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Department of Pharmacology The University of Iowa College of Medicine Iowa City, Iowa 52242-1109
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Since GPCR activation is an important determinant of the
agonist-induced GPCR phosphorylation (reviewed in Refs. 10, 11), the
effects of activating and inactivating mutations of the rLHR on the
internalization of hCG could be directly due to the effects of these
mutations on receptor activation or they could be more indirectly
mediated by changes in receptor phosphorylation. This question has been
difficult to address with the rLHR because the level of expression of
the rLHR in transiently transfected cells is rather low. Although this
low level of transient expression does not affect our ability to
conduct internalization assays (3, 5, 6), the phosphorylation of the
rLHR can only be studied in stably transfected cells (1, 2, 3, 12, 13).
Thus, structure-function studies on the phosphorylation of the rLHR are
very laborious because stably transfected cell lines with matched
receptor numbers (at least 
100,000 receptors per cell) must be
prepared for each mutant to be analyzed (2, 3, 12, 13). In addition,
the use of cotransfection strategies that are helpful in understanding
receptor phosphorylation and internalization cannot be readily
accomplished using stably transfected cell lines.
In recent comparative studies on the human LH receptor (hLHR) and the
rat LH receptor we noticed that the level of expression of the hLHR is
much higher than that of the rLHR (40,000 and 400,000 receptors
per cell, respectively) in 293 cells transiently transfected with
optimal amounts of plasmid. Since these high levels of expression of
the hLHR allow for the quantitation of receptor phosphorylation in
transiently transfected cells, we reasoned that this experimental
system would be more amenable to structure-function studies designed to
address the involvement of receptor activation and phosphorylation in
the process of internalization. In the studies presented herein we took
advantage of these properties of the hLHR and analyzed several
mutants of the hLHR to better understand the importance of receptor
activation and phosphorylation in the trafficking of the hLHR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Since many of the experiments described below necessitate robust surface expression, our choice of naturally occurring mutations that impair signaling was severely limited by the finding that many of these mutants are usually characterized by decreased expression and/or proper plasma membrane localization. We eventually settled on a point mutation in TM7 (I625K) that reduces cell surface expression only by approximately 50% (18). This mutant was initially identified in three brothers with a mild form of Leydig cell hypoplasia (18). When compared with cells expressing the wild-type hLHR (hLHR-wt), the agonist-induced cAMP response of cells expressing the I625K mutation (measured indirectly as an increase in the expression of a cAMP-driven reporter gene) is characterized by a rightward shift in the EC50 and a reduction in the maximal response. Because it is important to examine mutations that impair signaling, we also chose to analyze two laboratory-designed hLHR mutations (D405N in TM2 and Y546F in TM5) that, based on equivalent mutations of other GPCRs (7, 8, 19, 20, 21, 22), were predicted to result in an impairment in agonist-induced activation of the hLHR without affecting surface expression.
One additional mutant (designated 5S/A), in which five serine residues in the C-terminal tail of the hLHR (residues 657, 661, 670, 674, and 676) were simultaneously mutated to alanines, was constructed and analyzed. This mutant was predicted to be phosphorylation deficient based on the knowledge that four equivalent serines of the rLHR (residues 635, 639, 649, and 652) become phosphorylated upon agonist stimulation (2, 3, 12).1
Although the signaling properties of some of the hLHR mutants described
above have been previously characterized (14, 16, 17, 18), it was important
to document these properties under our experimental conditions. This
was done by measuring cAMP accumulation in transiently transfected 293
cells incubated with increasing concentrations of hCG. For simplicity,
however, the data presented in Table 1
summarize only the response obtained
with a concentration of hCG (3 nM) chosen to elicit maximal
cAMP accumulation in cells expressing hLHR-wt (c.f. Fig. 1). To correct for the inherent
variability associated with measuring the cAMP responses of transiently
transfected cells, the basal and hCG-induced cAMP responses mediated by
the different mutants were corrected by normalization to the cholera
toxin-induced cAMP response measured in the same experiment (see
columns labeled "Basal/Cholera Toxin" and "hCG/Cholera Toxin"
in Table 1). Thus, instead of using the absolute levels of cAMP, the
responsiveness of cells expressing the different mutants shown in Table 1 should be compared by using these ratios. Lastly, all cells were also
tested for 125I-hCG binding to ensure that
variations in signaling were not due to variations in receptor
expression. Table 1 shows that the cell surface expression of all
activating mutations (L457R, D578Y, and D578H) is comparable to that of
hLHR-wt. When incubated in the absence of agonist, the basal levels of
cAMP detected in cells expressing the three activating mutations are
15- to 30-fold higher than those detected in cells expressing an
equivalent density of hLHR-wt (see "Basal/Cholera Toxin" in Table 1). These elevated levels of basal cAMP account for 25–50% of the
amount of cAMP produced when cells expressing an equivalent density
hLHR-wt are stimulated with a maximally effective concentration of hCG.
