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J Biol Chem, Vol. 274, Issue 36, 25426-25432, September 3, 1999
,From the Department of Pharmacology, The University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Previous results from this laboratory have shown that human kidney (293) cells transfected with the rat follitropin receptor (rFSHR) internalize agonist (i.e. human follitropin, hFSH) at a rate similar to that of other agonist-G protein-coupled receptor complexes while 293 cells transfected with the rat lutropin/choriogonadotropin receptor (rLHR) internalize agonist (human choriogonadotropin, hCG) at a rate that is about 1 order of magnitude slower. Taking advantage of this difference and the high degree of homology between the rLHR and rFSHR, we have now used chimeras of these two receptors to begin to delineate structural features that influence their internalization.
Analysis of six chimeras that exchanged only the transmembrane domains (designated FLF and LFL), only the COOH-terminal domains (FFL or LLF) or both domains (FLL or LFF) show that the origin of the extracellular domain is at least as important, if not more, than the origin of the transmembrane and COOH-terminal domains in determining the rate of internalization of the gonadotropin receptors. Thus, the rates of internalization of agonist internalization mediated by FFL, FLF, and FLL more closely resemble rFSHR than rLHR, while the rates of agonist internalization mediated by LLF, LFL, and LFF more closely resemble rLHR than rFSHR.
The importance of the extracellular domain was also evident even upon
overexpression of arrestin-3, a protein that enhances the rate of
internalization of the wild-type receptors and chimeras by binding to
their intracellular regions.
The binding of agonists to G protein-coupled receptors
(GPCRs)1 triggers a number of
processes involved in signaling and desensitization (1-3). One of
these, the endocytosis of the agonist-receptor complex, is of
particular interest because it has been implicated in a variety of
functions, including the termination of cAMP signaling (4, 5), the
initiation of mitogenic signaling (6), the dephosphorylation and
resensitization (7, 8) or the down-regulation (9, 10) of GPCRs.
Despite the abundance of knowledge about structural motifs that mediate
endocytosis of receptors that span the membrane only once (11), little
information is available about the structural motifs that mediate the
endocytosis of the seven-transmembrane spanning GPCRs. Some studies
utilizing C-terminal truncations as well as receptor chimeras have
identified the C-terminal tail as being particularly important for
endocytosis (12-17), but a discrete definition of these motifs is not
yet available. Other studies indicate that a dileucine motif conserved
in the C-terminal tail may be involved in the endocytosis of some (18)
but not other GPCRs (17). A NPXY motif is also conserved in
transmembrane helix seven of the GPCRs. Although mutations of this
motif impair agonist-induced internalization they do so indirectly, by
preventing the agonist-induced activation rather than by a direct
participation in endocytosis (1).
The FSHR, LHR, and thyrotropin receptors, collectively known as the
glycoprotein hormone receptors, make up a small subfamily of the large
family of rhodopsin-like G GPCRs (19-21). They are among the largest
members of this family (~700 residues long), and about half of these
residues are located extracellularly. The extracellular amino-terminal
domains of the glycoprotein hormone receptors are encoded by multiple
exons and are responsible for the recognition and high affinity binding
of their large (28-38 kDa) glycoprotein hormone ligands. Their
transmembrane and cytoplasmic tails are encoded by a single exon and
are involved in signal transduction. Amino acid sequence alignments
among the glycoprotein hormone receptors from the same species (19, 20)
reveal a substantial degree of overall identity (40-50%). As expected
from their roles, there is a smaller degree of identity in the
extracellular domains (~40%) than in the transmembrane domains
(60-75%). Surprisingly, however, the COOH-terminal domains are the
most divergent, displaying an amino acid sequence identity of only
20-30%.
Like other GPCRs the binding of agonists to the gonadotropin receptors
(LHR or FSHR) triggers the endocytosis of the agonist-receptor complex
by a pathway that is dependent on a nonvisual arrestin, clathrin, and
dynamin (9, 22-30). Once internalized the agonist-LHR complex
accumulates in the lysosomes, where both the agonist and the receptor
are degraded (24-26). The fate of the internalized FSHR has not been
investigated, but it is clear that the internalized FSH also
accumulates in the lysosomes where it is eventually degraded to single
amino acids (22, 23).
