Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling by ß-Arrestin2
Serge L. Ferrari and
Alessandro Bisello
Division of Bone and Mineral Metabolism Harvard-Thorndike and
Charles A. Dana Research Laboratories Department of Medicine
Beth Israel Deaconess Medical Center and Harvard Medical School
Boston, Massachusetts 02215
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ABSTRACT
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PTH promotes endocytosis of human PTH
receptor 1 (PTH1Rc) by activating protein kinase C and recruiting
ß-arrestin2. We examined the role of ß-arrestin2 in regulating the
cellular distribution and cAMP signaling of two constitutively active
PTH1Rc mutants, H223R and T410P. Overexpression of a
ß-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells
inhibited constitutive cAMP accumulation by H223R and T410P in a
dose-dependent manner, as well as the response to PTH of both mutant
and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP
conjugates, fluorescent ligands, and ßarrestin2-GFP was analyzed
by fluorescence microscopy in HEK-293T cells. In cells expressing
either receptor mutant, a ligand-independent mobilization of
ß-arrestin2 to the cell membrane was observed. In the absence of
ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell
membrane, whereas intracellular trafficking of T410P was also observed.
While agonists promoted ß-arrestin2-mediated endocytosis of both
PTH1Rc mutants, antagonists were rapidly internalized only with T410P.
The protein kinases inhibitor, staurosporine, significantly decreased
internalization of ligand-PTH1Rc mutant complexes, although the
recruitment of ß-arrestin2 to the cell membrane was unaffected.
Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the
C-terminal cytoplasmic domain, agonists stimulated translocation of
ß-arrestin2 to the cell membrane followed by ligand-receptor
complex internalization without associated ß-arrestin2. In
conclusion, cAMP signaling by constitutively active mutant and
wild-type PTH1Rcs is inhibited by a receptor interaction with
ß-arrestin2 on the cell membrane, possibly leading to uncoupling from
Gs
. This phenomenon is independent from
protein kinases activity and the receptor C-terminal cytoplasmic
domain. In addition, there are differences in the cellular localization
and internalization features of constitutively active PTH1Rc mutants
H223R and T410P.
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INTRODUCTION
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PTH is a major regulator of serum calcium homeostasis and bone
metabolism (1). PTH-related protein (PTHrP), first described as the
hormone responsible for hypercalcemia of malignancy, is now recognized
as an autocrine/paracrine factor with various biological functions in
many tissues (2). Both PTH and PTHrP bind to and activate the PTH
subtype 1 receptor (PTH1Rc), a member of the class II subfamily of G
protein-coupled receptors (GPCRs), coupled to both
Gs
and Gq proteins (3).
Whereas the specific biological role of the
Gq-protein kinase C (PKC) signaling pathway is,
at present, unclear, the cAMP-protein kinase A (PKA) signaling pathway
seems to be responsible for the majority of the calciotropic and
skeletal actions of PTH and PTHrP (Ref. 4 and references therein).
Little is known, however, regarding the cellular and molecular
mechanisms that regulate PTH1Rc activation.
Point mutations that constitutively activate the PTH1Rc, namely a
heterozygous His-to-Arg mutation at position 223 (H223R) situated at
the boundary between the PTH1Rc first intracellular loop and second
transmembrane domain (5) and a heterozygous Thr-to- Pro mutation at
position 410 (T410P) situated in the sixth transmembrane domain (6),
have recently been identified. These PTH1Rc mutations are responsible
for Jansens metaphyseal chondrodysplasia, a rare autosomal dominant
disorder characterized by short-limbed dwarfism and hypercalcemia,
despite normal PTH and PTHrP levels (5, 6). Transient transfection of
Jansens PTH1Rc mutants into COS-7 cells is accompanied by a 2- to
8-fold increase in ligand-independent cAMP accumulation when
compared with wild-type PTH1Rc (5, 6, 7, 8). Interestingly, a number of
observations in vitro as in vivo suggest that
cAMP signaling by H223R and T410P mutants becomes desensitized (7, 8, 9).
Clarifying the molecular mechanisms responsible for the desensitization
of Gs
-mediated signaling by constitutively
active PTH1Rcs provides a unique opportunity to gain insights into the
regulation of PTH and PTHrP activity (4), and, more broadly, of
constitutively active GPCRs.
ß-Arrestins are a family of cytoplasmic adaptor molecules that
play a key role in G protein uncoupling (desensitization) and
endocytosis of agonist-activated GPCRs (10, 11). We have recently
demonstrated that agonist-human PTH1Rc complexes are internalized via
clathrin-coated vesicles in association with ß-arrestin2 and that
this mechanism is dependent on PKC activation (12). Indeed, the protein
kinase inhibitor, staurosporine, inhibited ßarrestin2-mediated
endocytosis of fluorescent agonists bound to the hPTH1Rc (12) and
increased cAMP accumulation in response to PTH (12, 13). These
observations led us to hypothesize that the cellular trafficking and
cAMP signaling by constitutively active human PTH1Rcs could be directly
regulated by ß-arrestins. In particular, we wanted to examine whether
the constitutive activity of Jansens receptor mutants might
result from impaired interaction with ß-arrestins and/or defective
receptor internalization. To test these hypotheses, we studied cAMP
accumulation by both H223R and T410P mutant as well as wild-type
PTH1Rcs after overexpression of a functional ß-arrestin2-green
fluorescent protein conjugate (ß-Arr2-GFP) (14). Using GFP-PTH1Rc
conjugates (15) and fluorescencelabeled PTH- and PTHrP-derived
agonists and antagonists (12), we were able to monitor independently
the cellular trafficking of constitutively active PTH1Rc mutants, their
ligands, and ß-Arr2-GFP by real-time fluorescence microscopy in
living cells. By correlating the level of expression and cellular
localization of the mutant receptors and ß-Arr2-GFP with their
Gs
-adenylyl cyclase activity, we have
delineated an essential molecular mechanism regulating cAMP signaling
by the human PTH1Rc.
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RESULTS
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Effects of ß-Arrestin2 on cAMP Signaling by Wild-Type and
Constitutively Active Mutant PTH1Rcs
To evaluate the influence of ß-arrestin2 on cAMP signaling, a
functional ß-Arr2-GFP conjugate (14) was coexpressed with either
wild-type, H223R, or T410P constitutively active mutant human PTH1Rcs
in COS-7 cells. Expression of ß-Arr2-GFP protein was verified by
measuring GFP fluorescence in whole-cell extracts 24 h after
transfection and increased proportionally to the amount of cDNA
transfected (Fig. 1A
). PTH1Rc expression
was evaluated by Scatchard analysis of radiolabeled
125I-PTH-(134) binding and did not
significantly differ between cells cotransfected with ß-Arr2-GFP or
control plasmid cDNA (data not shown). Moreover, the percentage of
cells expressing the PTH1Rcs, as evaluated by fluorescence microscopy
using a rhodamine-labeled antagonist ligand (see below), was similar in
cell preparations cotransfected with the lowest and highest amount of
ß-Arr2-GFP cDNA (64% ± 9% vs. 54% ± 7% of cells
expressing PTH1Rcs, with 0.1 and 0.8 µg ß-Arr2-GFP cDNA/well,
respectively).