A comparison of the hCG/cholera toxin ratio to the basal/cholera toxin
ratio in Table 1 shows that the cells expressing the activating mutants
respond poorly to hCG stimulation (D578Y) or not at all (L457R or
D578H). Full dose-response curves for cells expressing these mutants
have been published by others (14, 16, 17) and are in general agreement
with the data shown in Table 1.
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show that cells expressing these mutants display a reduced
cAMP responsiveness to a concentration of hCG (3 nM) that
is maximally effective in cells expressing hLHR-wt. The representative
dose- response curves presented in Fig. 1 show that this reduction
is due mostly to an approximately 10- to 30-fold increase in the
EC50 for hCG displayed by cells expressing the
D405N or Y546F mutants. The maximal hCG-induced cAMP response of cells
expressing the D405N or Y546F mutants were only approximately 15 to
25% lower that those expressing a comparable density of hLHR-wt (Fig. 1). Cells expressing the naturally occurring mutation that impairs
signaling (I625K) show an approximately 2-fold decrease in surface
expression compared with cells expressing hLHR-wt as expected from
published results (18). The reduced cAMP responsiveness to 3
nM hCG shown by the hCG/cholera toxin ratio in Table 1 for
cells expressing the I625K mutant is a reflection of a reduction in the
maximal response and an increase in the EC50 as
previously documented by other investigators (18). Although this
reduced hCG response mediated by the I625K mutant should be interpreted
with some caution (because of its reduced surface expression, see Table 1), we do not think that it can be solely explained by reduced surface
expression because at these levels of receptor expression a 2- to
5-fold decrease in the expression of hLHR-wt has little or no effect on
basal or hCG-stimulated cAMP accumulation (data not shown). Lastly, the
basal cAMP and hCG-induced cAMP responses (as judged by the response
ratios shown in Table 1) mediated by the 5S/A mutant were
indistinguishable from those of cells expressing a comparable density
of hLHR-wt.
Phosphorylation of hLHR Mutants
After labeling with 32P-orthophosphate,
transiently transfected cells expressing the different mutants were
stimulated with maximally effective concentrations of hCG or PMA for 15
min at 37 C. Since the concentrations of hCG needed to induce
phosphorylation of the LHR closely resemble those needed for receptor
occupancy, we used a saturating concentration of hCG (26
nM) in an attempt to fully saturate the receptors and
optimize the phosphorylation signal during the short incubation period
used [the dissociation constant (Kd) for hCG
binding to the hLHR is 1–3 nM, see Ref. 6 ]. Stimulation
of 293 cells stably expressing the rLHR-wt with 200 nM PMA
has been previously shown to result in robust phosphorylation of rLHR
(1), and it was included here as a positive control. Cell lysates were
prepared, equalized for receptor expression (based on parallel binding
assays performed in intact cells), immunoprecipitated with the 9E10
antibody, resolved on SDS gels, and visualized and quantitated with a
PhosphorImager as described in Materials and Methods.
Stimulation of cells expressing hLHR-wt with hCG or PMA results in an
approximately 3- and 4-fold increase in the incorporation of
32P into the receptor, respectively (Figs. 2 and 3).
