investigated: Asp
Lys/Lys
Asp (D397K, K583D)
LH/CG-R.
The mean K from multiple independent
transfections (n = 9) for hCG binding to wild-type
LH/CG-R on transfected intact COS-7 cells was 0.14 nM, with a
range of 0.07-0.25 nM (Table 1), there being no
difference between transfections with DEAE-dextran and Lipofectamine.
On the other hand, receptor numbers/cell (uncorrected for transfection
efficiencies) were greater with Lipofectamine transfection (mean of 2
10
) than with DEAE-dextran transfection (mean of
0.5
10
). The difference in receptor number/cell,
however, had no significant impact on the maximal cAMP production
elicited by hCG at 100 ng/ml in COS-7 cells transfected with the cDNA
to wild-type LH/CG-R; the mean was 20.6 (range =
12.3-31.7) pmol of cAMP/10
cells under the conditions
used and was independent of receptor density over the range
investigated (Table 1). Likewise, the effective mean
concentration of hCG necessary to increase the intracellular cAMP
concentration from basal values to 50% of the maximal value in COS-7
cells expressing wild-type LH/CG-R was 0.19 nM (range =
0.05-0.4 nM) (Table 1); this too was independent
of receptor density. For ease in comparing the different experiments,
the maximal hCG-mediated cAMP production over basal is normalized to
100% for each wild-type LH/CG-R control, and the value obtained with
the various receptor mutants, also corrected for basal, is expressed as
a percentage of that of wild-type. There was no evidence for a
functional endogenous LH/CG-R in nontransfected COS-7 cells: the mean
(range) of the basal cAMP production was 3.9 (1.6-6.7) pmol
cAMP/10
cells and that of control cells in the presence of
100 ng/ml hCG was 4.6(3.8-5.2) pmol cAMP/10
cells.
The Lys
Glu mutation resulted in a LH/CG-R that
specifically bound
I-hCG, and this binding was inhibited
by unlabeled hCG in a concentration-dependent manner (Fig. 2A). Although the estimated number of receptors
varied in the transfected cells, e.g. 2
10
for wild-type and 1
10
for mutant LH/CG-R
(uncorrected for transfection efficiency), the mutant LH/CG-R yielded a
binding affinity equivalent to that of wild-type LH/CG-R (Table 1). The Lys
Glu replacement resulted
in markedly decreased production of cAMP in response to added hCG, e.g. 15% that of wild-type LH/CG-R at 100 ng/ml hCG (Fig. 3A, Table 1). Since this result could be
attributed to the decreased number of cell surface receptors, this
mutant LH/CG-R was also transfected using Lipofectamine to obtain
higher efficiency and increased receptor expression. The mutant
receptor bound hCG with an affinity comparable to wild-type LH/CG-R (Table 1). There was an increase in cell surface expression, e.g. 2
10
receptors/cells (uncorrected for
transfection efficiency), for both wild-type and mutant LH/CG-R, but
the mutant receptor again stimulated cAMP production only 15% that of
wild-type LH/CG-R at 100 ng/ml hCG (Table 1).
Figure 2:
hCG
binding to COS-7 cells transfected with wild-type and mutant cDNAs to
LH/CG-R. Competition binding of I-hCG and hCG to
wild-type LH/CG-R and six single mutants of LH/CG-R are given in panels A-D. The binding of
I-hCG with no
added hCG was normalized to 100% in each
case.
Figure 3: cAMP levels in COS-7 cells transfected with wild-type and mutant cDNAs to LH/CG-R in the presence and absence of hCG. Panels A-D give data on six single mutant forms of LH/CG-R; in each case, results are provided for cells expressing wild-type LH/CG-R and for control cells.
To investigate
the side-chain specificity of Lys, replacements were also
made with Arg, Gln, and Pro. In each case the mutations yielded
LH/CG-Rs that bound
I-hCG with affinities comparable to
that of wild-type LH/CG-R (Fig. 2B, Table 1), but
these mutant receptors also resulted in cAMP accumulation <30% that
of wild-type LH/CG-R upon addition of hCG to transfected cells (Fig. 3B, Table 1).
Competitive binding assays
were unable to detect significant cell surface binding of I-hCG to cells transfected with cDNAs to the Lys
Asp single mutation and the Asp
Lys/Lys
Asp reciprocal mutation (Table 2).
