Multiple Domains Interacting with Gs in the Porcine Calcitonin Receptor
Philippe Orcel1,
Hirohisa Tajima1,
Yoshitake Murayama,
Toshiro Fujita,
Stephen M. Krane,
Etsuro Ogata,
Steven R. Goldring and
Ikuo Nishimoto
Department of Medicine (P.O., S.M.K., S.R.G., I.N.) Harvard
Medical School Cardiovascular Research Center and Arthritis
Research Massachusetts General Hospital-East Charlestown,
Massachusetts 02129
INSERM U349 (P.O.) Centre Viggo
Petersen Hopital Lariboisière 75010 Paris, France
Department of Pharmacology and Neurosciences (H.T., I.N.)
KEIO University School of Medicine Tokyo 160, Japan
Department of Medicine (Y.M., T. F.) University of Tokyo
School of Medicine Mejirodai, Bunkyo-ku Tokyo 113,
Japan
Cancer Research Center (E.O.) Toshima-ku, Tokyo
170, Japan
Department of Medicine (S.R.G.) Beth Israel
Deaconess Medical Center and New England Baptist Bone and Joint
Institute Harvard Institutes of Medicine Boston, Massachusetts
02215
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ABSTRACT
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The molecular basis for
Gs activation by the calcitonin (CT) receptor
was investigated. Based upon the analysis of conserved regions in G
protein-coupled receptors, two nonoverlapping regions in the
heptahelical porcine CT receptor (CTR) were selected as candidate
Gs-interacting domains: the third intracellular
loop residues 327344 (KLKESQEAESHMYLKAVR, P3 region) and the C-tail
residues 404418 (KRQWNQYQAQRWAGR, P4 region). To assess their
Gs-interacting function, we expressed these
sequences in hybrid insulin-like growth factor II receptors in which
the receptor native Gi-interacting domain was
converted to CTR sequences. In COS cells transfected with either P3- or
P4-substituted hybrid receptor, membrane adenylyl cyclase activity
significantly increased. The up-regulated activity of cAMP was
confirmed by measuring the transcriptional activity of the cAMP
response element in cells expressing either hybrid receptor. A
mutant CTR lacking the P4 region maintained positive cAMP response but
with an attenuated maximal capacity to produce cAMP. In contrast, we
could not assess the function of the P3 region using a conventional
deletion method, as CT bound poorly to cells transfected with either of
the two P3-deficient CTRs (one lacking the P3 region and the other
lacking P3 but having the P3 sequence in reverse orientation). These
data suggest that the third intracellular loop and the C-tail in CTR
have domain-specific roles in Gs activation and
that the hybrid receptor approach used here, combined with a
conventional mutagenesis approach, is useful for intact cell analysis
and functional dissection of G protein-coupled receptors.
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INTRODUCTION
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The calcitonin receptor (CTR) is a member of the superfamily of
heptahelical receptors, sharing common structural features with other G
protein-coupled receptors (GPCRs) (1 ). Binding of calcitonin (CT) to
this receptor activates membrane adenylyl cyclase (AC) and production
of cAMP through the G protein Gs, promotes
polyphosphoinositide turnover, and activates protein kinase C
(PKC) via G proteins of the Gq family; under
certain conditions, CT inhibits AC via the Gi
subclass of G proteins (2 3 4 ). While these results indicate
multipotential coupling of the CTR to various G protein-linked
pathways, a number of studies have shown that Gs
activation and subsequent cAMP accumulation transmit the major signal
for the physiological effects of CT in target cells (1 3 4 5 ). This
study was conducted to define the molecular basis for
Gs activation by CTR.
Various GPCRs share structurally similar sequences in their cytoplasmic
domains, which have been implicated in G protein interactions. By
examining the structure/function relationship of these regions, we
initially defined structural motifs underlying the G
protein-interacting function as: 1) at least two basic residues in the
N-terminal side; 2)
B1-B2-X-B3,
or
B1-B2'-X-X-B3
(B: basic residue, X: nonbasic residue in the C-terminal side); and 3)
length between 10 and 26 residues (6 ). Okamoto et al. (7 )
found that B2 can be substituted for alanine,
based upon the results from alanine residue substitution, and Ikezu
et al. (8 ) found that B3 can be
substituted for aromatic amino acid residue, based upon the identified
sequence in the
2-adrenergic receptor. As this is a screening method
for the G protein-coupling candidates followed by confirmation (or
exclusion) with actual experimentation, we considered reasonably broad
criteria to be most useful. To allow as broad a screening as possible,
we have therefore revised criterion 2 to 2'
B1-B2-X-B3,
or
B1-B2'-X-X-B3
(B1/B2'/B3:
basic or aromatic residue, B2: basic or aromatic
residue or alanine, X: non-basic residue). These criteria potentially
represent the structural characteristics of the regions shared by
various GPCRs as well as those of a small number of the regions that
have been shown to directly activate G proteins in vitro.