In general agreement with our analysis of phosphorylation sites of the
rLHR (2, 3, 12), we found that the simultaneous mutation of five serine
residues clustered toward the C-terminal end of the hLHR (a mutant
designated 5S/A) results in undetectable basal phosphorylation, an
approximately 90% inhibition of phosphorylation induced by hCG, and a
70–80% inhibition of the PMA-induced phosphorylation (Figs. 2 and 3).
Thus, we can readily conclude that most of the hCG-induced
phosphorylation of the hLHR occurs in one or more of these five
residues. We can also conclude that the sites phosphorylated in
response to PMA or hCG stimulation are overlapping but not
identical.
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). The addition of hCG to cells expressing
any of these activating mutations resulted in little or no additional
hLHR phosphorylation over that detected in the basal state (compare the
shaded and white bars shown for each mutant in
Fig. 3). The impairment in the ability of hCG to enhance the
phosphorylation of the L457R, -D578Y, and –D578H mutants parallels the
impairment in the hCG-induced cAMP response detected in cells
expressing these mutants (c.f. Table 1). Lastly, the addition of PMA to
cells expressing the L457R and D578Y mutants led to an increase in
receptor phosphorylation over that seen in the basal state (compare the
black and white bars shown for each mutant in Fig. 3), but
cells expressing D578H failed to respond to PMA with an additional
increase in phosphorylation above that detected in the basal state. The
lack of correlation between constitutive activation and increased basal
phosphorylation of the L457R, D578Y, and D578H mutants of the hLHR
(Figs. 2 and 3) agrees with recent findings reported for other GPCRs.
For example, constitutively active mutants of the 
1b- adrenergic
(24) or the angiotensin II type 1 receptor (25) do not uniformly
display an increase in agonist-independent phosphorylation. In
addition, agonist binding fails to enhance the phosphorylation of some
constitutively active mutants of these two GPCRs (24, 25).
As shown above, the magnitude of the effect of hCG on receptor
phosphorylation is not particularly strong, and phosphorylation assays
done in transiently transfected cells require robust expression of the
different receptor constructs. Unfortunately, the relatively low levels
of expression of the signaling-impaired I625K mutant (c.f. Table 1)
precluded the use of this mutant in phosphorylation experiments.
Therefore, the potential effects of signaling-impaired mutations on
receptor phosphorylation could only be analyzed with the two
laboratory-designed mutations that impair signaling (D405N and Y546F).
Cells expressing these mutants displayed normal levels of
agonist-independent receptor phosphorylation and an impairment in
hCG-induced phosphorylation (compare the shaded and
white bars shown for each mutant in Fig. 3) that paralleled
the impairment in their ability to mediate a cAMP response (Table 1).
Lastly, PMA was still able to enhance the phosphorylation of cells
expressing any of the inactivating mutations (compare the
black and white bars for each mutant in Fig. 3).
Overall then, the properties of the basal and hCG-dependent
phosphorylation of these mutations seem to parallel their activation
properties (as measured by cAMP accumulation and shown in Table 1).
Agonist-Induced Internalization of hLHR Mutants
Since the methods used to measure the t1/2
of internalization are rather sensitive (see Materials and
Methods) and this parameter is not dependent on the density of
cell surface receptors (26), the t1/2 values of
internalization of hCG could be reliably measured in cells expressing
any of the seven mutants described above.
The data summarized in Table 2 show that
cells expressing the three activating mutations (L457R, D578Y, and
D578H) internalize hCG with half-times that are 3- to 7-fold shorter
than that measured in cells expressing hLHR-wt. Cells expressing the
three signaling-impaired mutations (D405N, Y546F, and I625K)
internalize hCG with half-times that are 5- to 7-fold longer than those
measured in cells expressing hLHR-wt. Mutation of the phosphorylation
sites of the hLHR (5S/A) lengthened the t1/2 of
internalization of hCG less than 2-fold.
