Phosphorylation of the Lutropin/Choriogonadotropin Receptor Facilitates Uncoupling of the Receptor from Adenylyl Cyclase and Endocytosis of the Bound Hormone
Zheng Wang,
Xuebo Liu and
Mario Ascoli
Department of Pharmacology University of Iowa Iowa City,
Iowa 52242-1109
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ABSTRACT
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Stably transfected cell lines expressing the
wild-type rat LH/CG receptor (rLHR) or a full-length rLHR in which
S635, T638,
S639, S649, and
S653 were simultaneously mutated to alanine residues
(designated rLHR-5S/T
A) were used to probe the importance of
receptor phosphorylation on the regulation of receptor functions. The
mutant receptor binds hCG with high affinity and transduces the
hormonal signal into increases in cAMP and inositol phosphate
accumulation comparable in magnitude to those elicited by the wild-type
receptor.
In contrast to cells expressing rLHR-wt, which respond to hCG or
phorbol 12-myristate 13-acetate stimulation with an increase in rLHR
phosphorylation, the phosphorylation of rLHR in cells expressing
rLHR-5S/T
A is severely blunted. Likewise, the phorbol 12-myristate
13-acetate-induced desensitization of hCG-induced cAMP accumulation is
drastically reduced in cells expressing rLHR-5S/T
A. In contrast, the
hCG-induced desensitization of hCG-induced cAMP accumulation is
delayed, but not abolished, in cells expressing rLHR-5S/T
A. Lastly,
the rate of internalization of the receptor-bound hCG is slower in
cells expressing rLHR-5S/T
A than in cells expressing rLHR-wt.
These results show that phosphorylation of rLHR is necessary, but not
sufficient, for uncoupling of the receptor from adenylyl cyclase and
for endocytosis of the receptor-bound hormone.
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INTRODUCTION
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Like many other G protein-coupled receptors, the LH/CG receptor
(LHR) and the FSH receptor (FSHR) become phosphorylated upon agonist
stimulation (1, 2). In keeping with the knowledge that phosphorylation
is an important event in the agonist-induced desensitization of many G
protein-coupled receptors (reviewed in Refs. 36), we proposed that
the phosphorylation of LHR and FSHR is also involved in the
gonadotropin-induced desensitization of these receptors (1, 2). Other
investigators, however, have presented evidence that is not consistent
with this hypothesis (7, 8, 9).
In an ongoing series of experiments we set out to formally test the
hypothesis that phosphorylation is involved in the desensitization of
gonadotropin receptors. Using human kidney 293 cells stably transfected
with the rat LHR (rLHR) complementary DNA (cDNA), we showed that rLHR
becomes rapidly phosphorylated in serine residues when the cells are
exposed to hCG or a phorbol ester [phorbol 12-myristate 13-acetate
(PMA)], and that these two stimuli also induce desensitization of the
hCG-responsive adenylyl cyclase (1, 10, 11, 12, 13). Further analysis of three
progressive C-terminal truncations of the rLHR showed that most of the
hCG- or PMA-induced phosphorylation occurs in one or more of four
serine residues (S635, S639,
S649, and S652) located in the C-terminal
cytoplasmic tail of the rLHR (12, 13). Furthermore, our data showed
that two C-terminally truncated species of rLHR (designated rLHR-t628
and rLHR-t631) that cannot be phosphorylated also display a delay in
hCG-induced desensitization, a severe impairment in PMA-induced
desensitization, and an increase in the rate of hCG-induced receptor
down-regulation (10, 13). In contrast, another C-terminally truncated
species of rLHR (designated rLHR-t653) that can be phosphorylated
behaved similarly to the wild-type rLHR with regard to hCG- or
PMA-induced desensitization as well as hCG-induced receptor
down-regulation (13).
The experiments presented herein were designed to determine whether the
different functional properties of rLHR-t653, rLHR-t631, and rLHR-t628
described above are due to removal of the entire 632653 region or to
removal of the phosphorylation sites contained within this region. In
addition, we wanted to determine whether rLHR phosphorylation is needed
for the efficient endocytosis of the receptor-bound hormone. To this
end we constructed and analyzed a full-length rLHR mutant in which the
four serines and single threonine present in the 632653 region were
simultaneously mutated to alanines. This mutant was analyzed for hCG-
and PMA-induced phosphorylation, uncoupling, and down-regulation as
well as for its ability to mediate the endocytosis of the bound
ligand.