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Figure 1. Desensitization of PTH-Stimulated cAMP Signaling
by ß-Arrestin2 in COS-7 Cells Expressing Wild-Type PTH1Rc
COS-7 cells were cotransfected with wild-type PTH1Rc and either
ß-Arr2-GFP (closed bars and symbols) or a control plasmid
(open bars and symbols), as described in Materials and
Methods. cAMP accumulation was measured in the presence of IBMX (1
mM). A, ß-Arr2-GFP expression in cells
transfected with increasing amounts of ß-Arr2-GFP cDNA was measured
by spectrofluorimetry, as described in Materials and
Methods. B, cAMP accumulation in cells transfected with increasing
amounts of ß-Arr2-GFP or control plasmid cDNA, as indicated, and
stimulated with PTH-(134) (100 nM) for 30 min.
C and D, Kinetics of cAMP accumulation in response to PTH-(134) (100
nM) (C) or forskolin (10
µM) (D) in cells transfected with ß-Arr2-GFP
or control plasmid cDNA (0.4 µg/well). cAMP accumulation is expressed
as the percentage of maximal cAMP levels measured in the controls after
30 min [i.e. panel B, 16,452 ± 1,133 cpm/well; panel
C, 14,519 ± 1,391 cpm/well; panel D, 11,851 ± 998
cpm/well]. Each point represents the mean of three separate
measurements, with error bars denoting
SEM. (The experiments were repeated three times
with similar results.)
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ß-Arr2-GFP inhibited PTH-stimulated cAMP accumulation by wild-type
PTH1Rc in a dose-dependent manner: -28%, -34%, -56%, and -61%
with 0.1, 0.2, 0.4, and 0.8 µg/well ß-Arr2-GFP cDNA, respectively
(P < 0.0001 as compared with control plasmid, by
two-factor ANOVA) (Fig. 1B
). Kinetics of cAMP accumulation indicated
that the inhibitory effects of ß-Arr2-GFP were detectable within 2 to
5 min after exposure to PTH (Fig. 1C
). In contrast, direct stimulation
of adenylyl cyclase by forskolin was unaffected by ß-Arr2-GFP
overexpression (Fig. 1D
). Thus, ß-Arr2-GFP rapidly desensitized
PTH-stimulated cAMP signaling upstream of adenylyl cyclase.
The ligand-independent, constitutive cAMP accumulation in COS-7 cells
expressing T410P and H223R mutants was, respectively, 2.9 ± 0.1-
and 5.5 ± 0.2-fold higher compared with wild-type PTH1Rc
(mean ± SEM of three to five separate experiments
performed in triplicate). Coexpression of ß-Arr2-GFP inhibited
constitutive cAMP accumulation by H223R and T410P receptors in a
dose-dependent manner (Fig. 2A
). This
inhibition resulted from a slower rate of cAMP accumulation (Fig. 2B
).
The maximal cAMP accumulation in response to PTH-(134) (100
nM) was comparable for mutant and wild-type PTH1Rcs,
although the relative increase in cAMP accumulation above baseline was
markedly blunted in cells expressing H223R and T410P (Fig. 2C
), in
agreement with previous observations (7, 8). Similar to wild-type
receptors, ß-Arr2-GFP overexpression also significantly decreased the
maximal cAMP accumulation by both mutant receptors in response to PTH
(Fig. 2C
).

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Figure 2. Desensitization of Ligand-Independent and
PTH-Stimulated cAMP Signaling by ß-Arrestin2 in COS-7 Cells
Expressing Constitutively Active PTH1Rc Mutants
COS-7 cells were cotransfected with constitutively active PTH1Rc
mutants, H223R (left hand panels) or T410P (right
hand panels), and either ß-Arr2-GFP (closed bars and
symbols) or control plasmid (open bars and
symbols) cDNA, as described in Materials and
Methods. A, Ligand-independent, constitutive cAMP accumulation
after 30 min incubation with IBMX (1 mM) in cells
transfected with increasing amounts of ß-Arr2-GFP or control plasmid
cDNA, as indicated. B, Kinetics of constitutive cAMP accumulation in
cells transfected with ß-Arr2-GFP or control plasmid cDNA (0.4
µg/well) and incubated with IBMX (1 mM) for up to 30
min, as indicated. C, Kinetics of agonist-stimulated cAMP accumulation
in cells transfected as above. In this case, cells were incubated 30
min with IBMX (1 mM), followed by PTH-(134) (100
nM) for various periods of time, as indicated. Data
are expressed as percentage of the maximal cAMP levels measured in the
controls at the end of each experiment [i.e. panel A
(left), 6,711 ± 492 cpm/well; panel A
(right), 3,072 ± 147 cpm/well; panel B
(left), 6,474 ± 479 cpm/well; panel B
(right), 2,658 ± 25 cpm/well; panel C
(left), 9,026 ± 502 cpm/well; panel C
(right), 8,268 ± 298 cpm/well]. Each
point represents the mean of three separate
measurements, with error bars denoting SEM. (The
experiments were repeated three times with similar results.)
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Thus, overexpression of ß-arrestin2 in COS-7 cells significantly
inhibits cAMP signaling by both wild-type and constitutively active
mutant PTH1Rcs.
Distribution of ß-Arr2-GFP in HEK-293T Cells Expressing
Constitutively Active PTH1Rc Mutants
We have previously reported that in HEK-293T cells stably
expressing the human (h) PTH1Rc, receptor activation by PTH triggers
the recruitment of ß-arrestin2 to the cell surface, followed by
endocytosis of agonist-hPTH1Rc complexes associated with ß-arrestin2
(12). We hypothesized that ß-arrestin2 could inhibit cAMP signaling
by interacting with the constitutively active PTH1Rc mutants on the
cell membrane even in the absence of agonist stimulation. For this
purpose, the distribution of ß-Arr2-GFP was monitored by real-time
fluorescence microscopy in HEK-293T cells cotransfected with either the
H223R or T410P mutants. As shown in Fig. 3
, ß-Arr2-GFP was spontaneously
recruited to the cell surface in cells expressing H223R or T410P (Fig. 3
, a and d). This pattern differed markedly from cells expressing the
wild-type PTH1Rc, in which ß-Arr2-GFP was uniformly diffused in the
cytoplasm (Fig. 3g
) and was mobilized to the cell membrane only after
stimulation with the agonists PTH-(134) (Fig. 3h
) or PTHrP-(136)
(10100 nM). Agonist stimulation further promoted
redistribution of ß-Arr2-GFP from the cell membrane to the cytoplasm
in cells expressing H223R and T410P as well as wild-type PTH1Rcs (Fig. 3
, b, e, and i). In contrast, exposure to the antagonists
Bpa2-PTHrP-(136) and PTH-(734) (both at 1001,000 nM)
was followed by a rapid redistribution of ß-Arr2-GFP from the cell
membrane to the cytoplasm only in cells expressing T410P (Fig. 3f
), but
not H223R (Fig. 3c
) or the wild-type PTH1Rcs (not shown; Ref. 12).

