(Received for publication, September 22, 1995; and in revised form, January 22, 1996)
From the
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
-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 ()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).
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 values of all mutant
receptors were in the range of 408-827 pM (p
0.05) except for LH/CG-R
and
LH/CG-R
that showed an improved K
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
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
0.002)
>LH/CG-R
(p
0.026)>LH/CG-R
(p
0.015) > LH/CG-R
(p
0.003) > LH/CGR
(p
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
values as did the deletion of
Lys
(p
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
values of the mutant receptors in solution were
not significantly different from those of the wild type receptor (p
0.1) except for LH/CG-R
(p
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.
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
protein for the regulation of
receptor activation(18, 19, 20) . Therefore,
a physical linkage exists from exoloop 3 to cytoloop 3 and to G
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.
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
subunits of the glycoprotein hormones play important roles in receptor
activation. The roles are different in each
hormone(13, 15, 16) .
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.