In a recent series of experiments using a standardized assay to measure
the internalization of the rLHR and rFSHR expressed in transfected 293 cells, we have reported that the agonist-rFSHR complex is internalized
with a t1/2 of ~10 min (27, 28), while the
agonist-rLHR complex is internalized with a t1/2 of
~100 min (5, 9, 29, 31, 32). In the present study we have analyzed
six chimeras of the rFSHR and rLHR to begin to identify regions of
these receptors that influence their rates of internalization.
Plasmids and Cells--
Full-length cDNAs encoding for the
rFSHR and rLHR (33, 34) were subcloned into the pcDNAI/Neo
(Invitrogen) for expression. Six chimeras of these two receptors were
constructed using PCR strategies. Their identity was verified by
automated DNA sequencing (performed by the DNA core of The Diabetes and
Endocrinology Research Center of the University of Iowa). The overall
structure of these chimeras and the exact location of the junctions are
shown in Fig. 1. In order to name them we adopted a nomenclature in
which each receptor was subdivided into three domains,
NH2-terminal extracellular, transmembrane, and
COOH-terminal intracellular, and the presence of an L or and F in a
given position of each chimera indicates the origin of that region. For
example, LLF is a chimera in which the NH2-terminal
extracellular and the transmembrane domains are derived from the rLHR
and the COOH-terminal intracellular domain is derived from the
rFSHR.
An expression vector for arrestin-3 (35) was generously provided by Dr.
Jeff Benovic (Thomas Jefferson University).
Human embryonic kidney (293) cells were obtained from the American Type
Culture Collection (CRL 1573) and maintained in Dulbecco's modified
Eagle's medium containing 10 mM Hepes, 10% new born calf serum and 50 µg/ml gentamicin, pH 7.4. Transient transfections were
done using the calcium phosphate method of Chen and Okayama (36). Cells
were plated in 100-mm dishes and transfected with 10 µg of plasmid
when 70-80% confluent. After an overnight incubation the cells were
washed, trypsinized, and replated in 35-mm wells (5-10 × 105 cells/well), and used 24 h later.
Internalization Assays--
The endocytosis of
125I-hCG and 125I-hFSH were measured as follows
(37-39). Cells (plated in 35-mm wells) were preincubated in 1 ml of
Waymouth's MB752/1 containing 1 mg/ml bovine serum albumin and 20 mM Hepes, pH 7.4, for 30-60 min at 37 °C. Each well
then received 10 ng/ml 125I-hCG or 40 ng/ml
125I-hFSH (these concentrations are equivalent to the
Kd for the hCG-rLHR and the hFSH-rFSHR interaction,
respectively), and the incubation was continued at 37 °C. At the
times indicated cells were placed on ice and washed two to three times
with 2-ml aliquots of cold Hanks' balanced salt solution containing 1 mg/ml bovine serum albumin. The surface-bound hormone was then released by incubating the cells in 1 ml of cold 50 mM glycine, 150 mM NaCl, pH 3, for 2-4 min (24). This buffer was removed
and the cells were washed once more. The acid buffer washes were
combined and counted, and the cells were solubilized with 100 µl of
0.5 N NaOH, collected with a cotton swab, and counted to
determine the amount of internalized hormone.
Determinations of the rates of internalization were done using at least
five different data points collected at 3-10-min intervals (depending
on the chimera used). 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 (37-40). The half-life of
internalization (t1/2) is defined as
0.693/ke.
Hormones and Supplies--
Purified hFSH (AFP-5720D), hCG
(CR-127), and pregnant mare serum gonadotropin were kindly provided by
the National Hormone and Pituitary Agency of the National Institute of
Diabetes and Digestive and Kidney Diseases. 125I-hFSH and
125I-hCG were prepared as described elsewhere (41).
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. Other cell culture supplies and reagents were obtained from
Corning and Life Technologies, Inc., respectively. All other chemicals
were obtained from commonly used suppliers.