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Figure 3. Fluorescence Localization of ß-Arr2-GFP in
HEK-293T Cells Expressing Mutant PTH1Rcs
HEK-293T cells were cotransfected with mutants H223R (ac, and j),
T410P (df, k, and l) or wild type (gi) PTH1Rcs and ß-Arr2-GFP.
Distribution of ß-Arr2-GFP was monitored at 37 C by real-time
fluorescence microscopy as described in Materials and
Methods. With the H223R mutant, ß-Arr2-GFP was mobilized to
the cell surface in the absence of agonist stimulation (a), and
redistributed to the cytoplasm within 10 min after stimulation by
PTH-(134) (100 nM) (b), but not with the antagonist
Bpa2-PTHrP-(136) (1000 nM) (c). Similar observations
were made with the T410P mutant before (d) and after (e) agonist
stimulation, whereas Bpa2-PTHrP-(136) (1000 nM) also
translocated ß-Arr2-GFP to the cytoplasm (f). With wild-type PTH1Rcs,
ß-Arr2-GFP was uniformly diffused in the cytoplasm in the absence of
agonist stimulation (g), mobilized to the cell surface (h), and further
retranslocated to the cytoplasm (i) within 15 min after stimulation
with PTH-(134) (100 nM). Staurosporine (2
µM, 30 min) did not alter the ligand-independent
recruitment of ß-Arr2-GFP to the surface of cells expressing H223R or
T410P (j and k, respectively), but, in the latter, prevented
ß-Arr2-GFP remobilization to the cytoplasm in response to
Bpa2-PTHrP-(136) (1,000 nM) (l). Magnification,
x100200.
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The cell surface expression level of mutant and wild-type PTH1Rcs
[evaluated by Scatchard analysis of radiolabeled
125I-PTH-(134) binding] was similar in
HEK-293T and COS-7 cells (Table 1
).
However, the level of constitutive cAMP accumulation by the mutant
receptors was significantly lower in HEK-293T cells (1.1 ± 0.1-
and 2.4 ± 0.3-fold higher than wild-type PTH1Rc for,
respectively, T410P and H223R) compared with COS-7 cells (see above;
P < 0.001 for the difference between the two cell
lines, by two-factor ANOVA); the maximal PTH-stimulated (100
nM) cAMP accumulation in HEK-293T cells was also
significantly blunted for both mutant receptors (60 ± 5% of
maximal PTH-stimulated cAMP accumulation in wild-type-transfected
cells); and a small, but significant, increase in their
Kd values for the agonist PTH-(134) was
observed (P < 0.03 between HEK-293T and COS-7 cells,
by two-factor ANOVA) (Table 1
).
Thus, in HEK-293T cells, constitutively active PTH1Rc mutants trigger a
ligand-independent mobilization of ß-Arr2-GFP to the cell membrane.
This is paralleled by a blunted cAMP accumulation, both constitutively
and in response to PTH, as well as by a lower affinity of both mutant
receptors for their cognate agonists.
Colocalization of Fluorescent Ligands with ß-Arr2-GFP in
Cells Expressing Constitutively Active PTH1Rc Mutants
We next examined the endocytosis of specific fluorescent agonist
and antagonist ligands (12) in HEK-293T cells coexpressing PTH1Rc
mutants with ß-Arr2-GFP. The agonist Rho-PTH-(134) was rapidly
internalized and colocalized with ß-Arr2-GFP intracellularly in cells
expressing H223R (Fig. 4
, ac) and T410P (not shown), similarly to wild-type
PTH1Rcs (12). The antagonist Rho-Bpa2-PTHrP-(136) colocalized with
ß-Arr2-GFP on the cell membrane but was not rapidly internalized in
cells expressing H223R (Fig. 4
, df). Rho-Bpa2-PTHrP-(136) also
remained localized on the cell surface in cells expressing wild-type
PTH1Rcs, but did not colocalize with ß-Arr2-GFP (Fig. 4
, gi). In
contrast, in cells expressing the T410P mutant a rapid redistribution
of Rho-Bpa2-PTHrP-(136) to the cytoplasm in association with
ß-Arr2-GFP was observed (Fig. 4
, jl).

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Figure 4. Cellular Trafficking of Fluorescent PTH1Rc Ligands
with ß-Arr2-GFP
HEK-293T cells cotransfected with H223R (af), wild-type (gi), or
T410P (jo) PTH1Rcs and ß-Arr2-GFP were incubated with
rhodamine-labeled ligands (100 nM) and continuously
monitored at 37 C for 15 min as described in Materials and
Methods. In cells expressing H223R, the agonist Rho-PTH-(134)
was internalized within 15 min (a), ß-Arr2-GFP was redistributed into
the cytoplasm (b), and the two fluorescences colocalized
intracellularly (yellow spots on the overlay
image) (c). In contrast, the antagonist Rho-Bpa2-PTHrP-(136)
remained bound on the cell surface (d), where clusters of ß-Arr2-GFP
were also detectable (e) and colocalized with the fluorescent
ligand (yellow) (f). With wild-type PTH1Rc, the
antagonist Rho-Bpa2-PTHrP-(136) remained bound linearly to the cell
surface (g), whereas ß-Arr2-GFP was evenly distributed in the
cytoplasm (h). The overlay image shows the distinct
distribution of the fluorescent ligand and ß-Arr2-GFP in these cells
(i). With T410P, Rho-Bpa2-PTHrP-(136) was rapidly internalized (j),
ß-Arr2-GFP was redistributed to the cytoplasm (k) where it
colocalized with the fluorescent ligand (yellow spots)
(l). In these cells, staurosporine (2 µM, 30 min)
markedly decreased Rho-Bpa2-PTHrP-(136) internalization (m) and
ß-Arr2-GFP redistribution to the cytoplasm (n) while maintaining
their colocalization on the cell surface (yellow) (o).
Magnification, x100.