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RESULTS
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Preparation and Functional Properties of rLHR-5S/T
A
Phosphoamino acid analysis and phosphorylation experiments
involving three different C-terminally truncated species of the rLHR
have identified four serine residues in the 632653 region of the rLHR
as the major locus of hCG- or PMA-induced phosphorylation (12, 13). To
confirm and extend these observations, we constructed and analyzed a
full-length receptor mutant in which these four residues
(S635, S639, S642, and
S649) were simultaneously mutated to alanines. Although we
cannot detect phosphothreonine in the phosphorylated rLHR (12), the
single threonine residue (T638) located in the 632653
region was also mutated to alanine to account for the possibility that
the phosphorylation of a single threonine residue may not be detected
in the phosphoamino acid analysis. Thus, the new receptor mutant
characterized here (designated rLHR-5S/T
A) is a full-length mutant
in which S635, T638, S639,
S649, and S652 were simultaneously mutated to
alanines (Fig. 1
).

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Figure 1. Intracellular Regions of the rLHR
The amino acid sequence of the three cytoplasmic loops and the
C-terminal cytoplasmic tail of the rLHR is shown (for review, see Ref.
43). The five residues (S635, T638,
S639, S642, and S649) that were
simultaneously mutated to alanines are indicated. L628,
R631, and Q653 are also indicated as a point of
reference, as they represent the C-terminal end of three truncated
forms of rLHR that were previously characterized. Serine 659, another
potential phosphorylation site (see Discussion), and two
additional serines (365 and 617) that have been ruled out as potential
phosphorylation sites (12, 13) are also indicated.
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After transfection of 293 cells with the cDNA encoding for
rLHR-5S/T
A, several clonal cell lines were obtained expressing
different numbers of cell surface receptors. One of these cell lines
that expressed a high density of mutant receptors [designated
293L(5S/T
A-2); binding capacity, 237,000 ± 13,000 molecules of
hCG/cell] and a cell line that expressed a comparable density
wild-type rLHR [designated 293L(wt-17); binding capacity, 232,000
± 15,000 molecules of hCG/cell] were chosen for further analysis. The
Kd values for the binding of hCG to 293L(wt-17)
and 293L(5S/T
A-2) cells were 301 ± 1 and 365 ± 60
pM, respectively.
As shown in Table 1
, the basal levels of cAMP, the
EC50 for cAMP accumulation, and the maximal cAMP response
to hCG are somewhat higher in 293L(5S/T
A-2) cells than in
293L(wt-17) cells. To correct for the possibility of clonal variation
we also measured the cAMP response to cholera toxin and calculated a
ratio for the maximal cAMP response to hCG and the maximal cAMP
response to cholera toxin. This ratio is somewhat lower in
293L(5S/T
A-2) cells than in 293L(wt-17). As this response ratio is
comparable to those previously reported for other cell lines expressing
rLHR-wt (12, 13), we conclude that the 5S/T
A mutation of rLHR has
only a minimal effect on the transduction of the cAMP signal. The same
can be said for the effects of this mutation on the transduction of the
inositol phosphate signal. When corrected for the inositol response
induced by NaF, the hCG-induced inositol phosphate response is
comparable in 293L(wt-17) and 293L(5S/T
A-2) cells (data not
shown).
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Table 1. Human CG- and Cholera Toxin-Stimulated cAMP
Accumulation in Stably Transfected 293 Cells Expressing rLHR-wt or
rLHR-5S/T- A
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Human CG and PMA-Induced Phosphorylation
The data presented in Fig. 2A
show that basal,
hCG-induced, and PMA-induced phosphorylations of rLHR are severely
impaired in cells expressing rLHR-5S/T
A. Longer exposures and
densitometic scanning of the autoradiograms indicated that
phosphorylation is not completely abolished, however. In two
independent experiments quantitated by densitometry, hCG-induced
phosphorylation was reduced by 92 ± 2%, and PMA-induced
phosphorylation was reduced by 89 ± 1%. These findings are
consistent with previous data showing that phosphorylation is largely
preserved in a C-terminal truncation of rLHR at residue 653, but is
completely abolished when truncations are performed at residue 631 or
628 (12, 13). The reduced phosphorylation in cells expressing
rLHR-5S/T
A cannot be explained by differential immunoprecipitation
of rLHR-wt and rLHR-5S/T
A, as both receptors can be readily detected
in immunoprecipitates of [35S]cysteine/methionine-labeled
cells (Fig. 2B
). As previously reported, immunoprecipitates from cells
metabolically labeled with [35S]cysteine/methionine
reveal not only the cell surface 85-kDa rLHR, but also a 68-kDa
immature intracellular precursor of the rLHR (12, 13, 14).