Construction and Signaling Properties of Gonadotropin Receptor
Chimeras--
Six chimeras of the rLHR and the rFSHR were constructed
as shown in Fig. 1. Exchanging only the
COOH-terminal cytoplasmic domains produced LLF and FFL; exchanging only
the transmembrane domains produced LFL and FLF; and exchanging the
transmembrane and COOH-terminal cytoplasmic domains produced LFF and
FLL.
The identity of the rLHR/rFSHR chimeras was confirmed by sequencing
(see "Materials and Methods") and by measuring their ability to
bind hCG and hFSH and to respond to them with increased cAMP accumulation (Table I), because these
properties should be entirely dependent on the origin of the
extracellular domain (42, 43). In these experiments the levels of cell
surface expression of the different chimeras containing the
extracellular domains of the rLHR or the rFSHR were matched (as
measured by binding of their respective agonists, see column labeled
"Ligand-bound" in Table I), and the cAMP responses mediated by the
chimeras were corrected by normalizing the data to an internal control
obtained by measuring the cAMP response of the transfected cells to
cholera toxin (see column labeled "Response ratio" in Table I). All
chimeras containing the extracellular domain of the rFSHR responded
well to a maximally effective concentration of hFSH, but this response was somewhat lower in FFL than in the other chimeras. The cAMP response
to hCG was minimal in all the chimeras containing the extracellular
domain of the rFSHR. The cAMP response of the chimeras containing the
extracellular domain of the rLHR to a maximally effective concentration
of hCG seemed to be more variable, and two chimeras (LLF and LFF)
displayed a substantial reduction in this response when compared with
rLHR. The cAMP response to hFSH was minimal in all the chimeras
containing the extracellular domain of the rLHR.
Internalization of hFSH and hCG--
When 293 cells transiently
transfected with the rLHR are exposed to 125I-hCG they
internalize the bound hormone slowly (Fig.
2, top panels) while cells
transiently transfected with the rFSHR internalize the bound
125I-hFSH quickly (Fig. 2, bottom panels). Using
these experimental conditions one can calculate an endocytotic rate
constant (ke) from the slopes of two kinds of
plots as shown in Fig. 2, B and E (39), or in
Fig. 2, C and F (37, 38, 40). In either case the
half-life of internalization (t1/2) is defined as
0.693/ke. While both plots give similar results (compare Fig. 2, B and C, and Fig. 2,
E and F), we routinely used plots like those
shown in Fig. 2, C and F, as opposed to those shown in Fig. 2, B and E, to calculate rates of
internalization because they tended to be linear with more data points,
and the results obtained were more reproducible.
In doing these experiments care must be taken to avoid the loss of the
internalized ligand (which occurs as consequence of its degradation and
subsequent release of degradation products into the medium) during the
time course of measurement (39, 40). Since degradation products of
125I-hCG begin to be released within 90 min (24, 31), the
longest time point used to estimate the t1/2 of
internalization of 125I-hCG was 60 min (see Fig.
2A). As shown in Fig. 2, B and C, the t1/2 of internalization of 125I-hCG is
~100 min, and since the 60-min time course used is shorter than the
calculated t1/2 of internalization, it can be argued
that using this method to estimate the t1/2 of
internalization may yield erroneous results. We don't believe that is
the case, however, because measurements of the rate of internalization
of 125I-hCG in 293 cells transfected with the rLHR using
other methods that follow the fate of the bound 125I-hCG
for up to 4 h have resulted in estimates of the
t1/2 of internalization that are similar
(i.e. 80-140 min) to those obtained with the method used
here (5, 29, 31, 32). Degradation products of 125I-hFSH
begin to be released within 45 min (data not shown), and the longest
time point used for the experiments utilizing 125I-hFSH was
18 min (Fig. 2D). As shown in Fig. 2, E and
F, this time course is about twice as long as the
t1/2 of internalization of the
125I-hFSH.