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Internalization of agonist and antagonist ligands was further assessed
by a radioligand assay. As shown in Table 2
, a major fraction (
70%) of the
radiolabeled agonist 125I-PTH-(134)
specifically bound to wild-type, H223R, and T410P PTH1Rcs was
internalized within 30 min, with no significant differences between
these receptor types. In contrast, a significantly higher fraction of
the bound radiolabeled antagonist
125I-Bpa2-PTHrP-(136) was internalized in cells
expressing the T410P mutant compared with cells expressing wild-type
and H223R mutant PTH1Rc. Internalization of both agonist and antagonist
radioligands (the latter bound to T410P) was significantly reduced in
the presence of sucrose, a known inhibitor of clathrin-coated vesicles
(12, 16, 17).
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Table 2. Radioligand Internalization in HEK-293T Cells
Expressing Wild-Type and Mutants, H223R and T410P,
PTH1Rcs
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Thus, in HEK-293T cells, ß-Arr2-GFP colocalizes on the cell
membrane with fluorescent ligands bound to constitutively active mutant
PTH1Rcs. This is followed by internalization of agonists with
ß-Arr2-GFP in cells expressing either mutant receptor, similarly to
the wild-type PTH1Rc. However, the cellular trafficking of antagonist
ligands markedly differs in cells expressing H223R or T410P mutants:
whereas fluorescent antagonists bound to T410P are rapidly internalized
by a ß-arrestin-2-, clathrin-coated vesicle-dependent mechanism,
antagonists bound to H223R (or wild-type PTH1Rcs) are not.
Cellular Trafficking of GFP-PTH1Rc Conjugates
To directly monitor by real-time fluorescence microscopy the
cellular distribution of constitutively active mutant PTH1Rcs,
GFP-receptor conjugates [H223R-GFP and T410P-GFP] were generated and
transiently transfected in HEK-293T cells. Cell surface receptor
expression as well as constitutive and PTH-stimulated activity were
verified by, respectively, radioligand binding and cAMP assays and were
similar to those of the original receptor mutants (data not shown). A
human wild-type PTH1Rc-GFP (generously provided by Dr. Caroline Silve,
INSERM, Paris) was also used and has been characterized previously
(15).
In transiently transfected HEK-293T cells, H223R-GFP was clearly
localized on the cell membrane, and an intense fluorescent signal was
also detected in the vicinity of the nucleus (Fig. 5
, a and d). A similar
distribution was observed in cells expressing wild-type PTH1Rc-GFP
(Fig. 5
g). The intracellular localization of receptor-GFP
conjugates is compatible with the presence of a PTH1Rc pool in a
Golgi compartment, as previously observed by indirect fluorescence
microscopy (12). In contrast, in cells transfected with T410P-GFP, in
addition to the intracellular receptor pool, green fluorescence was
mainly detectable as multiple dots localized beneath the cell surface
and within the cytoplasm (Fig. 5
j). In keeping with the
radioligand internalization assays (Table 2
), the fluorescent agonist
Rho-PTH-(134) was rapidly internalized in association with H223R-GFP
(Fig. 5
, b and c), T410P-GFP, and wild-type PTH1Rc-GFP (not shown). The
fluorescent antagonist Rho-Bpa2-PTHrP-(136) colocalized with
H223R-GFP and wild-type PTH1Rc-GFP on the cell surface, with no
evidence of endocytosis of the ligand-receptor complex after 15 min at
37 C (Fig. 5
, e, f, h, and i). In contrast, Rho-Bpa2-PTHrP-(136)
initially colocalized with T410P-GFP on the cell surface (Fig. 5
k), and subsequently in the cytoplasm (Fig. 5
l).

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Figure 5. Cellular Trafficking of PTH1Rc-GFP Conjugates and
Colocalization with Their Fluorescent Ligands
HEK-293T cells were transfected with H223R (af), wild type (gi), or
T410P (jo) PTH1Rcs-GFP conjugates and their cellular distribution
monitored at 37 C before and after binding of specific fluorescent
ligands, as described in Materials and Methods. In the
absence of ligand, H223R-GFP was linearly distributed on the cell
surface and was also present in an intracellular pool (a). The agonist
Rho-PTH-(134) at first colocalized with H223R-GFP on the cell
membrane (in yellow on the overlay image) (b), followed
by internalization of agonist-receptor complexes after 15 min (c). The
antagonist Rho-Bpa2-PTHrP-(136) also colocalized with M223R-GFP on the cell membrane
(yellow) immediately after binding (e), but the
ligand-receptor complex was not internalized after 15 min (f). The
wild-type PTH1Rc-GFP was similarly distributed before ligand binding
(g), and antagonist Rho-Bpa2-PTHrP-(136)PTH1Rc-GFP complexes
(yellow) also remained mostly localized on the cell
surface immediately (h) and 15 min (i) after binding. In contrast,
T410P-GFP was detectable as distinct fluorescent spots throughout the
cytoplasm, particularly in the submembranal region, in the absence of
ligand (j). Clusters of antagonist Rho-Bpa2-PTHrP-(136)-T410P-GFP
complexes (yellow) were detectable on the cell surface
immediately after binding (k) and intracellularly after 15 min (l). In
these cells, staurosporine (2 µM, 30 min) markedly
decreased the cytoplasmic distribution and increased the cell surface
localization of T410P-GFP in the absence of ligand (m). It also
decreased clustering and internalization of
Rho-Bpa2-PTHrP-(136)-T410P-GFP complexes (yellow)
immediately after binding (n) and after 15 min at 37 C (o).
Magnification, x100.
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These observations confirm that internalization of agonist-receptor
complexes is similar for wild-type and Jansens mutant PTH1Rcs. They
also indicate that the cellular distribution of the T410P mutant in the
absence of ligand and after antagonist binding markedly differs from
wild-type and H223R PTH1Rcs, suggesting an increased level of
spontaneous intracellular trafficking of the T410P mutant.
Influence of Protein Kinases Activation on Cellular Trafficking and
cAMP Signaling of Constitutively Active PTH1Rc Mutants
We have previously reported that the protein kinases inhibitor
staurosporine, but not the selective PKA inhibitor H89, markedly
decreases ß-arrestin2-mediated internalization of fluorescent
agonists bound to the human PTH1Rc and significantly increases cAMP
accumulation in response to its cognate agonists (12, 13). We therefore
examined the effects of these pharmacological agents on the cellular
distribution of constitutively active mutant PTH1Rcs, their ligands,
and ß-arrestin2, as well as on cAMP signaling in HEK-293T cells.