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Figure 2. Human CG and PMA-Induced Phosphorylation of rLHR-wt
and rLHR-5S/T A
Immunoprecipitates were prepared from cells that had been metabolically
labeled with 32P for 3 h (A) or
[35S]Translabel for 24 h. In A, the
32P-prelabeled cells were also incubated with buffer only,
1000 ng/ml hCG for 5 min, or 200 nM PMA for 30 min before
immunoprecipitation. These concentrations and incubation times were
chosen to elicit a maximal response (1, 12, 13). In both panels,
equivalent amounts of cellular protein were used for
immunoprecipitation. The results presented are from a densitometric
scan of a representative autoradiogram.
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Agonist and PMA-Induced Desensitization
The results summarized in Fig. 3A
show that
preincubation of 293L(wt-17) cells with hCG results in a time-dependent
decrease in cAMP responsiveness to a subsequent challenge with hCG
(desensitization). The results summarized in Fig. 3B
show that this
loss of responsiveness cannot be accounted for by a decrease in cell
surface receptors (down-regulation) because these decline at a much
slower rate. As this loss of responsiveness also cannot be explained by
changes in the functional properties of Gs or
adenylyl cyclase (11, 13, 15, 16, 17, 18), these results operationally define a
type of desensitization (which we call uncoupling) that must be due to
a change in the functional properties of the rLHR rather than to a
change in the density of cell surface rLHR.
The results presented in Fig. 3
also show that although hCG-induced
down-regulation of rLHR is preserved in the phosphorylation-negative
mutant, hCG-induced uncoupling is delayed. The delay in uncoupling
detected in cells expressing rLHR-5S/T
A agrees well with previous
data obtained for receptor truncations. Thus, cells expressing
rLHR-t653, a rLHR truncation that retains the phosphorylation sites
mutated here, uncouple normally when incubated with hCG (12, 13). In
contrast, cells expressing rLHR-t631 or rLHR-t628, two rLHR truncations
that remove the phosphorylation sites mutated here, show a delay in
uncoupling indistinguishable from that shown in Fig. 3A
for cells
expressing rLHR-5S/T
A (10, 12, 13). The delay in the time course of
uncoupling detected in cells expressing rLHR-5S/T
A is obvious at
early time points. During a 15-min preincubation with a saturating
concentration of hCG, 293L(wt-17) cells lose 4060% of their ability
to respond to a subsequent challenge with any concentration of hCG,
whereas the ability of 293L(5S/T
A-2) cells to respond to a
subsequent challenge with any concentration of hCG is not impaired
(Fig. 4
).

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Figure 4. Effect of Prior Exposure to hCG on the cAMP
Response Induced by Increasing Concentrations of hCG
293L(wt-17) and 293L(5S/T A) cells were initially divided into two
groups and preincubated without (open bars) or with 100
ng/ml hCG (dark bars) for 15 min to induce receptor
uncoupling. At the end of this preincubation, the free and
receptor-bound hormone were removed (see Materials and
Methods), and each group of cells was divided into four
subgroups. One subgroup of cells was further incubated without hormone
for 15 min at 37 C to determine the residual levels of cAMP present
after the preincubation. The other three subgroups were restimulated
with increasing concentrations of hCG (as indicated on the
abscissa) for 15 min at 37 C. The bars
shown represent the levels of intracellular cAMP measured at the end of
the restimulation period after correction for residual levels of cAMP.
Basal levels of cAMP were 4.4 ± 0.4 and 23.1 ± 3.3
pmol/106 cells for 293L(wt-17) cells preincubated without
or with hCG, respectively. The corresponding values for
293L(5S/T A-2) cells were 0.7 ± 0.1 and 7.4 ± 0.8
pmol/106 cells. Each bar represents the
average ± SEM of three independent experiments. The
numbers in parentheses above each pair of bars depict the cAMP response
in cells preincubated with hCG expressed as a percentage of the
response in cells preincubated in the absence of hCG.
