(Received for publication, August 22, 1996, and in revised form, December 10, 1996)
From the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944
The lutropin/choriogonadotropin receptor is a
seven-helix transmembrane (TM) receptor. A unique feature of TM helices
is the content of Pro, which generally is absent in helices of
globular proteins. Because Pro disrupts helices and introduces a
~26° kink, it has been speculated that Pro plays a crucial role in
the structure of TM helices, exoloops, and cytoloops of TM receptors.
To examine the roles of the five TM Pros of the
lutropin/choriogonadotropin receptor, these residues were individually
substituted. Mutant receptors were examined for surface expression,
hormone binding, and cAMP induction. Surface expression was monitored
after introducing the flag epitope into the receptors. Flag epitopes
slightly affected cAMP induction but not hormone binding or surface
expression of receptors as monitored by immunofluorescence microscopy
and 125I-anti-flag antibody. The results indicate that
Pro479 in TM 4 and Pro598 in TM 7 play
important yet contrasting roles. Pro479 is crucial for
hormone binding at the cell surface but not after solubilization of the
receptor. This is more likely due to the Pro side chain than the
Pro-induced kink. Pro598 is important for surface
expression. The kinks of Pro463 of TM 4, Pro562
of TM 6, or Pro591 of TM 7 are not important because the
substitution of Phe for these residues did not significantly impact
surface expression, hormone binding, and cAMP induction.
The LH/CG1 receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. Unlike most seven-TM receptors, it is comprised of a long extracellular N-terminal exodomain and a membrane-associated C-terminal endodomain containing 7 TM helices (1, 2). The exodomain is capable of high affinity hormone binding (3-5) without hormone action (5, 6). In contrast, the endodomain is capable of low affinity hormone binding (5, 7) and receptor activation (6, 8-10). Low affinity hormone binding and initial receptor activation are likely to involve the exoloops and TM helices of the endodomain. The structure of these domains probably is affected by the organization of TM helices.
A unique feature of TM helices is the presence of Pro (11), in contrast to the general lack of Pro in helices of globular proteins (12). Pro disrupts helices (13) and introduces a ~26° kink (14) in the helix backbone (15). The kink in TM helices is thought to orient so that the Pro-containing convex side is adjacent to neighboring TM helices, whereas the concave side faces the lipid phase (12). As a result, TM Pros are thought to influence the packing and organization of TM helices and thus, the mechanism of receptor activation (16). Despite their potential significance, the five TM Pros of the LH/CG receptor have not been examined together. Herein, we report the importance of two of the Pros in hormone binding and targeting of the receptor to the surface membrane.
Mutant LH/CG receptor cDNAs were prepared in the pSELECT vector using the Altered Sites Mutagenesis System (Promega) and subcloned into pcDNA3 (Invitrogen) as described previously (9). Mutated LH/CG receptor plasmids were transfected into human embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines were established in minimum essential medium containing 10% horse serum and 500 µg/ml of G418. They were used for hormone binding, cAMP production, antibody binding, and fluorescence microscopy.
125I-hCG Binding and Intracellular cAMP AssayStable cells were assayed for 125I-hCG binding in the presence of 150,000 cpm of 125I-hCG (17) and increasing concentrations of unlabeled hCG. The Kd 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 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 × g for 10 min at 4°, 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 an 125I-cAMP assay kit (Amersham Corp.) following the manufacturer's instruction and validated for use in our laboratory.
125I-hCG Binding to Solubilized LH/CG ReceptorTransfected cells were washed twice with ice-cold 150 mM NaCl, 20 mM HEPES, pH 7.4 (buffer A). Cells
were scraped on ice, collected in buffer A containing protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 10 mM
EDTA), and pelleted by centrifugation at 1300 × g for
10 min. Cells from a 10-cm plate were resuspended in 0.6 ml of buffer A
containing 1% Nonidet P-40, 20% glycerol, and the above protease
inhibitors (buffer B), incubated on ice for 15 min, and diluted with
5.4 ml of buffer A containing 20% glycerol plus the protease
inhibitors (buffer C). The mixture was centrifuged at 100,000 × g for 60 min. The supernatant (500 µl) was mixed with
125I-hCG and 6.5 µl of solution containing increasing
concentrations of unlabeled hCG. Varying concentrations of unlabeled
hCG were dissolved in 0.9% NaCl and 10 mM
Na2HPO4 at pH 7.4. Binding assay mixtures were
incubated at 4 °C for 12 h. This solution was, then, thoroughly
mixed with 250 µl of buffer A containing bovine -globulin (5 µg/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 × g for 30 min, and supernatants were
removed. Pellets were resuspended in 1.5 ml of buffer A containing 20%
polyethylene glycol 8000, centrifuged, and counted for
radioactivity.