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). Arrestin-3(284–409), is a construct that encodes for the
clathrin-binding domain of arrestin-3 and behaves as a
dominant-negative inhibitor of nonvisual arrestin-mediated endocytosis
(31). Overexpression of this construct also lengthened the
t1/2 of internalization of hCG mediated by
hLHR-wt, -5S/A, and all three activating mutants but had only a small
effect on the already long t1/2 of
internalization of hCG mediated by the signaling-impaired mutations
(Table 2). It should also be noted that, in spite of the inhibitory
effects of arrestin-2(284–309) on the t1/2 of
internalization of hCG mediated by any of the activating mutations of
the hLHR, the t1/2 of internalization of hCG
measured in cells cotransfected with the activating mutants of the hLHR
and arrestin-3(284–409) was similar (i.e. 11–31 min) to
that detected in cells cotransfected with hLHR-wt and an empty vector
(i.e. 20 min). Conversely, arrestin-3 shortened the
t1/2 of internalization of hCG mediated by
hLHR-wt, -5S/A, and all three signaling-impaired mutations but had
little or no effect on the already short t1/2 of
internalization of hCG mediated by the activating mutations. The
effects of arrestin-3 on the inactivating mutations were rather
pronounced, and the t1/2 of internalization of
hCG in cells co-transfected with the D405N, Y546F, or I625K mutants and
arrestin-3 (i.e. 8–18 min) was similar to that detected in
cells cotransfected with hLHR-wt and an empty vector (20 min).
Since the D405N, Y546F, and I625K mutations impair receptor
activation2 (Table 1), phosphorylation
(Figs. 2 and 3), and agonist internalization (Table 2), we cannot
determine whether the impairment in internalization is due to the
impairment in receptor activation or in the phosphorylation of the
receptor. This is an important issue because the involvement of hLHR
phosphorylation on internalization appears to be minimal as documented
by the finding that the internalization of hCG is barely affected by
mutation of five serine residues in the C-terminal tail of the hLHR
(i.e. the 5S/A mutant in Table 2) that drastically impair
phosphorylation (Figs. 2 and 3) without impairing receptor activation
(Table 1). An independent assessment of the relative importance of
activation and phosphorylation in internalization was conducted by
analyzing double mutants in which the phosphorylation sites of the
D405N, Y546F, and I625K mutants were simultaneously mutated to alanine
residues. Since the phosphorylation of signaling-impaired mutants can
often be rescued by overexpression of one of the G protein-coupled
receptor kinases (GRKs) (22, 33), we also analyzed the behavior of
these mutants in cells cotransfected with GRK2.
The data presented in Fig. 4 show that
cotransfection with GRK2 enhances the hCG-promoted
phosphorylation3 of the hLHR-wt, and it
rescues the impairment in hCG-promoted phosphorylation displayed by the
D405N and Y546F mutants. Figure 4 also shows that mutation of the
phosphorylation sites of hLHR-wt, hLHR-D405N, and
hLHR-Y546F4 largely prevents
phosphorylation even in cells cotransfected with GRK2. The results
summarized in Fig. 5 show that 1)
mutation of the phosphorylation sites of hLHR-D405N, -Y546F, and
-I625K5 had no measurable
effect on the already slow t1/2 of
internalization of hCG mediated by these mutants (compare white
bars in the left and right panels of Fig. 5); 2) overexpression of GRK2 shortens the t1/2
of internalization of hCG mediated by D405N, Y546F, and I625K (compare
white and black bars on the left panel
of Fig. 5) to levels that are similar to those detected in cells
expressing hLHR-wt; and 3) mutation of the phosphorylation sites of
hLHR-D405N, -Y546F, and -I625K prevents the ability of GRK2 to rescue
the slow rate of internalization of hCG mediated by these mutants
(compare the white and black bars on the
right panel of Fig. 5).
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shows that mutation of the
phosphorylation sites of hLHR-L435R, -D578Y, and -D578H lengthens the
t1/2 of internalization of hCG approximately
3-fold, bringing it to values that are comparable to the
t1/2 of internalization mediated by hLHR-wt.
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, streptavidin blots of 9E10
immunoprecipitates of lysates obtained from biotinylated cells result
in the visualization of a prominent (85 kDa) band that is present in
293 cells transfected with hLHR-wt but absent from untransfected cells
or from cells transfected with the empty expression vector. This band
represents the mature, cell surface LHR as shown previously (37, 38).