The internalization of 125I-hCG and 125I-hFSH
mediated by the different chimeras was next analyzed using transiently
transfected 293 cells. In contrast to the agonist-induced cAMP
responses, which are dependent on receptor density, the rate of
internalization of agonist was found to be independent of receptor
density (data not shown, also see "Discussion"). In the experiments
presented in Fig. 3; however, the levels
of cell surface expression of the different chimeras containing the
extracellular domains of the rLHR or the rFSHR were matched as
described for the experiments presented in Table I. The internalization
data summarized in Fig. 3 are grouped based on the origin of the
extracellular domain, because all chimeras containing the extracellular
domain of the rFSHR internalized 125I-hFSH at a rate that
was similar to that of the rFSHR (Fig. 3A), while all
chimeras containing the extracellular domain of the rLHR internalized
125I-hCG at a rate that was similar to that of the rLHR
(Fig. 3B). Within each of these major groups, however, the
rate of internalization was also affected by the origin of the
transmembrane and/or COOH-terminal cytoplasmic domains. Fig.
3A shows that substitution of only the COOH-terminal domain
of the rLHR into the rFHSR (i.e. the FFL chimera) lengthened
the t1/2 of internalization of
125I-hFSH, while substitution of only the transmembrane
domain (i.e. the FLF chimera) had little or no effect on the
internalization of 125I-hFSH. The combined substitution of
these two domains (i.e. the FLL chimera) was more effective
in lengthening the t1/2 of internalization than the
individual substitution of the COOH-terminal domain, however. Fig.
3B shows that exchanging only the COOH-terminal tail of the
rLHR for the rFSHR (i.e. LLF) had little or no effect on the
internalization of 125I-hCG, while exchanging only the
transmembrane domain of the rLHR for the rFSHR (i.e. LFL)
shortened the t1/2 of internalization. The combined
substitution of both of these domains (i.e. LFF) was only
slightly more effective in shortening the t1/2 of
internalization than the individual substitution of the transmembrane
domain, however.
Previous results from this laboratory have shown that overexpression of
arrestin-3 enhances the internalization of agonists mediated by the
rLHR or the rFSHR and that the magnitude of this effect is more
pronounced with 125I-hCG than with 125I-hFSH
(28-30). This phenomenon is illustrated by the results presented in
Table II. Overexpression of arrestin-3
shortens the t1/2 of internalization of
125I-hFSH mediated by rFSHR less than 2-fold, but it
shortens the t1/2 of internalization of
125I-hCG mediated by rLHR ~4-fold. Since the arrestins
are known to bind to the intracellular regions of GPCRs (reviewed in
Ref. 2), we expected that the overexpression of arrestin-3 would have a
greater effect on the internalization of agonist mediated by FLL than
on the internalization of agonist mediated by LFF. As shown in Table II
this was in fact found to be the case. Arrestin-3 shortened the
t1/2 of internalization of agonist mediated by FLL
and by LFF ~7- and ~4-fold, respectively. Even in cells
cotransfected with arrestin-3, the t1/2 of
internalization of agonist mediated by LFF was till ~4-fold longer
than the t1/2 of agonist internalization mediated by
FLL, however.
Thus, all results presented show that the origin of the extracellular
domain has a substantial effect on the rate of internalization of the
gonadotropin receptors.
Six chimeras of the rLHR and rFSHR were used in experiments
designed to determine the structural basis of the different rates of
agonist internalization mediated by the gonadotropin receptors.
Results on the specificity of gonadotropin responsiveness show that the
abilities of the different chimeras to recognize and respond to a given
ligand (i.e. hCG or hFSH) are entirely dictated by the
origin of the extracellular domain. Thus, all rLHR/rFSHR chimeras that
have the extracellular domain of the rLHR display a robust cAMP
response to hCG and a minimal response to hFSH, while those that have
the extracellular domain of the rFSHR display a robust cAMP response to
hFSH and a minimal response to hCG (Table I).
In order to avoid potential problems with a possible effect of receptor
density on internalization, all the experiments presented here were
conducted using transiently transfected cells with matched receptor
density. It should be emphasized, however, that the rates of
internalization of agonist mediated by the rLHR or the rFSHR are
independent of receptor density and of the mode of transfection (i.e. transient versus stable transfections).