I-hCG binding to detergent-soluble extracts of
transfected cells was found for the reciprocal mutant but not the point
mutant (Table 2). However, Western blot analysis indicated that
the (Lys
Asp) LH/CG-R was expressed (Fig. 4). Wild-type and mutant LH/CG-Rs were about equally
distributed among three bands of apparent molecular mass 101, 93, and
82 kDa; the total protein in the mutant LH/CG-R was much less than that
of wild-type LH/CG-R.
Figure 4:
Western blots of lysates from COS-7 cells
transfected with cDNAs to wild-type LH/CG-R (WT) and
(Lys
Asp)LH/CG-R (K401D). Equivalent
amounts of cellular protein were analyzed in each case; control cells, i.e. nontransfected, exhibited no immunostaining bands (data
not shown). The apparent molecular mass values of the three
immunoreactive bands are indicated; these were based on comparisons of
the mobilities with those of prestained standards (rabbit muscle
myosin, 205 kDa; Escherichia coli
-galactosidase, 116
kDa; BSA, 66 kDa; chicken egg ovalbumin, 45 kDa; bovine erythrocyte
carbonic anhydrase, 29 kDa; soybean trypsin inhibitor, 20 kDa; bovine
milk
-lactalbumin, 14.2 kDa; bovine milk aprotinin, 6.5
kDa).
Arg and Lys
were
each replaced with Glu; both mutations yielded LH/CG-Rs that bound
I-hCG with affinities comparable to that of wild-type
LH/CG-R (Fig. 2, C and D, Table 1).
These mutant LH/CG-Rs were capable of stimulating cAMP production in a
dose-dependent manner, quite similar to wild-type LH/CG-R (Fig. 3, C and D, Table 1).
Our results on the substitution of Lys of the
rat LH/CG-R with 4 amino acid residues, positively charged Arg,
negatively charged Glu, polar but nonionizable Gln, and nonpolar Pro
(an imino acid), show that the mutant LH/CG-Rs bind hCG as well as
wild-type receptor, but coupling to adenylate cyclase is greatly
diminished. These findings are particularly intriguing since
Lys
is located extracellularly, at the boundary of ECL
III and TMH VII, and is unable to interact directly with G
.
Thus, one can conclude that ligand-mediated transmembrane signaling
involves a conformational change of the LH/CG-R in which
Lys
participates in some manner. Furthermore, this
observation provides additional evidence that receptor
activation/signaling can be dissociated from high affinity hormone
binding in LH/CG-R. A similar conclusion was recently reached in
reference to another portion of the rat LH/CG-R, namely the
extracellular region just prior to TMH I(25) . In that study we
showed that individual replacements of Glu
and
Asp
with Lys yielded mutant receptors that also bound hCG
like wild-type LH/CG-R but exhibited greatly diminished signaling.
Interestingly, recent results with FSH indicate that different sites
are involved in receptor binding and signal transduction(26) .
The finding that the [Arg]LH/CG-R mutant, in
which one positively charged side chain is replaced with another, does
not signal effectively indicates a stringent specificity for the lysine
side chain. Interestingly, a charged residue at the interface of ECL
III and TMH VII is not required for proper membrane localization since
the replacements of Lys
with Gln and Pro yielded mutants
that gave levels of cell surface expression comparable to that of
wild-type receptor.
There is no information on the type of
interaction between Lys of LH/CG-R and either the ligand
or another portion of the receptor. Since replacements of Lys
have no appreciable effect on hCG binding, one would conclude
that any such interaction between the hormone and Lys
makes a negligible contribution to the overall free energy of
binding. However, high affinity hormone binding may occur to the ECD,
followed by a conformational change that ``presents'' the
hormone to Lys
in ECL III where low affinity binding may
occur. Using overlapping synthetic peptides, Roche et al.(15) found that a peptide based on the sequence of rat ECL
III inhibited the binding of
I-hCG to rat luteal
membranes with an IC
of 0.2 mM. The most obvious
interaction of Lys
would be electrostatic, e.g. an ion-dipole or an ion-ion bond between the positively charged
amino group and a negatively charged carboxyl group on hCG or on the
receptor ECD or ECLs. Alternatively, a more hydrophobic environment
could result in an abnormal pK of Lys
, which
could lead to the formation of a strong hydrogen bond. Since our
results suggest some form of ligand-mediated conformational change of
the receptor, it is reasonable to expect that Lys
of the
LH/CG-R may flip from one type of interaction to another concomitant
with hormone binding and that this change accompanies, or perhaps even
triggers, a change in the conformation or relative interhelical
position of TMH VII.