Treatment with staurosporine (2 µM for 30 min) prevented
neither the ligand-independent recruitment of ß-arr2-GFP to the cell
membrane in cells expressing H223R or T410P (Fig. 3
, j and k), nor its
mobilization in response to PTH (and PTHrP) in cells expressing
wild-type PTH1Rcs (not shown). However, staurosporine decreased
internalization of the fluorescent agonist Rho-PTH-(134) associated
with ß-Arr2-GFP and with either mutant or wild-type PTH1Rc-GFP (data
not shown). In addition, in cells expressing T410P, staurosporine
inhibited the rapid redistribution from the cell membrane to the
cytoplasm of ß-Arr2-GFP in response to the antagonist
Bpa2-PTHrP-(136) (Fig. 3
l) as well as of the fluorescent
antagonist Rho-Bpa2-PTHrP-(136) itself (Fig. 4
, mo). Moreover,
staurosporine decreased intracellular trafficking and increased the
cell surface localization of T410P-GFP, while inhibiting
internalization of Rho-Bpa2-PTHrP-(136)-T410P-GFP complexes (Fig. 5
, mo).
Table 3
summarizes the effects of
staurosporine on receptor expression and radioligand internalization,
as well as constitutive and PTH-stimulated cAMP accumulation in
HEK-293T cells expressing mutant and wild-type PTH1Rcs. Thus,
staurosporine (2 µM, 30 min) modestly increased the cell
surface expression of mutant PTH1Rcs, more significantly so for T410P
(in keeping with our observations by fluorescence microscopy, above);
it significantly decreased internalization of agonists with all
receptor types and of antagonists with T410P; eventually, it
significantly increased both constitutive and PTH-stimulated (100
nM) cAMP accumulation (up to 4-fold in cells expressing
T410P) compared with untreated cells. In contrast, incubation with the
selective PKA inhibitor, H89 (up to 50 µM), did not
affect any of the parameters evaluated above.
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Table 3. Effects of Staurosporine on Receptor
Expression, cAMP Activity, and Internalization of Wild-Type and Mutant,
H223R and T410P, PTH1Rcs
|
|
These observations suggest that protein kinases activity is not
required for the ligand-independent recruitment of ß-arrestin2 to the
cell membrane by constitutively active PTH1Rc mutants. However, protein
kinases, particularly PKC, may play a role in the internalization of
ligand-receptor complexes and in the constitutive intracellular
trafficking of the T410P mutant receptor.
Role of PTH1Rc C-Terminal Cytoplasmic Domain on ß-Arrestin2
Mobilization and Ligand-Receptor Complex Endocytosis
It has been shown that, upon agonist activation, the PTH1Rc is
phosphorylated at multiple sites within its intracellular C-terminal
domain (18, 19). Also, PTH1Rc phosphorylation in response to PTH is
markedly inhibited by staurosporine (19). To examine whether the C
terminus of the human PTH1Rc is involved in the interaction with
ß-arrestin2 and the internalization of ligand-receptor complexes, we
generated C-terminally-truncated receptor constructs containing only an
eight-amino acid residue tail (17) [H223R-del, T410P-del, and wild
type PTH1Rc-del]. Cell surface expression levels and maximal
PTH-stimulated (100 nM) cAMP accumulation of the wild-type
PTH1Rc-del in HEK-293T cells were comparable to intact wild-type
PTH1Rcs (as evaluated by, respectively, radioligand binding and cAMP
accumulation assays; data not shown). In contrast, neither binding of
radioiodinated PTH-(134) nor cAMP accumulation in response to
PTH-(134) was detectable in HEK-293T cells transfected with H223R-del
or T410P-del, indicating that these truncated receptor mutants were
either not expressed or not functional (data not shown). Accordingly,
fluorescence microscopy analysis was performed in HEK-293T cells
coexpressing wild-type PTH1Rc-del and ß-Arr2-GFP: upon stimulation
with the fluorescent agonist Rho-PTH-(134) (100 nM),
ß-Arr2-GFP was normally recruited to the cell membrane where it
colocalized with the agonist bound to the truncated receptor (Fig. 6
, ac). Rho-PTH-(134) was then
rapidly internalized (Fig. 6
, gi). In this case however, in
contrast to cells expressing intact PTH1Rcs (12), ß-Arr2-GFP
rediffused immediately into the cytoplasm and did not remain associated
intracellularly with the fluorescent agonist-receptor complex (Fig. 6
df).

View larger version (18K):
[in this window]
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|
Figure 6. Cellular localization of a Fluorescent Agonist with
ß-Arr2-GFP in Cells Expressing a C-Terminally-Truncated Wild-Type
PTH1Rc
HEK-293T cells cotransfected with a wild-type PTH1Rc lacking the
C-terminal cytoplasmic domain and ß-Arr2-GFP were incubated with
Rho-PTH-(134) (100 nM) and monitored after 2 (a, b,
and c) and 15 (d, e, and f) min at 37 C, as described in
Materials and Methods. After 2 min, Rho-PTH-(134) was
bound on the cell surface (a), ß-Arr2-GFP was recruited to the cell
membrane (b), and the overlay image shows the
colocalization (yellow) of the fluorescent ligand with
ß-Arr2-GFP in these cells (c). After 15 min, Rho-PTH-(134) was
internalized (d), and ß-Arr2-GFP has rediffused into the cytoplasm
(e) and was no more colocalized with the fluorescent ligand on the
overlay image (f). Magnification, x100.
|
|
In summary, these observations indicate that the presence of the PTH1Rc
C-terminal cytoplasmic domain is not required for an interaction
between the agonist-activated receptor and ß-arrestin2 on the cell
membrane. However, while agonists bound to PTH1Rc-del are internalized,
this occurs without associated ß-arrestin2.
 |
DISCUSSION
|
---|
The goal of this study was to investigate the role of
ß-arrestin2 in regulating the cellular distribution and cAMP
signaling of constitutively active mutant PTH1Rcs, H223R and T410P,
recently identified as the cause of hereditary Jansens metaphyseal
chondrodysplasia (5, 6, 7). Our study led to four major findings. First,
both the constitutive activity of Jansens mutant receptors and their
responsiveness to PTH (and PTHrP) is dependent on the level of
ß-arrestin2 expression (Fig. 2
). Similarly, agonist-stimulated cAMP
accumulation by the wild-type PTH1Rc is inhibited by increasing
expression of ß-arrestin2 (Fig. 1
). Second, inhibition of cAMP
signaling by the H223R and T410P mutants correlates with a
ligand-independent mobilization of ß-arrestin2 to the cell membrane
(Figs. 3
and 4
). Third, both mutant receptors are internalized by a
ß-arrestin2- and clathrin-coated vesicles-mediated process in
response to their cognate agonists. However, the cellular distribution
of the T410P mutant and its internalization features in response to
antagonists are different from those of the H223R mutant and wild-type
PTH1Rcs (Fig. 5
). Fourth, neither PTH1Rc-mediated activation of protein
kinases nor the receptor C-terminal cytoplasmic domain (which contains
the major phosphorylation sites) is required for the receptor
interaction with ß-arrestin2 on the cell membrane.