For fluorescence labeling of
LH/CG receptors, the flag epitope (18), Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
(5-GAC TAC AAG GAC GAT GAC GAT AAG-3
), was inserted between the C
terminus (Ser26) of the signal sequence and the N terminus
(Arg27) of mature receptors. The flag epitope (19) has
successfully been used as a marker to identify, trace, and purify
recombinant proteins carrying the tag without significantly impairing
their biological activities (20, 21). Intact cells were cultured on
coverslips in 6-well plates for 2 days and fixed with 4% formaldehyde in PBS for 10 min at 25°. For labeling permeabilized cells, cells were fixed with 4% formaldehyde in PBS for 5 min at 4° and treated with 0.1% Triton X-100 in PBS for 5 min at 4°. Fixed intact or permeabilized cells were washed five times with PBS for 2.5 min each at
25°. They were sequentially treated with 0.4% type IV gelatin
isolated from calf skin (Sigma) in modified Eagle's medium free of
phenol red for 10 min at 25° and then with 5% goat serum and 1%
fetal calf serum in the same medium for 20 min at 25°. The treated
cells were incubated with 500 µl/well of primary antibody solution
(25 µg of mouse anti-flag antibody in 1 ml of modified Eagle's
medium containing 5% goat serum and 1% fetal calf serum) for 2 h
at 37°. The cells were washed three times with PBS for 2.5 min each
at 25 °C and treated with a 400-fold dilution of Texas Red
conjugated to goat anti-mouse IgG (Molecular Probes). Finally, the
cells were washed with PBS six times for 2.5 min each at 25 °C. The
coverslip containing processed cells was mounted on glass slides using
50% glycerol in PBS and sealed using nail polish. Specimens were
examined under a Leica TCS-4D laser scanning confocal microscope
equipped with Scanware analysis software. The entire experiments were
completed in a day to prevent increasing background fluorescence.
Mouse anti-flag monoclonal antibody M2 (Eastman Kodak Co.) was iodinated with 125I according to the published procedure for radioiodination of hCG (17), and 125I-anti-flag antibodies were purified on a Sephadex G-150 column. Binding of 125I-anti-flag to 293 cells expressing flag-LH receptors was carried out following the 125I-hCG binding assay described above.
There are five Pros in the seven TM helices of the LH/CG receptor,
Pro463 and Pro479 in TM 4, Pro562
in TM 6, and Pro591 and Pro598 in TM 7 (Fig.
1). These TM Pros were individually substituted with Phe
to produce LH/CG-RP463F, LH/CG-RP479F,
LH/CG-RP562F, LH/CG-RP591F, and
LH/CG-RP598F. The mutant receptor plasmids were transfected
into 293 cells. Stably transfected cells were assayed for
125I-hCG binding and hCG induced cAMP synthesis. Counts of
empty tubes (background) were ~50 CPM and nonspecific binding was
~100 CPM including background. Maximum specific binding counts/min are included in parentheses in the table section of the figure. Intact
cells expressing LH/CG-RP463F, LH/CG-RP562F or
LH/CG-RP591F bound hCG with Kd values
44-100% higher than the Kd value of the wild type
receptor (Fig. 2A). They were also capable of
inducing cAMP synthesis with EC50 values 37-89% higher
than that of the wild type receptor (Fig. 2C). These results
suggest that the high EC50 values are due to the
corresponding high Kd values. Also these hCG binding
and hCG-dependent cAMP induction are specific for the
receptor since nontransfected cells and mock transfected cells did not
bind 125I-hCG nor induced hCG-dependent cAMP
production. In contrast to the functional mutants,
LH/CG-RP479F and LH/CG-RP598F did not bind hCG
or induce cAMP production at the normal hCG concentrations. However, at
>µM hCG concentrations, LH/CG-RP479F showed
some hCG binding but not cAMP induction (data not shown). This result
does not clearly distinguish whether the apparent low affinity hormone binding of LH/CG-RP479F is due to a reduction in binding
affinity or to low surface expression.
Solubilized Receptors
Since these two mutant receptors might be synthesized and trapped within cells, 125I-hCG binding studies were performed with cells solubilized in Nonidet P-40. hCG binding to solubilized LH/CG-RP479F was barely detectable. In contrast, solubilized LH/CG-RP598F bound hCG (Fig. 2B) with affinity similar that of solubilized wild type receptor, LH/CG-RP463F, LH/CG-RP562F and LH/CG-RP591F. However, the Kd values of solubilized receptors are ~2-fold higher than those of the corresponding receptors on intact cells. This small loss in apparent binding affinities of solubilized receptors is often observed for wild type and mutant receptors (22). It may reflect either the low sensitivity of the assay or slight denaturation of receptors during the solubilization and/or assay steps. In any event, our data indicate that LH/CG-RP598F is synthesized and capable of hCG binding. Furthermore, the result suggests that the mutant receptor might be trapped inside of the cells. Alternatively, LH/CG-RP598F may be expressed on the cell surface but is incapable of binding hCG in situ, perhaps due to defective folding. In this case, solubilization in the nonionic detergent may facilitate proper folding and promote hormone binding. In contrast, LH/CG-RP479F is either not synthesized or synthesized but incapable of binding hormone.