Previous experiments from this laboratory have shown that the
t1/2 of internalization of 125I-hFSH is
~10 min in clonal lines of 293 cells stably expressing the rFSHR at a
density of 40,000 to 400,000 molecules/cell (27, 28). The experiments
reported here for the rFSHR were done using transiently transfected 293 cells expressing an average density of ~40,000 molecules/cell and the
t1/2 of internalization of 125I-hFSH was
also ~10 min (Figs. 2 and 3 and Table II). Increasing the expression
of the rFSHR to ~400,000 molecules/cell did not affect the rate of
internalization, however. Likewise, the t1/2 of
internalization of 125I-hCG mediated by the rLHR is ~100
min in clonal lines of 293 cells stably expressing the rLHR at a
density of 3,000-200,000 molecules/cell (5, 29, 31, 32). This value
compares well with the t1/2 of internalization 125I-hCG mediated by the rLHR reported here (Figs. 2 and 3
and Table II) for transiently transfected 293 cells expressing rLHR at
an average density of ~40,000 molecules/cell.
Like many other GPCRs (2, 3), the agonist-induced activation of the LHR
and FSHR are necessary for the efficient endocytosis of the
agonist-receptor complex. Thus, the rate of internalization of the
hCG-LHR complex is faster than that of the free LHR (5, 37); the rate
of internalization of an antagonist-LHR complex is slower than that of
the agonist-LHR complex (44); signaling impairing mutations of the rLHR
(5, 31) or the rFSHR (28) decrease internalization of the
agonist-receptor complex; and activating mutations of the rLHR enhance
the internalization of the free and/or agonist-occupied receptor (5).
Cyclic AMP accumulation per se is not needed for
internalization, however, because the slow rate of internalization of
the antagonist-LHR complex is not enhanced by the addition of cAMP
analogs, and the internalization of the hCG-LHR complex is not impaired
in cells that express a cAMP-resistant phenotype (44). Likewise, the
internalization of the hFSH-rFSHR complex is also not impaired in cells
that express a cAMP-resistant
phenotype.2
All the chimeras used here are capable of being activated by their
appropriate agonists, as judged by cAMP accumulation (Table I). While
their ability to become activated in response to agonist binding may
vary, there is no correlation between this parameter and the rate of
internalization of agonist. If the extent of agonist-induced activation
was an important determinant of the rate of internalization of a given
chimera one would expect that the chimeras that do not respond well
with an increase in cAMP accumulation would display a long
t1/2 of internalization. This is certainly not the
case, as shown by the finding that the t1/2 of
internalization of agonist mediated by FFL and FLL are both longer than
the t1/2 of internalization of agonist mediated by
rFSHR (Fig. 3), yet only the agonist-induced activation of FFL is
reduced compared with that of rFSHR (Table I). Likewise, the
t1/2 of internalization of agonist mediated by LLF
is similar to that or rLHR (Fig. 3), but the agonist-induced activation
of LLF is substantially lower than that of rLHR (Table I). Last, the
t1/2 values of internalization of agonist mediated
by LFL and LLF are both shorter than that of rLHR (Fig. 3), but the
agonist-induced activation of LFL is similar to that of rLHR, while the
agonist-induced activation of LFF is less than that of rLHR (Table
I).
One reason why there is a correlation between the agonist-induced
activation and internalization of some GPCRs is that the agonist-activated GPCRs are better substrates for the G protein-coupled receptor kinases (GRKs) than the free receptors. The GRK-phosphorylated GPCRs in turn have a higher affinity for a family of proteins called
arrestins and some of these (i.e. the nonvisual arrestins) are known to act as adapter molecules bridging the phosphorylated GPCR
to clathrin for subsequent endocytosis via coated pits (2, 3). This
general pathway applies to the gonadotropin receptors as illustrated by
the following findings. First, dominant-negative mutants of GRK2,
nonvisual arrestins or dynamin inhibit internalization (28-30).