Several lines of evidence indicate that the constitutive activity of
GPCRs is regulated and their responsiveness to agonists is desensitized
(7, 8, 9, 21, 22, 23). Thus, in COS-7 cells transiently transfected with
Jansens PTH1Rc mutants, ligand-independent cAMP accumulation
decreases rapidly after its initial onset, whereas the relative
increase of cAMP accumulation in response to PTH is blunted compared
with wild-type receptors (6, 7, 8) (Fig. 2B
). Moreover, transgenic models
suggest that the expression and/or constitutive activity of Jansens
receptor mutants could be downregulated in vivo (9). In
this study we report that an interaction between these mutant receptors
and ß-arrestin2 is one of the mechanisms regulating their
constitutive activity. The ß-Arr2-GFP conjugate used in this study
has previously been shown to be functionally equivalent to wild-type
ß-arrestin2 in COS-7 cells (14). Kinetics of PTH-stimulated cAMP
accumulation indicate that ß-arrestin2 inhibits cAMP signaling by
both constitutively active mutant and wild-type PTH1Rcs through similar
mechanisms. ß-Arr2-GFP overexpression had no effect on the activation
of adenylyl cyclase by forskolin, suggesting that ß-arrestin2 could
directly uncouple activated PTH1Rcs from Gs
proteins. Recently, binding of G protein-coupled receptor kinases
(GRKs) to the PTH1Rc has been shown to inhibit signaling through
Gq (24). However, the acute stimulation of
adenylyl cyclase by PTH was only marginally affected (24). Our
observations support a distinct role for ß-arrestins in the rapid
inhibition of cAMP signaling. Similarly, overexpression of
ß-arrestin2 in COS cells expressing the ß2-adrenergic receptor
desensitized the response to its cognate agonists (25). Hence, these
results provide additional evidence for a common role of ß-arrestins
in the regulation of Gs
-mediated signal
transduction by a number of GPCRs, including ß2-adrenergic receptors
(10, 11, 25, 26), m2 muscarinic acetylcholine receptors (27), FSH
receptors (28), A2b adenosine receptor, and
PGE2 receptor (29). It should be noted, however,
that other mechanisms such as the PKA-mediated activation of
cAMP-phosphodiesterase and the PTH-stimulated up-regulation of
regulators of G protein signaling (RGS) may also contribute to this
process (30, 31).
We have recently reported that agonist stimulation of wild-type PTH1Rc
promotes a rapid relocalization of ß-arrestin2 from the cytoplasm to
the cell membrane (12). To support the hypothesis that constitutively
active PTH1Rc mutants induce a ligand-independent mobilization of
ß-arrestin2, we monitored the cellular localization of ß-Arr2-GFP
by fluorescence microscopy in living cells (12, 14). Expression of
Jansens receptor mutants in HEK-293T cells is indeed associated
with a ligand-independent recruitment of ß-Arr2-GFP to the cell
membrane (Figs. 3
and 4
). Similarly, the
1b
adrenergic receptor mutant A293E, which constitutively activates
Gq in COS-7 cells, promotes ß-arrestin2
mobilization independently of the agonist, epinephrine, in HEK-293
cells (32). These findings support the emerging idea that
desensitization in response to spontaneous receptor activity may occur
as a result of ligand-independent interactions between constitutively
active GPCRs and ß-arrestins on the cell membrane. Thus, rapid
inhibition of Gs
-mediated signal transduction
and targeting of ligand-PTH1Rc complexes to clathrin-coated vesicles
(12), both mediated by ß-arrestin2, may be two chronologically and
mechanistically related, but functionally distinct, phenomena.
Two sets of experiments were performed to gain insights into the
molecular basis of the interaction between ß-arrestin2 and the
PTH1Rc. First, pharmacological inhibition of protein kinases activity
by staurosporine [which is known to inhibit phosphorylation of the
activated PTH1Rc (19)] did not prevent the constitutive colocalization
of Jansens receptor mutants with ß-Arr2-GFP on the cell membrane.
Second, truncation of the cytoplasmic tail of the wild-type human
PTH1Rc [which contains most of the receptors phosphorylation sites
(17, 18, 19)] did not affect agonist-induced translocation of ß-Arr2-GFP
to the cell membrane (Fig. 6
). Of note, C-terminal deletions of the rat
and opossum PTH1Rc have previously been reported to result in
functional receptors (17, 20). Taken together, our data suggest that
recruitment of ß-arrestin2 by activated PTH1Rcs is independent from
receptor phosphorylation and, more broadly, from the receptor
C-terminal cytoplasmic domain. Moreover, a fluorescent agonist was
rapidly internalized in cells expressing the
Cterminally-truncated, wild-type human PTH1Rc (Fig. 6
). In
agreement with the latter observation, proximal deletions of the
opossum PTH1Rc C terminus decreased, but did not abolish,
internalization of agonist radioligands (17). However, we found that in
cells expressing PTH1Rc-del, ß-Arr2-GFP did not colocalize with the
fluorescent agonist intracellularly, suggesting that the receptor
cytoplasmic tail may provide stabilization of
ligand-receptor-ß-arrestin2 complexes during endocytosis. Similar
conclusions have been drawn regarding the secretin receptor (33, 34)
and a chimeric angiotensin II type 1A receptor containing the
cytoplasmic tail of the ß2-adrenergic receptor (35).
Using GFP-tagged PTH1Rc mutants and fluorescent agonists and
antagonists, we were able to monitor independently the cellular
distribution of both constitutively active PTH1Rcs and their ligands by
fluorescence microscopy in living HEK-293T cells. Our results indicate
important similarities, but also differences, between the H223R and
T410P mutants. In the absence of ligand, wild-type and H223R PTH1Rcs
are mostly localized on the cell membrane as well as in an
intracellular pool. The latter observation is in agreement with our
previous data obtained by indirect fluorescence microscopy in HEK cells
stably expressing a human C-Tag-PTH1Rc (12). The precise function of
this cytoplasmic pool of receptors is presently unknown. Agonist
binding to either receptor mutant is followed by rapid and extensive
endocytosis, as shown by both fluorescence microscopy and radioligand
internalization, and this process can be inhibited by disrupting
clathrin-coated lattices with sucrose (Table 2
). Therefore, the usual
mechanisms of ß-arrestin2-mediated endocytosis of agonist-PTH1Rc
complexes previously described (12) appear to be operative with both
constitutively active mutant PTH1Rcs. In agreement with these findings,
rapid internalization of constitutively active LH/human CG receptor
mutants (36, 37) and
1b adrenergic receptor
mutants (32) also occurs in response to their cognate agonists.