Antibody BindingTo further analyze expression and
localization of the Pro479 Phe and Pro598
Phe mutants, two independent immunological methods were utilized. Cells were transfected with plasmids encoding receptors carrying the
flag epitope, flag wild type LH/CG receptor,
flag-LH/CG-RP479F, and flag-LH/CG-RP598F. For
immunofluorescent labeling studies, cells were either examined intact
or after treatment with Triton X-100 to permeabilize the plasma
membrane and to allow the antibody to enter the cytosol. Confocal laser
fluorescence microscopy shows bright fluorescence of the flag wild type
receptor on intact cells and in permeabilized cells (Fig.
3). Cells expressing the wild type receptor lacking the
flag tag did not showed fluorescence, regardless of permeabilization.
In addition, the cells expressing flag wild type receptor did not show
fluorescence when treated for fluorescence labeling without anti-flag
antibody. These controls demonstrate that the flag wild type receptor
is expressed on the cell surface and within cells.
Flag-LH/CG-RP479F was also observed on intact and
permeabilized cells, indicating it is expressed on the cell surface and
within cells. On the other hand, flag-LH/CG-RP598F was
observed in permeabilized cells only, indicating that
flag-LH/CG-RP598F was not transported to the cell
surface.
The other approach used to test expression of the receptors was
radioimmune ligand binding using radioiodinated monoclonal anti-flag
antibody. 125I-anti-flag antibody bound to the cells
expressing the flag wild type receptor or flag-LH/CG-RP479F
but not to the cells expressing flag-LH/CG-RP598F (Fig.
4). The Kd values were 29 nM and 147 nM for the flag wild type receptor
and flag-LH/CG-RP479F, respectively. The number of binding
sites on the cell surface are significant, 27,000 and 138,000/cell,
excluding the possibility that the binding observed resulted from
nonspecific binding. Taken together, these and the fluorescence
microscopy indicate that 125I-anti-flag was capable of
specifically detecting the flag epitope in the receptors. More
importantly, the results demonstrate that the Flag-wild type receptor
and flag-LH/CG-RP479F were expressed on the cell surface,
whereas flag-LH/CG-RP598F was not.
Activities of Flag Receptors
To test whether the flag epitope
might interfere with processing and activities of the flag-LH/CG
receptors, hCG binding and cAMP induction were examined. The flag wild
type receptor and the wild type receptor on intact cells and in
solution bound hCG with similar affinities (Fig. 5,
A and B). In addition, the flag wild type
receptor was capable of hCG-dependent cAMP induction, although the EC50 value for cAMP induction was ~3-fold
higher than the value of the wild type receptor (Fig. 5C).
Furthermore, the Kd values of solubilized
LH/CG-RP598F and flag-LH/CG-RP598F were similar
(Figs. 2B and 5B). These data show that the
flag-LH/CG receptors are active, although their potency is somewhat
different from LH/CG receptors lacking the flag epitope. With this in
mind, we examined flag-LH/CG-RP479F. While hCG did not bind
to flag-LH/CG-RP479F on intact cells, hCG bound to the
receptor in detergent solution with low affinity (Fig. 5B).
This is not entirely surprising since hCG binding to
LH/CG-RP479F in solution was barely detectable.
The loss of binding on intact cells and the extremely low affinity
binding in solution of flag-LH/CG-RP479F could be due to the introduction of the bulky phenyl side chain or the loss of the Pro
induced kink at this position in TM helix 4. To distinguish these
possibilities, Pro479 was substituted with Gly and Ala to
produce Flag-LH/CG-RP479G and
Flag-LH/CG-RP479A, respectively. Both mutants failed to
bind hCG on intact cells and induce hCG dependent cAMP synthesis (Fig. 6, A and B). On the other hand,
they were capable of binding hCG in detergent solution (Fig. 6,
C and D). Kd values were ~2-fold higher than that of the flag wild type receptor. In addition, receptor concentrations were greater than that of the flag wild type
receptor. Again, it appeared possible that both mutants may not be
expressed on the cell surface or may be expressed on the surface but
defective in hCG binding. In the latter case, they may have undergone
changes in the structure when solubilized in the detergent solution. To
resolve these possibilities, cells transfected with plasmids carrying
the mutant cDNAs were treated with anti-flag antibodies and
examined under confocal microscopy. The results in Fig.