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and the quantitation of several experiments
is shown in Fig. 8. During the 4-h
incubation period used here, there is little or no change in the
complement of surface LHR in cells expressing the hLHR-wt or the 5S/A
mutant. The density of surface receptors in cells expressing the three
activating mutations decreased by 25–75% during the same time period,
whereas the density of surface receptors in cells expressing the
signaling-impaired mutations decreased by 10–40%. Thus, the faster
turnover of the L435R, D578Y, and D578H mutants is not a property
uniquely associated with constitutive activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and Ref. 2). Second, the
mutation of these phosphorylation sites lengthens the
t1/2 of internalization of hCG mediated by the
hLHR or the rLHR 1.5- to 2.5-fold (Table 2 and Refs. 2, 3). It
should be noted that although the magnitude of this change is roughly
the same, the absolute value of the t1/2 of
internalization of the phosphorylation-deficient mutant of the rLHR is
approximately 10 times longer than that of the equivalent mutant of the
hLHR simply because the t1/2 of internalization
of the rLHR-wt is also several fold longer than that of the hLHR-wt
(6). Third, equivalent mutations of the hLHR (D405N in TM3 and Y546F in
TM6) and the rLHR (D383N in TM3 and Y524F in TM6) that impair
agonist-induced activation of the receptor lengthen the
t1/2 of internalization of hCG (Tables 1 and 2
and Refs. 7, 8), whereas equivalent mutations of the hLHR (L435R in
TM3 and D578Y in TM6) and the rLHR (L435R in TM3 and D556Y in TM6) that
induce agonist-independent receptor activation shorten the
t1/2 of internalization of hCG (Tables 1 and 2
and Refs. 8, 9). Fourth, overexpression of a dominant-negative
mutants of dynamin or a dominant-negative mutant of the nonvisual
arrestins lengthen the t1/2 of
internalization of hCG mediated by the hLHR and the rLHR (Table 2 and
Refs. 3, 5, 6), whereas overexpression of the nonvisual arrestins
shortens the t1/2 of internalization of hCG
mediated by the hLHR and the rLHR (Table 2 and Refs. 3, 5, 6, 26, 39). Our studies also highlight two major differences between the
hLHR and the rLHR. First, the t1/2 of
internalization of hCG mediated by the rLHR-wt is much slower (120
min) than that mediated by the hLHR-wt (20 min). We have already
shown that this difference can be fully accounted for by seven
noncontiguous residues that are not phosphorylated and are present in
topologically distinct domains (the second and third intracellular
loops and the juxtamembrane region of the C-terminal tail) of the
receptor (6). The second difference is on the effects of GRK2 on
internalization. Overexpression of this kinase shortens the
t1/2 of internalization of hCG mediated by the
rLHR-wt approximately 2-fold (39), but it has only a minimal effect on
the t1/2 of internalization of hCG mediated by
the hLHR-wt (Fig. 5, left panel).
When considered together these results clearly show that the rLHR and
the hLHR internalize hCG by a pathway that is facilitated by
agonist-induced activation and phosphorylation of the receptor and
requires the participation of the nonvisual arrestins and dynamin. More
importantly, our ability to analyze activation, internalization, and
phosphorylation of the hLHR in transiently transfected cells (as shown
in this paper) has now allowed us to conduct more extensive
structure-function studies that provided novel mechanistic information
about the relative importance of receptor activation and
phosphorylation to the process of internalization. A summary of the
most relevant data regarding this issue is presented in Table 3 to facilitate the following discussion. The overall
importance of receptor activation and phosphorylation can be readily
documented by the finding that three distant mutations (i.e.