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In previous experiments we had also shown that the hCG-induced
down-regulation was normal in cell lines expressing rLHR-t653, but it
was faster in cells expressing rLHR-t631 or rLHR-t628 (13). The data
presented in Fig. 3B
clearly show that the enhanced down-regulation
detected in rLHR-t631 and rLHR-t628 is not due to the deletion of
phosphorylation sites, because the hCG-induced down-regulation is
similar in cells expressing rLHR-wt and those expressing rLHR-5S/T
A.
Thus, we must now conclude that other amino acids in the 632653
region are important to the process of down-regulation.
The addition of PMA to 293 cells transfected with rLHR-wt also leads to
receptor phosphorylation and desensitization of the cAMP response to
hCG that is due to uncoupling rather than receptor down-regulation (1, 12, 13). In contrast to the hCG-induced uncoupling, which results in a
reduction in the cAMP response to all concentrations of hCG tested
(5200 ng/ml; as illustrated in Fig. 4
), the PMA-induced uncoupling
leads to a reduction in the cAMP response only when the cells are
rechallenged by low concentrations of hCG (i.e. <5 ng/ml;
see Fig. 5
). As the concentrations of hCG used in the
rechallenge increase, the inhibitory effect of PMA wanes to the point
where there is no effect or even an increase in the cAMP response (Fig. 5
). The data presented in Fig. 5
also show that, in keeping with the
impairment in PMA-induced phosphorylation, 293L(5S/T
A-2) cells
display a substantial impairment in the magnitude of PMA-induced
uncoupling. This finding is consistent with previous data for receptor
truncations, which showed that a reduction in PMA-induced uncoupling
correlates with the removal of the phosphorylation sites mutated here
(13).

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Figure 5. Effect of Prior Exposure to PMA on the cAMP
Response Induced by Increasing Concentrations of hCG
293L(wt-17) and 293L(5S/T A-2) cells were divided into two groups and
preincubated without (open bars) or with 200
nM PMA (dark bars) for 30 min. At the end of
this preincubation, each group of cells was divided into six subgroups.
One subgroup of cells was further incubated without hormone for 15 min
at 37 C, and the other five subgroups were further incubated with
increasing concentrations of hCG (as indicated on the
abscissa) for 15 min at 37 C. The bars
shown represent the levels of intracellular cAMP measured at the end of
this incubation after subtraction of the amount of cAMP present in the
cells incubated without hCG. Basal levels of cAMP were 0.9 ± 0.2
and 0.4 ± 0.1 pmol/106 cells for 293L(wt-17) cells
preincubated without or with PMA, respectively. The corresponding
values for 293L(5S/T A-2) cells were 1.4 ± 0.1 and 0.7 ±
0.1 pmol/106 cells. Each bar represents the
average ± SEM of three independent experiments. The
numbers in parentheses shown above each pair of bars depict the cAMP
response in cells preincubated with PMA expressed as a percentage of
the response in cells preincubated in the absence of PMA.
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Receptor-Mediated Endocytosis
Although it is clear that the receptor-bound
[125I]hCG is internalized and degraded when it binds to
the LHR expressed in gonadal or transfected cells (19, 20, 21), little is
known about the regulation of this process. Since it is known, however,
that progressive truncations of the C-terminal cytoplasmic tail that
remove the four serine residues identified as phosphorylation sites
also enhance the rate of hormone internalization (20), the availability
of a rLHR-5S/T
A allows us to determine whether the increased
internalization rate of the truncated receptors is due to the removal
of phosphorylation sites.
The data summarized in Fig. 6
show that the
internalization of [125I]hCG is slower in
293L(5S/T
A-2) cells than in 293L(wt-17) cells. Additional
experiments in which the rate constants for endocytosis (ke) were
measured in these two cell lines indicated that [125I]hCG
is internalized with a half-life of 74 ± 4 min (n = 3) in
293L(wt-17) cells and 103 ± 8 min (n = 3) in
293L(5S/T
A-2) cells. Thus, we can now conclude that the enhanced
rate of internalization previously detected in C-terminally truncated
forms of rLHR is not due to the removal of phosphorylation sites. In
fact, our data suggest that the agonist-induced phosphorylation of rLHR
facilitates its internalization.

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Figure 6. Internalization and Degradation of the
Receptor-Bound [125I]hCG in 293L(wt-17) and
293L(5S/T A-2) Cells
Cells were preincubated with 40 ng/ml [125I]hCG for 15
min at 37 C. At the end of this incubation (time 0 in the figure), the
free hormone was removed by washing, the cells were placed in warm
fresh medium without hormone, and the incubation was continued at 37 C.