7 demonstrate that flag-LH/CG-RP479G and
Flag-LH/CG-RP479A are present both on the surface of intact
cells and within permeabilized cells.
In this study, the five TM Pros of the LH/CG receptor were individually substituted with Phe and the resulting mutant receptors were examined for hormone binding and cAMP induction. In addition, surface expression of nonbinding receptors were characterized with flag-LH/CG receptors using immunofluorescence microscopy and antibody binding.
The Pro479 Phe and Pro598
Phe
substitutions caused dramatic, yet contrasting effects (Table
I). The Pro598
Phe substitution
prevented surface expression without affecting hormone binding. In
contrast, the Pro479
Phe substitution allowed surface
expression but impaired hCG binding to the receptor on the cell surface
as did Pro479
Gly and Pro479
Ala
substitutions. Surprisingly, solubilization in Nonidet P-40 rescued the
ability of the mutants to bind the hormone. This novel observation
demonstrates a striking difference in hormone binding on intact cells
and in nonionic detergent solution. A simple explanation is that,
during solubilization, the mutant receptors undergo structural changes
leading to a structure that more closely resembles the native receptor.
Alternatively, the structure of the mutant receptors may be subtly yet
critically different depending on whether they are embedded in the
lipid bilayer or associated with the nonionic detergent in solution. It
is also possible that hormone binding of the mutants might be
interfered with due to other membrane molecules which were present in
the membrane but removed by detergent solubilization.
|
Although substitutions of Gly, Ala, and Phe for Pro479
impacted hormone binding to solubilized receptors, the extents were
significantly different. The Kd value of
flag-LH/CG-RP479F is significantly higher than those of
flag-LH/CG-RP479G and flag-LH/CG-RP479A. One
wonders whether the bulky phenyl side chain of Phe479
interferes the interaction of TM 4 with a neighboring TM, hormone binding, and/or structural restoration during solubilization. A similar
result was observed when Pro556 of the thyrotropin
receptor, which is equivalent to Pro479 of the LH/CG
receptor, was replaced with Leu in the hypothyroid hyt/hyt mouse (23,
24). The thyrotropin receptor mutant was expressed on the cell surface
but was incapable of binding hormone. Therefore, the substitution of
bulky amino acids for the TM 4 Pro appears to be intolerable for
hormone binding at the cell surface. Also, the
Pro479-induced kink does not appear to play a significant
role on hormone binding, since the Pro479 Gly and
Pro479
Ala substitutions had the same effect, although
the helix is expected to bend by Gly but not by Ala. Taken together,
these results indicate that the side chain of Pro479 is
more important than the Pro479-induced kink for hormone
binding, particularly at the cell surface. On the other hand,
Pro479 is not required for surface expression of the
receptor.
The substitution of Phe for Pro463 of TM 4, Pro562 of TM 6, and Pro591 of TM 7 slightly increased the Kd values for hCG binding on intact cells and in detergent solution as well as the EC50 values for cAMP induction. However, the substitutions did not significantly impact the maximal cAMP level. The results indicate that these three TM Pros play marginal roles on hormone binding and cAMP induction, and have no effect on surface expression.
The marginal effect of the Pro463 Phe substitution is
not surprising. Pro463 in TM 4 is variant among the LH/CG
receptors of different species and is generally absent in other
glycoprotein hormone receptors and seven TM receptors. On the other
hand, Pro479 of TM 4 and Pro598 of TM 7 are
conserved throughout seven TM receptors. Likewise, Pro562
of TM 6 and Pro591 of TM 7 are conserved among the
glycoprotein hormone receptors and are partially conserved among other
seven TM receptors. Pro463 may be nonessential because it
is located at the third amino acid position from the N terminus of the
TM 4 helix. Although internal Pros are helix destabilizers, a Pro
within the first three positions does not destabilize an
helix
(12), because amide nitrogens of the first four residues in an
helix do not form hydrogen bonds.
Pro562 is located in the middle of TM 6 and
Pro591 in the N-terminal one-third of TM 7. Consequently,
the loss of either Pro induced kink may affect exoloop 3. Since these
Pro Phe substitutions only slightly reduced the high affinity for
hormone binding, exoloop 3 may not play a crucial role in high affinity
hormone binding. These findings are consistent with the observation
that exoloop 3 of the LH/CG receptor is essential for receptor
activation but is only marginally important for high affinity hormone
binding2 (22). Also the results are in
accord with studies showing that a synthetic peptide corresponding to
the exoloop 3 sequence of the LH/CG receptor inhibits
125I-hCG binding to the LH/CG receptor only slightly
(25).
Finally, the data in this study also demonstrate the utility of flag epitopes for the localization, transport, and quantification of glycoprotein hormone receptors. Introduced epitopes do not significantly impact the biological activity of the receptor, which in turn provides a sound methodology for studying their surface expression and activation.