D405N in TM2, Y546F in TM5, and I625K in TM7) of the hLHR that impair
hCG-induced receptor activation and hCG-induced phosphorylation
lengthen the t1/2 of agonist internalization to
values (100–150 min) that are close to the t1/2
of internalization of hCG mediated by the hLHR-wt in cells in which
endocytosis has been blocked with a dominant- negative mutant of
dynamin (160 min). The results summarized in Table 3 also show that
1) phosphorylation is relatively unimportant to internalization if
activation is normal because the 5S/A mutant internalizes hCG with a
t1/2 (30 min) that is only slightly longer
than that of hLHR-wt (20 min); and 2) activation is relatively
unimportant to internalization if phosphorylation is normal because the
long t1/2 of internalization of hCG displayed by
the signaling-impaired mutants can be shortened to 30–50 min when the
phosphorylation of these mutants is rescued by cotransfection with
GRK2. When considered together, these results are consistent with a
model whereby hCG-induced activation and phosphorylation of the hLHR
play redundant roles in internalization. Ultimately, however, it is
obvious that in the absence of phosphorylation or receptor activation,
the long t1/2 of internalization of hCG can be
readily rescued by overexpression of arrestin-3 (Table 2). Thus, the
data presented here also document the paramount importance of the
interaction of the hLHR with a nonvisual arrestin to the process of
internalization.
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The reasons behind the ability of activating mutations of the hLHR
(L457R, D578Y, and D578H) to internalize hCG faster than hLHR-wt (Table 2) remain elusive. As shown here these mutants cannot be further
activated or phosphorylated by hCG (Table 1 and Figs. 1 and 2). The
involvement of receptor phosphorylation in the internalization of hCG
can still be documented by mutation of the phosphorylation sites
of these activating mutants, however (Fig. 6). Thus, when occupied by
hCG, these three activating mutations seem to assume a conformation
that bypasses many of the requirements (i.e. activation,
phosphorylation, and/or arrestin binding) needed for the
internalization of agonist.
In summary, the studies presented herein and elsewhere (2, 3, 7, 8, 9) show a remarkable association between the activation and the internalization of the LHR, and the new studies presented here imply that the activation and phosphorylation of the hLHR play redundant roles in the process of internalization. The intimate association between receptor activation and internalization suggests that internalization is part of the process of activation or, conversely, that it represents a short feedback loop involved in the termination of hormone action. Either scenario argues for continued research to fully understand the molecular and cellular basis of this pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Vectors encoding for GRK2 (45) arrestin-3 and arrestin-3(284–409) (31) were generously provided by Dr. Jeff Benovic (Thomas Jefferson University, Philadelphia, PA). An expression vector for dynamin-K44A (27) was generously provided by Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA). All of these were subcloned into pcDNA3.1. The expression of these constructs in our experimental system has been previously documented (34, 39).
Human embryonic kidney (293) cells were maintained in DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4. Transient transfections were done using the calcium phosphate method of Chen and Okayama (46). Cells were plated in 100-mm dishes that had been coated with gelatin and transfected (using 5 µg of plasmid for the 100-mm dishes or 0.2 µg of plasmid for the 35-mm wells) when 70–80% confluent. After an overnight incubation, the cells were washed, trypsinized, and, depending on the assay to be performed, plated in gelatin-coated dishes or wells and incubated for an additional 24 h before use.
Binding, Internalization, and cAMP Assays
The expression of the different receptor constructs was
ascertained by measuring the binding of a saturating (13
nM) concentration of 125I-hCG (the
Kd for hCG binding to the hLHR is 1–3
nM; see Ref. 6) to intact cells. All binding assays were
done during a 1-h incubation at room temperature using transfected
cells plated in gelatin-coated 35-mm wells (see above). They were all
corrected for nonspecific binding, which was measured in the presence
of 50 IU/ml of partially purified hCG (3,000 IU/mg). The methods used
to measure the internalization of 125I-hCG have
been described previously (5, 26). Determinations of the rates of
internalization were done using at least five different data points
collected at 3- to 10-min intervals (depending on the construct
transfected) after the addition of a concentration of
125I-hCG (3 nM) equivalent to the
Kd. The endocytotic rate constant (ke) was
calculated from the slope of the line obtained by plotting the
internalized radioactivity against the integral of the surface-bound
radioactivity (6, 26, 47). The half-time of internalization is defined
as 0.693/ke.
Hormonal responsiveness was assessed by measuring cAMP accumulation in
intact transfected cells plated in gelatin-coated 35-mm wells (see
above). Total cAMP was measured at the end of a 2-h incubation (37 C)
in medium devoid of phosphodiesterase
inhibitors6 and supplemented with
buffer only or with concentrations of hCG (3 nM) or cholera
toxin (0.6 nM) that are known to be maximally effective in
transiently transfected cells expressing hLHR-wt (14, 48).