With a small number of GPCRs, the usefulness of these criteria have
been noted by ourselves (9 10 ) and by other groups (11 12 13 14 15 16 17 18 19 ), when
these researchers specified domain-specific functions in GPCRs or a
non- or atypical receptor type of G protein-linked proteins. Analysis
of the predicted amino acid sequence of the porcine CTR revealed the
presence of two nonoverlapping regions, referred to here as P3 and P4,
as assessed by these criteria. The first sequence KLKESQEAESHMYLKAVR
(P3) is located in the third cytoplasmic loop (residues 327344; the
numbering is for CTR-1a); the second sequence KRQWNQYQAQRWAGR (P4) is
located in the C-tail of the receptor (residues 404418). While there
are two alternative splicing isoforms of the porcine CTR, termed CTR-1a
and -1b (20 ), these sequences are completely conserved between them. It
should be emphasized that a few GPCRs so far investigated with the
aforementioned criteria bear the E/DRY motif in the juxtamembrane
region of the second intracellular loop, whereas CTR belongs to a
different subfamily of GPCRs, bearing no E/DRY motif in the
corresponding site. Therefore, it was further necessary to investigate
whether these regions in CTR can interact with Gs
in intact cells.
Short polypeptides of length less than 30 amino acids, such as the P3
and P4 regions, are generally hard to express in cells and, even if
expressed, may not have effective access to the plasma membrane where G
proteins reside. We therefore developed a novel strategy using an
insulin-like growth factor II (IGF-II) receptor (IGF-IIR) hybrid system
as a sequence-expressing vector for monitoring the stimulation of AC.
Increased AC activity is one of the most reliable indices of
Gs stimulation in intact cells. The IGF-IIR is
the first single-transmembrane receptor identified that interacts with
and activates G proteins, especially Gi, in
cell-free systems (21 ), semi-cell-free systems (22 ), or whole-cell
systems (23 24 ). Although failure of IGF-IIR coupling to G proteins
was reported once (25 ), a subsequent paper (23 ), with two of the same
authors, demonstrated the Gi coupling function of
IGF-IIR in vivo. While most of the GPCRs consist of a
heptahelical structure, such a structure is therefore not an absolute
prerequisite for interactions with G proteins. Subsequent studies by
ourselves (26 ) and by other groups (14 16 17 ) have lent additional
credence to this notion. In the case of IGF-IIR, its cytoplasmic
R2410-K2423 region has been consistently shown to be the domain
necessary and sufficient for Gi activation in all
systems so far analyzed (6 22 24 ). It has been shown that the
Gs-activating function of a specific amino acid
sequence can be examined by replacing this R2410-K2423 region in the
IGF-IIR, expressing the hybrid IGF-IIR, and checking whether the
activity of the cAMP system is enhanced (22 ).
We constructed two hybrid IGF-IIR cDNAs, each containing the P3 or P4
sequence in place of the native R2410-K2423 region (the hybrid
receptors are referred to as P3/IGF-IIR or P4/IGF-IIR, respectively).
These constructs were transiently expressed in COS cells and examined
for activation of the AC pathway by monitoring for stimulation of the
membrane AC activity and increased production of cAMP in intact cells.
We also investigated the intramolecular role of these domains by
examining CTR mutants lacking either the P3 or P4 domains.
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RESULTS
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The primary sequence of the cloned porcine CTR-1a (pCTR) was
analyzed for regions that satisfy the aforementioned criteria for G
protein-interacting candidate domains (see Introduction). We
identified two such domains, both located in the intracellular regions.
The first sequence, KLKESQEAESHMYLKAVR (P3), is located in the
predicted third intracellular loop (amino acids 327344); the second
sequence, KRQWNQYQAQRWAGR (P4), is located in the C-terminal tail of
the receptor (amino acids 404418) (Fig. 1A
). These sequences are completely
conserved in the corresponding sites in the third intracellular loop
and the C-tail in CTR-1b.