However, in contrast to the H223R mutant, T410P-GFP was also broadly
localized in the cytoplasm of unstimulated HEK-293T cells. Similarly,
localization of the constitutively active
1d-adrenoreceptor in intracellular
compartments has been recently reported (38). In addition, occupancy of
the T410P mutant by antagonists, i.e. in the absence of
stimulation of intracellular signaling, was rapidly followed by the
appearance of antagonist-receptor-ß-arrestin2 complexes in the
cytoplasm (Figs. 4
and 5
). Taken together, these observations indicate
that the T410P mutation of the PTH1Rc is functionally unique and
clearly differs from the H223R mutation (although both result in
constitutive receptor activity). They suggest that the T410P mutant
PTH1Rc has an increased level of spontaneous intracellular trafficking.
These findings support the hypothesis that an altered intracellular
trafficking of T410P might explain its lower constitutive activity
compared with H223R (7).
The molecular mechanisms underlying the higher constitutive trafficking
of the T410P mutant are, at present, not fully clear. Current models of
GPCR signaling and distribution suggest that constitutive
activity might be associated with constitutive phosphorylation
(39, 40, 41). Unfortunately, in the absence of high-affinity
antibodies against the human PTH1Rc, our attempt to directly
investigate phosphorylation of Jansens receptor mutants remained
elusive. Moreover, C terminus truncation of H223R and T410P mutants
resulted in severely reduced receptor expression and/or functionality,
precluding their direct examination by fluorescence microscopy.
Nevertheless, staurosporine apparently decreased intracellular
trafficking of the T410P mutant, both spontaneously and after
antagonist binding, resulting in its predominant localization on the
cell membrane (Figs. 4
and 5
). In turn, staurosporine, but not the
specific PKA inhibitor H8M89, significantly increased both constitutive
and agonist-stimulated cAMP accumulation by the PTH1Rc mutants, more
significantly so with T410P (Table 3
). Accordingly, one might speculate
that PTH1Rc-mediated activation of protein kinases, particularly PKC,
plays a role in the constitutive intracellular trafficking of the T410P
mutant. In this regard, it is interesting to note that T410P has been
reported to signal normally through Gq, whereas H223R does not
(5, 6, 7, 8).
In conclusion, this study addresses the role of ß-arrestin2 in
the regulation of both cAMP signaling and cellular localization of
human PTH1Rcs. Taken together, our observations provide important
insights into the regulatory mechanisms of PTH (and PTHrP) activity. In
addition, they support an emerging concept about the direct role of
ß-arrestins in the regulation of constitutive GPCRs activity. The
fact that naturally occurring, constitutively active GPCR mutants are
likely subjected to regulation in vivo (9, 21, 22, 42)
highlights the potential physiological relevance of this regulatory
mechanism. These results may lead to the development of novel molecular
strategies to address a number of disorders associated with
constitutively active GPCR mutants (22, 29).
 |
MATERIALS AND METHODS
|
---|
Peptide Synthesis and Radioligand Preparation
The syntheses, purification, and characterization of
PTHrP-(136)NH2 [PTHrP-(136)],
[Nle8,18,Tyr34]bPTH-(134)NH2
[PTH-(134)],
[Nle8,18,Lys13(N
-5-carboxymethylrhodamine),L-2-Nal23,Arg26,27,Tyr34]bPTH-(134)NH2
[Rho-PTH-(134)],
[Bpa2,Ile5,Arg11,Lys13,Tyr36]PTHrP-(136)NH2
[Bpa2-PTHrP-(136)], and
[Bpa2,Ile5,Arg11,Lys13(N
-5-carboxymethylrhodamine),Tyr36]PTHrP-(136)NH2
[Rho-Bpa2-PTHrP-(136)], were carried out as previously described
(12). The pure products were characterized by analytical HPLC,
electron-spray mass spectrometry, and amino acid analysis.
Radioiodination and HPLC purification of PTH-(134) and
Bpa2-PTHrP-(136) were carried out as reported previously (43).
Expression Vectors Encoding Constitutively Active and Wild-Type
Human PTH1Rcs, ß-Arr2-GFP
Expression plasmids encoding Jansens mutant receptors T410P
and H223R were the generous gift of Dr. H. Juppner (Massachusetts
General Hospital, Boston, MA) (5, 6). T410P PTH1Rc mutant full-length
cDNA was subcloned into the mammalian expression vector pZeoSv2(+)
(Invitrogen, Carlsbad, CA) using the ApaI and
EcoRI polylinker restriction sites. H223R PTH1Rc mutant
full-length cDNA was amplified by PCR using primers forward
5'-GCGATGGGGACCGCCCGG and reverse 5'-TCACATGACTGTCTCCCA. The 2-kb PCR
product was directly ligated into the mammalian expression vector
pCDNA3.1-TOPO (Invitrogen). The previously cloned
full-length wild-type human PTH1Rc cDNA (44) was subcloned into the
HindIII and NotI restriction sites of pZeoSv2(+).
Receptor-GFP conjugates (H223R-GFP and T410P-GFP) were generated by
subcloning the mutant receptors cDNA into HindIII and
SacII restriction sites of the pEGFP-N1 vector
(CLONTECH Laboratories, Inc. Palo Alto, CA). A human
wild-type PTH1Rc-GFP (generously provided by Dr. Caroline Silve,
INSERM, Paris) was also used and has previously been characterized
(15). Truncated receptors lacking the whole C-terminal cytoplasmic
domain but for eight proximal amino acid residues, i.e.
del472
593stop (H223R-del, T410P-del, and
wild-type PTH1Rc-del) were generated by PCR using primers forward as
above and reverse 5'-CTTGATCTCAGCTTGTAC. The 1.6-kb PCR product was
directly ligated into the mammalian expression vector pCDNA3.1-TOPO
(Invitrogen). Integrity and orientation of the final
constructs were verified by dideoxynucleotide sequencing and
endonuclease restriction of unique SphI and SpeI
sites in the H223R and T410P mutants, respectively (H. Juppner,
personal communication). A p(S65T)-N3 plasmid containing the sequence
of ß-Arr2-GFP was a generous gift from Dr. M. Caron (Duke University,
Durham, NC) and has been previously characterized (14, 45).