Ji et al.(27) reported that
Asp of the LH/CG-R, located at the boundary of TMH II and
ECL I, formed an ion pair with Lys
of the
-subunit of
hCG. They postulated that ligand binding may alter the conformation of
the unoccupied receptor by reorienting TMH II (28) and
concluded that this residue was important in signaling but was not
essential for hormone binding(29) , i.e. as we found
for Lys
.
Whatever the exact role of Asp in LH/CG-R function, we asked whether it and Lys
could participate in an ion-ion interaction. This was based in
part on the close proximity of TMHs II and VII suggested by projection
maps of rhodopsin (13) and by reciprocal ion-pair mutations in
the gonadotropin-releasing hormone receptor(30) . Moreover,
interhelical interactions of TMH I, in proximity to II and VII, have
been shown for the adrenergic receptors(31) . We found that the
reciprocal mutation failed to express in a functional form at the cell
surface, although hormone binding could be detected in
detergent-solubilized cells. Thus, Asp
and Lys
can be individually replaced with oppositely charged amino acid
residues and cell surface expression is obtained, but replacing both
residues results in intracellular trapping.
Lys is
invariant in the gonadotropin receptors, i.e. LH/CG-R and
FSH-R, but in TSH-R the equivalent position is occupied by
Gly(2) . In a revealing study, ECL III of the TSH-R was
replaced with that from the
-adrenergic receptor; it
was found that the mutant TSH-R bound TSH with high affinity, but the
sensitivity of TSH-stimulated cAMP production and the maximal level of
TSH-mediated cAMP production were significantly diminished in the
mutant TSH-R(32) . Coupled with our studies on Lys
in ECL III of the LH/CG-R, these results indicate an important
role of ECL III in glycoprotein hormone signaling and a possible region
of specificity delineating gonadotropin receptors from the TSH-R.
Two other positively charged residues of LH/CG-R, which are
invariant in the glycoprotein hormone receptors, Arg
(located in the ECD at the interface with TMH I) and Lys
(ECL II) were each replaced with Glu with no observable effect on
hCG binding or receptor activation. Lys
(ECL I) was
replaced with Asp, and the mutant receptor failed to exhibit
significant hCG binding to intact or detergent-solubilized cells,
suggesting that this amino acid residue may be involved in hormone
binding. However, binding may occur but is difficult to detect if there
is a low level of mutant receptor expression, if the receptor is
rapidly degraded or if the K
is significantly
increased. Lys is present at this position in LH/CG-R and FSH-R, while
in TSH-R it is occupied by His; depending upon the pK of the His, the
potential exists for retention of a positive charge at this location in
ECL I of all glycoprotein hormone receptors.
Our results on Western
analysis of wild-type LH/CG-R and (Lys
Asp)
LH/CG-R revealed the presence of three bands of apparent molecular mass
101, 93, and 82 kDa. The original Western blot experiments on expressed
LH/CG-R were performed using human 293 cells stably transfected with
the wild-type LH/CG-R cDNA(24) . A major 85-kDa form,
considered the mature receptor, and a minor 68-kDa form, which appeared
to be the incompletely glycosylated form, were found. The apparent
93-kDa band observed in transiently transfected COS-7 cells corresponds
to the M
of purified rat ovarian
LH/CG-R(2) , although wide variations have been reported in the M
of LH/CG-R from various sources. In another
study from our laboratory using a slightly different set of protein
standards, we found major and minor bands of apparent molecular mass 93
kDa (
80%) and 78 kDa for expressed wild-type LH/CG-R(25) .
The greater amount of protein loaded in that study could easily prevent
resolution of the bands of 93 and 101 kDa reported herein.
Interestingly, the Lys
Asp LH/CG-R mutant also
gave the same apparent M
forms as wild-type
LH/CG-R, but at much lower levels.
In summary, these results enable
us to conclude that Lys of the rat LH/CG-R is critical in
receptor activation following hormone binding. Since Lys
(ECL III) is located on the opposite side of the membrane from G
proteins and does not appear to be involved in hormone binding, these
findings offer considerable weight to the concept that receptor binding
and activation are dissociable phenomena. Of interest are other
observations we recently made that certain amino acid residues in TMH
VII are also critical for signal transduction (33) . Thus, this
region of the receptor appears to be important in transmembrane
signaling subsequent to hormone binding.