At the times indicated, the cells were placed on ice and used to
determine the amount of surface-bound, internalized, and degraded
hormone as described in Materials and Methods. The
bottom panels show the same data, except that the
internalized and degraded radioactivity were manually combined to
simplify the data into two, rather than three, components. Each
point represents the average ± SEM of
three independent experiments. The initial counts per min bound were
58,000 ± 1,000 and 38,000 ± 4,000 for 293L(wt-17) and
293L(5S/T A-2) cells, respectively.
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DISCUSSION
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Previous studies from this and other laboratories have shown that
the hCG- or PMA-induced desensitization of hCG-stimulated cAMP
synthesis may be due to changes in the functional properties of the
rLHR and/or to changes in the density of cell surface rLHR, rather than
to a change in the functional properties of Gs or
adenylyl cyclase (11, 13, 15, 16, 17, 18). Under the experimental conditions
employed herein, the hCG- or PMA-induced desensitization detected in
transfected 293 cells appear to be due to changes in the functional
properties of the rLHR (a phenomenon that we call uncoupling), rather
than to changes in the density of cell surface rLHR (a phenomenon that
we call down-regulation) for the following reasons. First, within the
range of receptor expression used in these experiments, the maximal
cAMP response to hCG is basically independent of receptor number (13).
Second, when PMA is used as a stimulus, desensitization occurs in the
absence of receptor down-regulation (13), and when hCG is used as a
stimulus, the time course of down-regulation is much slower than that
of desensitization (13). Thus, this experimental paradigm should allow
us to determine the molecular basis of the hCG- or PMA-induced
uncoupling of the rLHR from adenylyl cyclase.
As only phosphoserine is detectable in the phosphorylated rLHR (12),
there are only seven potential phosphorylation sites present in the
intracellular regions of this receptor. Two of these (S365
and S617) can be readily excluded by the finding that
receptor phosphorylation is completely abolished in two downstream
truncations (rLHR-t628 and rLHR-t631) that retain these two residues
(12, 13). Studies with these two truncations and an additional
truncation at residue 653 where phosphorylation is minimally affected
(12, 13) have indicated that the four serines mutated here
(S635, S639, S649, and
S653) are the major loci of phosphorylation. A small
decrease in the phosphorylation signal detected in rLRH-t653 (12, 13)
led us to suspect that S659 may represent an additional
phosphorylation site. The fact that the phosphorylation of 5S/T
A is
severely impaired, but not lost, confirms the conclusion that
S635, S639, S649, and/or
S653 represent the major loci of phosphorylation and
further indicates that S659 represents the residue
responsible for the residual phosphorylation detected in this
mutant.
The time course of hCG-induced uncoupling in cells expressing rLHR-wt
suggests the existence of two phases (13). These consist of a fast
phase that occurs within 5 min of hCG addition and leads to a 4060%
reduction in hCG-stimulated cAMP synthesis and a slower phase that
ensues for the next 5060 min and leads to a further 2040%
reduction in hCG-stimulated cAMP synthesis. The removal (12, 13) or
mutation of phosphorylation sites delays the early phase of hCG-induced
uncoupling, but has little or no effect on the slow phase. We would
argue that this delay in the onset of uncoupling is caused by the
phosphorylation of S635, S639,
S649, and/or S652, and that the phosphorylation
of S659 (discussed above) is functionally silent. Thus, the
functional properties of the agonist-induced uncoupling of rLHR-t631
and rLHR-t628 (both of which show no residual phosphorylation and lack
S635, S639, S649, S652,
and S659) (12, 13) are identical to those of rLHR-5S/T
A,
in which only S635, S639, S649, and
S652 were mutated and show minimal residual
phosphorylation. Clearly then, agonist-induced uncoupling is delayed
regardless of the presence or absence of S659. The converse
is also true, in that the removal of S659 without removing
S635, S639, S649, and
S652 (which was accomplished in rLHR-t653) results in a
receptor that displays a minimal reduction in phosphorylation and a
normal time course and magnitude of agonist-induced uncoupling (12, 13). Taken together, our results indicate that phosphorylation is
necessary, but not alone sufficient, for the hCG-induced uncoupling of
rLHR.