Transfection Procedure
COS-7 cells (a generous gift from Dr. S. Goldring, Beth Israel
Deaconess Medical Center, Boston, MA) and human embryonic kidney cells
(HEK-293T, a generous gift from Dr. O. Behar, Massachusetts General
Hospital, Boston, MA) were plated at 1.5 x
105 cells per coverslip on 25-mm glass coverslips
for fluorescence microscopy experiments and at 1.0 x
105 cells per well in 24-well plastic dishes
(Corning, Inc., Corning, NY) for adenylyl cyclase,
radioligand binding, and radioligand internalization assays, as
previously described (12). Twenty-four hours after plating, transient
transfections were carried out using Fugene (Roche Molecular Biochemicals, Indianapolis, IN) reagent and constructs cDNA in a
2:1 to 3:1 ratio (vol/wt). Typically, 2.0 µg and 0.33 µg of,
respectively, PTH1Rc and ß-Arr2-GFP plasmid cDNA, or 0.51.0 µg of
PTH1Rc-GFP plasmid cDNA were transfected per coverslip, whereas 0.4
µg of PTH1Rc plasmid cDNA and variable amounts of ß-Arr2-GFP or
control plasmid cDNA, as indicated in the figure legends, were
transfected per 24-well dish. All subsequent experiments were performed
24 h after transfection.
ß-Arr2-GFP Conjugate Protein Expression
COS-7 cells grown in 24-well plastic dishes and transfected with
increasing amounts of ß-Arr2-GFP cDNA, as described above, were lysed
24 h later with 100 µl of a solution containing
Tris-PO4 (25 µM)/dithiothreitol (2
µM)/EDTA (2 µM) (Sigma,
St. Louis, MO)/Glycerol 10%/Triton X-100 1%. The lysate was briefly
centrifuged and GFP fluorescence measured in the supernatant by
spectrofluorimetry (Analyst 96.384, LJT Biosystems, Sunnyvale, CA)
using 475-nm and 530-nm excitation and emission wavelengths,
respectively.
Radioreceptor Binding and Internalization Assays
Radioreceptor binding assay was carried out as reported (46)
using HPLC-purified
125I-[Nle8,18,Tyr34]bPTH-(134)NH2
[125I-PTH-(134)] as radioligand. Curves were
drawn by CA-Cricket Graph III (version 1.0; Computer Associates,
Islandia, NY), and cell surface receptor expression was
estimated from Scatchard analysis, as described previously (47).
Radioligand internalization assays (17) were performed by incubating
the cells for 30 min at room temperature in 250 µl of DMEM/10% FBS
containing approximately 100 x 103 cpm (0.1
nM) of either the agonist 125I-PTH-(134) or
the antagonist
125I-[Bpa2,Ile5,Arg11,Lys13,Tyr36]PTHrP-(136)NH2
[125I-Bpa2-PTHrP-(136)]. Under these conditions,
specific binding represented 1020% of the total radioligand.
Cells were then washed twice with ice-cold PBS, and surface-bound
radioligand was extracted by two consecutive treatments with 500 µl
of an ice-cold solution containing 50 mM glycine/100
mM NaCl (pH 3). Cells were then lysed with 0.1
M NaOH to measure internalized radioligand. In some
experiments, cells were preincubated 30 min and maintained in
hypertonic medium (0.45 M sucrose) during incubation with
the radioligands, to inhibit clathrin-coated vesicles-mediated
internalization, as previously demonstrated (12). Nonspecific binding
and internalization were determined in parallel experiments in which
radioligand binding was competed by 1 µM unlabeled
PTH-(134).
Adenylyl Cyclase Assay
cAMP accumulation was determined in subconfluent cell cultures
in the presence of the phosphodiesterase inhibitor,
isobutylmethylxanthine (IBMX) (1 mM), as previously
described (46). Kinetics of constitutive cAMP accumulation in cells
expressing Jansens mutant receptors were obtained by incubating the
cells with IBMX (1 mM) at 37 C for various periods of time,
as indicated in the figure legends. Kinetics of PTH-stimulated cAMP
accumulation were evaluated in cells preincubated 30 min with IBMX (1
mM) and subsequently exposed to PTH-(134) (100
nM) in the presence of IBMX for variable periods of time.
In all cases, the reaction was stopped by adding 1.2 M
trichloroacetic acid, and cAMP was isolated by the two-column
chromatographic method.
Fluorescence Microscopy
Cellular distribution of fluorescent ligands, receptor-GFP
conjugates, and ß-Arr2-GFP was assessed by real-time fluorescence
microscopy as previously described (12). Briefly, transfected cells
grown on glass coverslips were rinsed in PBS and mounted in an
open-air, temperature-controlled block on a Nikon
epifluorescence microscope (Diaphot 300, Nikon, Melville,
NY). ß-Arr2-GFP or PTH1Rcs-GFP distribution was first analyzed in the
absence of ligand by monitoring the cells maintained in PBS/BSA 0.1%
at 37 C for up to 30 min. Effects of agonists and antagonists on
ß-Arr2-GFP or PTH1Rcs-GFP localization were evaluated by subsequently
adding the various ligands (100 to 1,000 nM) in the same
buffer and by monitoring redistribution of the green fluorescence after
2, 5, 10, and 15 min. Dual fluorescence microscopy to colocalize
ß-Arr2-GFP (and PTH1Rcs-GFP, respectively) and rhodamine-labeled
ligands was performed by preincubating cells with ice-cold PBS/BSA 1%
(blocking of nonspecific binding) followed by incubation with the
fluorescent ligand for 10 min on ice or at room temperature, as
indicated. The unbound ligand was then removed by carefully rinsing the
coverslips with PBS, and the cells were warmed to 37 C for continuous
microscopy monitoring. Images of the distribution of ß-Arr2-GFP
(PTH1Rcs-GFP, respectively) and rhodamine-labeled ligands were acquired
sequentially, i.e. within a 10-sec interval and in the same
cellular plan, using fluorescein and rhodamine filters, respectively.
Overlay images were generated using the Image-Pro Plus software (Media
Cybernetics, Silver Spring, MD).
Statistics
Comparisons between experimental groups were performed by
two-factor ANOVA using Statview (SAS Institute, Inc.,
Cary, NC).
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank particularly Dr. M. Rosenblatt for his
intellectual, human, and financial support to the authors and their
work. We thank Drs. L. Suva, M. Chorev, and J. Alexander for helpful
discussions and critical reading of this manuscript. We also thank Drs.
M. Caron for kindly providing us with ß-Arr2-GFP cDNA; H. Juppner for
Jansens mutant receptor cDNAs; and Caroline Silve for hPTH1Rc-GFP
cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. S. Ferrari, Beth Israel Deaconess Medical Center, Division of Bone and Mineral Metabolism (HIM 944), 77 Ave Louis Pasteur, Boston, Massachusetts 02215. E-mail:
sferrar2{at}caregroup.harvard.edu
This work was supported, in part, by Grant RO1-DK-47940 from the
National Institute of Diabetes, Digestive and Kidney Diseases to
M.R. S.L.F. is supported by a postdoctoral fellowship grant from
the Swiss National Science Foundation and the Fondation des Bourses de
Recherche en Médecine et Biologie.
Received for publication March 17, 2000.
Accepted for publication October 11, 2000.
 |
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