Second, cotransfections with arrestin-3 or several members of the GRK
family (i.e. GRK2, GRK4 Since agonist-induced activation promotes the phosphorylation of serine
residues present at the COOH-terminal cytoplasmic tail of the rLHR (29,
32) or serine and threonine residues present in the first and third
intracellular loops of the rFSHR (27, 28), and the arrestin binding
sites of GPCRs have been mapped to intracellular regions (2), we
predicted that the structural determinants for endocytosis of the
gonadotropin receptors must reside in their intracellular loops and/or
COOH-terminal cytoplasmic tails. The analysis of the six rLHR/rFSHR
chimeras show indeed that the transmembrane and/or cytoplasmic domains of these receptors affect the rate of internalization (Fig. 3). It
appears that the cytoplasmic tail of the rLHR is more important for
internalization than the transmembrane domain because the FLF chimera
internalized agonist at about the same rate as the rFSHR, while the FFL
chimera internalized agonist slower than the rFSHR. Some kind of
interaction between these two domains of the rLHR may occur during
internalization, however, because grafting the cytoplasmic and
transmembrane domains of the rLHR onto the extracellular domain of the
rFSHR resulted in a chimera, FLL that internalized agonist at a slower
rate than FFL or FLF. In contrast, the transmembrane domain of the
rFSHR seems to be more important for internalization than the
cytoplasmic tail because the LLF chimera internalized agonist at about
the same rate as rLHR, while the LFL chimera internalized agonist at a
slightly faster rate than rLHR wild type. Moreover, the combined
substitution of both of these domains resulted in a chimera, LFF that
internalized agonist at about the same rate as the chimera containing
only the transmembrane domain.
While these findings provide a necessary first step in defining
structural motifs that modulate the rate of internalization of agonist
mediated by the gonadotropin receptors, the most interesting and
surprising finding reported here is that the extracellular domain is at
least as important, if not more, in determining the rate of agonist
internalization. Thus, while there is a ~10-fold difference in the
t1/2 of internalization of agonist mediated by the
rFSHR and rLHR (Table II), grafting the transmembrane and cytoplasmic
domains of the rFSHR onto the extracellular domain of the rLHR shortens
the t1/2 of agonist internalization only ~2-fold
(compare rLHR and LFF in Table II). Likewise, grafting the
transmembrane and cytoplasmic domains of the rLHR onto the
extracellular domain of the rFSHR lengthens the t1/2
of agonist internalization only ~ 2-fold (compare rFSHR and FLL
in Table II). Even in cells overexpressing arrestin-3, a manipulation
that enhances the internalization of agonist mediated by the
gonadotropin receptors, the rate of agonist internalization mediated by
a given chimera is more dependent on the origin of the extracellular
domain than on the origin of the transmembrane and cytoplasmic domains
(Table II). Thus, in cells overexpressing arrestin-3 the
t1/2 of agonist internalization mediated by FLL
(~4 min) is closer to that of the rFSHR (~7 min) than to that of
LFF (~16 min). Conversely, the t1/2 of agonist
internalization mediated by LFF (~16 min) is closer to that mediated
by rLHR (~24 min) than to that mediated by rFSHR (~7 min). These
findings contrast those of previous studies that have shown that the
structural features of other GPCRs that determine the rate of
endocytosis are located in their COOH-terminal and transmembrane
domains (13, 15, 16).
In summary, the results presented here underscore the uniqueness of the
process of agonist-induced activation and agonist-induced internalization of the two gonadotropin receptors (and perhaps the
three glycoprotein hormone receptors in general) when compared with
other GPCRs and argue for the existence of a major conformational change that starts with the binding of agonist to the extracellular domain and is relayed to the transmembrane and cytoplasmic domains. Interactions among these domains have been previously proposed to
explain signal transduction by the glycoprotein hormone receptors (21,
45). The data presented here clearly show that such interactions are
equally important in determining the fate of the agonist-activated receptor. Our data also provide the basis for additional mutagenesis experiments designed to define structural motifs present in the intracellular regions of these receptors that affect internalization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overall structure of the gonadotropin
receptor chimeras. The overall structures of the rLHR and the
rFSHR are shown in black and gray, respectively.
Chimeras were constructed by exchanging one or two of the three major
receptor domains (extracellular, transmembrane, and intracellular) as
indicated. The predicted boundaries for each of these domains were
taken from Refs. 33 and 34. The amino acid sequences around the
junctions of each chimera are also shown. Note that the mature rLHR and
rFSHR are 674 and 675 residues long, respectively. The apparent gap of
a few residues shown in each junction is not real, it is simply due to
introduction of gaps for optimal alignment (19), which results in an
artificial shift of the extracellular/transmembrane domain and
transmembrane domain/COOH-terminal tail boundaries.