The delay in the time course of hCG-induced uncoupling (as opposed to a
change in magnitude) observed in the phosphorylation-deficient mutants
of rLHR is not peculiar to this receptor, nor unexpected from previous
reports on the ß2-adrenergic receptor (22). In fact, the
removal (by truncation) or mutation of the C-terminal phosphorylation
sites of the ß2-adrenergic receptor also results in a
delay in the time course of agonist-induced desensitization, but has
little or no effect on the magnitude of desensitization detected upon
prolonged incubation with agonist (22).
In contrast to the data discussed above for hCG-induced
uncoupling, the magnitude of PMA-induced uncoupling is severely
impaired by the removal (13) or mutation of phosphorylation sites. We
would again argue that this decrease in the magnitude of PMA-induced
uncoupling is caused by the phosphorylation of S635,
S639, S649, and/or S652, and that
the phosphorylation of S659 (discussed above) is
functionally silent. Thus, there is little or no PMA-induced
phosphorylation or uncoupling in cells expressing rLHR-t631 and
rLHR-t628 (both of which lack S635, S639,
S649, S652, and S659) (12, 13) or
in cells expressing rLHR-5S/T
A, in which only S635,
S639, S649, and S652 were mutated.
In contrast, the removal of S659 without removing
S635, S639, S649, and
S652 (which was accomplished in rLHR-t653) results in a
receptor that displays a minimal reduction in PMA-induced
phosphorylation and a normal magnitude of PMA-induced uncoupling (12, 13). Taken together our data argue that the PMA-induced phosphorylation
of this locus is the most important (if not the only) posttranslational
modification involved in the PMA-induced uncoupling of the rLHR.
The data presented here also show that the hCG-induced phosphorylation
of the rLHR at residues S635, S639,
S649, and/or S652 is needed for the efficient
endocytosis of the receptor-bound hormone. This is the only case where
previous data obtained with rLHR truncations that remove or maintain
phosphorylation sites do not match the data obtained with the
simultaneous mutation of the phosphorylation sites. Thus, rLHR-t653
(phosphorylation-positive truncation) and rLHR-t631
(phosphorylation-negative truncation) were previously shown to exhibit
a faster rate of internalization than rLHR-wt (20), whereas as shown
here, rLHR-5S/T
A exhibits a slower rate of internalization. These
results are also interesting because of recent reports of the possible
importance of activation and/or phosphorylation of G protein-coupled
receptors in the process of internalization. There is a growing body of
evidence indicating that the activation of G protein-coupled receptors
in general (23, 24, 25, 26, 27) is needed for the efficient internalization of the
hormone-receptor complex. Likewise, although initial experiments argued
against a role for phosphorylation on the sequestration of
ß2-adrenergic receptors (22, 28, 29), emerging models for
this (30, 31, 32) as well as the M2 muscarinic receptor (33) indicate that
agonist-induced phosphorylation facilitates the agonist-induced
sequestration of these two receptors.
We have previously reported that the rate of endocytosis of the
antagonist-occupied mouse LHR is slower than the rate of endocytosis of
the agonist-occupied mouse LHR (26) and that two mutations of the rLHR
that impair signal transduction also slow down the endocytosis of the
bound agonist in transfected 293 cells (27). Although we interpreted
these data to indicate that an agonist-induced conformational change in
the LHR and/or a physical association of the LHR with its cognate G
protein(s) were needed for efficient endocytosis of the bound ligand,
the data presented here argue that we should also consider the
possibility that LHR phosphorylation is an important component of this
process.
In summary, the data presented herein clearly show that phosphorylation
of the rLHR at S635, S639, S642,
and/or S649 is necessary for hCG- and PMA-induced
uncoupling and for the efficient endocytosis of the receptor-bound
hormone. Additional studies are currently underway to determine the
phosphorylation state and functional properties of additional mutants
of the LHR in which S635, S639,
S642, and S649 as well as S659 and
T638 are individually mutated to alanine. Although we
predict that the S659A or T638A mutations would have little or no
impact on phosphorylation, uncoupling, or internalization, the analysis
of the other four mutants will allow us to more accurately determine
the identity of the phosphorylation sites and their impact on function.
Additional experiments are also being performed to determine whether
other posttranslational modifications of the rLHR are involved in
hCG-induced uncoupling and to determine whether the previously
described transduction-deficient mutants of rLHR do not internalize
efficiently simply because they are not phosphorylated in response to
hCG binding.