Dose-response curves were generated by incubating transiently
transfected cells with increasing concentrations of hCG as shown in
Fig. 1. The parameters that describe these dose responses
(i.e. EC50 and maximal response) were
calculated from these data as described elsewhere (7, 12, 13).
Phosphorylation Assays
Cells were plated in 100-mm dishes that had been coated with
gelatin and transfected when 70–80% confluent. After an overnight
incubation, the cells were washed, trypsinized, and plated in
gelatin-coated 100-mm dishes and in gelatin-coated 35-mm wells and
incubated in DMEM containing 10 mM HEPES, 1% BSA, and 50
µg/ml gentamicin, pH 7.4, for 24 h.
The cells plated in 35-mm wells were used to assess cell surface receptor expression by 125I-hCG as described above. The cells plated in the 100-mm dishes were used for phosphorylation assays as follows. The medium was aspirated and the cells were incubated for 3 h at 37 C in DMEM devoid of phosphate but containing 10 mM HEPES, 1% BSA, 50 µg/ml gentamicin, and 200 µCi/ml 32P-orthophosphate. At this point the cells received buffer only, hCG (final concentration, 26 nM), or PMA (final concentration, 200 nM) and the incubation was continued for 15 min at 37 C. These concentrations and times were chosen based on the maximal effects of hCG and PMA on the phosphorylation of the rLHR (1, 2, 3, 13). Cells were placed on ice and washed once with buffer A (0.15 M NaCl, 20 mM HEPES, 5 mM EDTA, 3 mM EGTA, 50 mM ß-glycerophosphate, 10 mM NaF, 100 µM sodium orthovanadate, 1 mM phenylmethyl sulfonyl fluoride, 1 µM leupeptin, 0.08 µg/ml okadaic acid, 1 nM cypermethrin, and 1 µM pepstatin A, pH 7.4). After addition of 1 ml of lysis buffer (1% NP40, 4 mg/ml dodecyl-ß-D-maltoside. 0.8 mg/ml cholesteryl hemisuccinate in buffer A) the dishes were rocked on ice for 30 min. The lysate was clarified by centrifugation, and aliquots containing the same amount of receptor (calculated from the binding experiments done in parallel as described above) were partially purified on a wheat germ agglutinin column as described before (37). The eluant from the wheat germ agglutinin column was incubated with a monoclonal antibody to the myc epitope (9E10) that had been preabsorbed to agarose-conjugated protein G (see below) for 90–120 min at 4 C. After extensive washing the material absorbed to the beads was eluted by vigorous mixing of the beads in SDS sample buffer for 15 min at room temperature. The eluted material was then resolved on SDS gels, the gels were visualized and quantitated using a PhosphorImager, and the images were captured in a digital format for presentation.
In more recent experiments we have been able to omit the wheat germ
agglutinin column and use crude lysates for immunoprecipitation. This
protocol is less laborious and more economical but it often reveals
faint bands of a mol wt similar to that of the hLHR in the negative
controls (c.f. Fig. 4). These are nonspecific bands, however, that can
also be detected in untransfected cells or in cells transfected with
pcDNA3.1.
Prebinding of the 9E10 antibody to the protein G agarose beads was accomplished by incubating 50 µl of a 20–25x dilution of concentrated antibody (a concentrated supernatant from cultured 9E10 cells) with 25 µl of a 50% slurry of protein G agarose (purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. The beads were then washed by centrifugation and used as described above.