Expression and signaling properties of rFSHR/rLHR chimeras
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Fig. 2.
Rates of internalization of
125I-hFSH or 125I-hCG in 293 cells transiently
transfected with the rFSHR or rLHR. 293 cells transiently
transfected with the rLHR (top panels) or rFSHR
(bottom panels) were preincubated in warm medium for 1 h. At the end of this incubation (time = 0 in the figure) the
cells expressing rLHR received 125I-hCG (10 ng/ml), and the
cells expressing rFSHR received 125I-hFSH (40 ng/ml), and
the incubation was continued at 37 °C. The concentrations of
125I-hCG and 125I-hFSH chosen are equivalent to
those concentrations that give half-maximal binding at equilibrium.
A and D, surface-bound and internalized
radioactivity were measured at the times indicated using the acid
release procedure described under "Materials and Methods."
B and E, plots of the ratio of
internalized/surface ligand versus time are shown. The
straight lines shown were obtained using a linear least
square fit of the data points shown. The endocytotic rate constant,
ke, can be calculated from the slopes of these
lines (39). Note that while all the points were used for cells
expressing the rFSHR (E), only the first three points of the
data obtained with the rLHR could be used in this plot (B)
to obtain a good regression coefficient. C and F,
the integral of the surface-bound radioactivity was calculated at each
time point as described under "Materials and Methods," and a plot
of the internalized hormone versus the integral of the
surface-bound hormone was generated as shown. The straight
line shown through the points was obtained using a linear least
square fit of the data points. The endocytotic rate constant,
ke, can be calculated from the slopes of these
lines (37, 38, 40). The results of a representative experiment are
shown. Note that the scales are different in each panel.
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Fig. 3.
Half-lives of internalization of
125I-hFSH and 125I-hCG by rFSHR/rLHR
chimeras. 293 cells transiently transfected with the indicated
chimeras were preincubated in warm medium for 1 h. Cells then
received 125I-hFSH (top panel) or
125I-hCG (bottom panel), and the incubation was
continued at 37 °C. Determinations of the rates of internalization
were done using at least five different data points collected at
3-10-min intervals as described under "Materials and Methods."
Each value represents the mean ± S.E. of three to six independent
transfections. In the top panel the asterisks
indicate statistically significant differences (p < 0.05) from rFSHR. In the bottom panel the
asterisks indicate statistically significant differences
(p < 0.05) from rLHR.
Effect of arrestin-3 on the internalization of agonist mediated by
selected rFSHR/rLHR chimeras
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, or GRK6) enhance the
internalization of agonists mediated by the rLHR and/or rFSHR (28-30).
Last, the slow internalization of hFSH mediated by signaling-impaired
mutants of the FSHR can be rescued by cotransfection with arrestin-3
(28).
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ACKNOWLEDGEMENTS |
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We thank Dr. Deborah L. Segaloff for useful suggestions and for her critical reading of the manuscript. We also thank Dr. Jeffrey L. Benovic (Thomas Jefferson University) for providing us with the arrestin-3 expression vector and Dr. Steve Wiley (University of Utah) for providing us with the spreadsheet to determine ke. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa are also gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD-28962 and CA-40629 (to M. A.) and DK-25295 (which supported the services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Partially supported by a fellowship from the Lalor Foundation.
§ To whom correspondence should be addressed: Dept. of Pharmacology, 2-319A BSB, The University of Iowa, Iowa City, IA 52242-1109. Tel.: 319-335-9907; Fax: 319-335-8930; E-mail: mario-ascoli@uiowa.edu.
2 M. Ascoli, unpublished observations.
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ABBREVIATIONS |
|---|
The abbreviations used are: GPCR, G protein-coupled receptor; FSH, follitropin; rFSHR, rat follitropin receptor; hFSH, human follitropin; LHR, lutropin/choriogonadotropin receptor; rLHR, rat lutropin/choriogonadotropin receptor; hCG, human choriogonadotropin; GRK, G protein-coupled receptor kinase.
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