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MATERIALS AND METHODS
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Plasmids and Cells
The cloning of the rat luteal LH/CG receptor cDNA and the
template plasmid containing the full-length coding region plus portions
of the 5'- and 3'-untranslated regions of the wild-type rLHR cDNA have
been previously described (34). The mutant rLHR-5S/T
A, in which
S635, T638, S639, S649,
and S652 were simultaneously mutated to alanines in the
full-length LH/CG receptor, was created by PCR with overlap extension
(35) to alter the nucleotides coding for these residues. The sequence
of the entire region of each mutant cDNA generated by PCR was verified
by dideoxy sequencing (36). The mutant and wild-type rLHR cDNAs were
subcloned into the eukaryotic expression vector pcDNAI/Neo (Invitrogen,
San Diego, CA) for transfection.
The origin and handling of the parental human embryonic kidney (293)
cells and the methods used for transfection and isolation of clonal
cell lines stably transfected with the wild-type or mutant rLHR cDNAs
have been described in detail previously (12, 27, 37, 38).
Other Methods
Metabolic labeling of cells and subsequent immunoprecipitation
of the rLHR were achieved as previously described (1, 2, 12, 13, 14).
Autoradiograms of the dried gels were obtained using intensifying
screens, and the autoradiograms were scanned using a Bio-Rad Molecular
Imaging System (Bio-Rad Laboratories, Richmond, CA). All of the
autoradiograms shown here are reproductions from the scanned images.
Equilibrium binding parameters for hCG were measured during an
overnight incubation (4 C) of intact cells with a fixed concentration
of [125I]hCG and increasing concentrations of hCG as
described previously (37, 38). Concentration-response curves for
the hCG-induced increases in cAMP and inositol phosphate accumulation
were performed during a 30- and 60-min incubations at 37 C,
respectively. These were analyzed as described previously (12, 13).
Measurements of hCG-induced desensitization were performed after
incubation of cells with a fixed concentration of hormone (100 ng/ml)
for increasing periods of time. After removal of the free and bound
hormone, the cAMP response of the cells was assessed during a 15-min
incubation with the indicated concentrations of hCG (12, 13).
PMA-induced desensitization was measured after incubating the cells
with 200 nM PMA for 30 min. The cAMP response of the cells
was then assessed during a 15-min incubation with the indicated
concentrations of hCG (12, 13). The methodology used to asses the
endocytosis of hCG and to measure the rates of internalization have
also been described (27, 39, 40). For these experiments, the
surface-bound hormone was removed after a brief exposure of the cells
to an isotonic pH 3 buffer. The radioactivity that remains cell
associated was considered to be internalized hormone. Degraded hormone
was measured by solubility in trichloroacetic acid.
Hormones and Supplies
Purified hCG (CR-127) was obtained from the National Hormone and
Pituitary Agency of the NIDDK. [125I]hCG was prepared as
described previously (41), to give a specific radioactivity of
25,00030,000 cpm/ng. [32P]orthophosphate was obtained
from DuPont-New England Nuclear (Boston, MA). Methionine/cysteine- and
phosphate-free DMEM as well as Tran35S-Label were purchased
from ICN Biomedicals (Irvine, CA). Nonidet P-40, protease inhibitors,
N,N',N'-triacetylchitotriose, protein
A-agarose, fibronectin-like engineered polymer, and BSA were obtained
from Sigma Chemical Co. (St. Louis, MO). Okadaic acid and cypermethrin
were purchased from LC Laboratories (Woburn, MA). Wheat germ agglutinin
was obtained from Vector Laboratories (Burlingame, CA), and Geneticin
was obtained from Life Technologies (Grand Island, NY). The rabbit
antibody to the rLHR (Bugs) has been previously described (42). Cell
culture supplies were obtained from Corning (Corning, NY) and Life
Technologies, respectively. All other materials were obtained from
commonly used suppliers.
 |
ACKNOWLEDGMENTS
|
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We thank JoEllen Fabritz for assistance with the cell culture,
and Drs. Bill Hipkin and Deborah Segaloff for helpful discussions.
 |
FOOTNOTES
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Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2512 BSB, University of Iowa, Iowa City, Iowa 52242-1109.
This work was supported by a grant from the NIH (CA-40629). The
services and facilities provided by the Diabetes and Endocrinology
Research Center of the University of Iowa (supported by NIH Grant
DK-25295) are also gratefully acknowledged.
Received for publication October 3, 1996.
Revision received November 13, 1996.
Accepted for publication November 14, 1996.
 |
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