Analysis of the Turnover of the Free hLHR by Surface
Biotinylation
Transfected cells plated in gelatin-coated 35-mm wells (see
above) were washed four times with ice-cold PBS (10 mM
sodium phosphate, 150 mM NaCl, pH 8) and then biotinylated
during two consecutive 15 min incubations (at room temperature) with
freshly prepared 0.5 mg/ml solutions of
sulfosuccinimidyl-6-(biotinamido)hexanoate (from Vector Laboratories, Inc., Burlingame, CA) in the same buffer. The
cells were then washed once with DMEM containing 10 mM
HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4, and
twice with PBS (35, 36). Some cells were saved on ice and processed
immediately (t = 0 samples), while others were incubated in warm
DMEM containing 10 mM HEPES, 10% newborn calf serum, and
50 µg/ml gentamicin, pH 7.4, for 4 h. At the indicated times the
cells were placed on ice, lysed, and immunoprecipitated with the 9E10
antibody prebound to protein G agarose. Lysates and
immunoprecipitations were prepared as described in
Phosphorylation Assays, except that okadaic acid and
cypermethrin were not included in the solutions and the lysates were
not purified on wheat germ agglutinin before immunoprecipitation. Also,
no attempt was made to normalize the amount of receptor
immunoprecipitated among the different constructs used. The
immunoprecipitates were resolved on SDS gels as described above, and
the resolved proteins were electrophoretically transferred to
polyvinylidene fluoride membranes as described elsewhere (49).
After blocking (49), the blots were incubated for 1 h with 100
ng/ml of horse-radish peroxidase-conjugated streptavidin (from
Vector Laboratories, Inc.), and the proteins were finally
visualized using the Super Signal West Femto Maximum Sensitivity system
of detection from Pierce Chemical Co. (Madison, WI). The
blots were visualized and quantitated using a Fluo-S MAX system
(Bio-Rad Laboratories, Inc., Hercules, CA). These images
were also captured in a digital format for presentation (c.f. Fig. 7).
Hormones and Supplies
Human embryonic kidney (293) cells and the 9E10 hybridoma cell
line were obtained from the American Type Culture Collection (Manassas, VA). Purified hCG (CR-127, 13,000
IU/mg) was kindly provided by Dr. A. Parlow and the National Hormone
and Pituitary Agency of the National Institute of Diabetes and
Digestive and Kidney Diseases. 125I-hCG was
prepared as described elsewhere (50). Partially purified hCG (3,000
IU/mg) was purchased from Sigma (St. Louis, MO), and it
was used only for the determination of nonspecific binding (see above).
125I-cAMP and cell culture medium were obtained
from the Iodination Core and the Media and Cell Production Core,
respectively, of the Diabetes and Endocrinology Research Center of the
University of Iowa. Concentrated supernatant from the 9E10 cells was
prepared by the Hybridoma Facility of the Cancer Center of the
University of Iowa. Other cell culture supplies and reagents were
obtained from Corning, Inc. (Corning, NY) and Life Technologies, Inc. (Gaithersburg, MD), respectively. All other
chemicals were obtained from commonly used suppliers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by a grant from the NIH: CA-40629 to MA. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by NIH grant DK-25295.
1 Note that the mature rLHR and hLHR highly
homologous proteins are composed of 674 and 677 residues, respectively
(23 ). The different numbers assigned to equivalent residues are
artificially caused by differences in the numbering of amino acids.
Since the N terminus of the mature hLHR is not known, residue number 1
is taken to be the methionine present at the N terminus of the signal
peptide. In contrast, since the N terminus of the mature rLHR is known,
this residue (which corresponds to residue 23 of the hLHR) is taken to
be residue number 1. 
2 Note that although we use cAMP as a measurement
of receptor activation we do not imply that cAMP per se
is necessary for internalization. In fact, a role for cAMP in this
process has already been excluded (32 ).
3 In agreement with previous results (22 33 34 ),
we found that GRK2 cotransfections did not enhance receptor
phosphorylation in cells incubated without hCG.
4 Mutation of the phosphorylation sites of the
activating or inactivating mutations of the hLHR had little or no
effect on their signaling properties (not shown).
5 As noted above, the low level of expression of
the I625K mutant made it difficult to reliably measure phosphorylation.
Throughout the rest of this paper we assume that the properties of the
phosphorylation of the I625K mutant are similar to those of the D405N
and Y546F mutants.
6 Experiments performed in medium with
isobutylmethylxanthine (a phosphodiesterase inhibitor) gave
qualitatively similar results.
Received for publication June 7, 2000. Revision received July 25, 2000. Accepted for publication August 11, 2000.
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