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Figure 1. Hybrid Receptor Construction
A, The amino acid sequence of the porcine CTR from position 297 to 424,
and location of the putative G protein-coupling sequences in the third
intracellular domain (residues 327344) and in the C-tail of CTR
(residues 404418). B, Construction of the hybrid
receptors. The IGF-IIR-XhoI has two XhoI
sites, corresponding to Leu-Glu (LE), between codons for wild-type
IGF-IIR residues 2409 and 2410, and residues 2423 and 2424 (the
inserted LEs are therefore at positions 2410/2411 and 2426/2427). The
hybrid P3/IGF-IIR or P4/IGF-IIR thus consists of the P3 or P4 sequence,
respectively, LE before and after these sequences, and IGF-IIR lacking
the native amino acids 24102423.
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Hybrid P3/and P4/IGF-IIRs were constructed utilizing the
XhoI sites, which had been inserted into the original human
IGF-IIR cDNA as described previously (22 ). Sequencing of the region
encoding the inserts between these sites verified that P3/and
P4/IGF-IIRs have the correct P3 or P4 sequences between the native
amino acids 2410 and 2423 of the IGF-IIR (Fig. 1B
). Hybrid IGF-IIRs
with reverse-oriented P3 or P4 sequences were obtained in other clones.
No stop codon was present in these reverse sequences, thus allowing the
expression of the entire hybrid receptors. The deduced sequences of
these reverse-oriented constructs were as follows: revP3,
SNSFKIHRFSFLRFFKF; revP4, STSPSLSLVLIPLSF. Each consisted of the same
residue numbers as the original P3 or P4 region. The corresponding
hybrid receptors, referred to as revP3/and revP4/IGF-IIR, were used as
controls in the following experiments.
Mock-transfected COS cellscells transfected with two different empty
vectors (pECE or pcDNA1)exhibited low basal membrane AC activity
(Fig. 2
, A and B). When cells were
transfected with either P3/IGF-IIR or P4/IGF-IIR cDNA, membrane AC
activity significantly increased. In contrast, transfection of either
hybrid receptor with reverse-oriented regions, revP3/IGF-IIR or
revP4/IGF-IIR, was without effect on AC activity (Fig. 2A
). In these
experiments, the hybrid receptors, P3/IGF-IIR, P4/IGF-IIR,
revP3/IGF-IIR, and revP4/IGF-IIR, were expressed to similar levels, as
assessed with anti-IGF-IIR antibody immunostaining or IGF-II binding
(data not shown). In cells expressing P3/IGF-IIR or P4/IGF-IIR, low
concentrations of IGF-II exerted a small additional effect on AC
activity. The wild-type IGF-IIR had no positive effect on AC activity
in the presence or absence of IGF-II (Fig. 2B
).

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Figure 2. AC Activity of the Membranes from Cells Transfected
with the Wild-Type pCTR or with P3/IGF-IIR or P4/IGF-IIR
A, Various amounts of either P3/IGF-IIR or P4/IGF-IIR hybrid receptor
cDNAs were transfected into 106 COS cells, and the cell
membranes were prepared for the measurement of the AC activity, as
described in Materials and Methods. As controls,
activity of the membranes prepared from cells transfected with 5 µg
of the empty vector used for hybrid receptor constructions (pECE), with
5 µg of the wild-type pCTR cDNA in the absence (-) or presence (+)
of 100 nM sCT, or with the non-sense revP3/IGF-IIR (10
µg) or revP4/IGF-IIR (2.5 µg) cDNA. The 100% activity is the
activity stimulated by 100 µM isoproterenol in parental
COS cells, 49.6 ± 3.9 pmol/min/mg protein. B, Effect of IGF-II on
AC activity in P3/IGF-IIR- or P4/IGF-IIR-expressing membranes. Membrane
AC activity of COS cells transfected with 10 µg of P3/IGF-IIR, 2.5
µg of P4/IGF-IIR, or wild-type IGF-IIR cDNA was measured in the
absence (-) or presence (+) of 3 nM of IGF-II (10
nM IGF-II was used in case of IGF-IIR transfection). As
controls, we used activity of the membranes prepared from cells
transfected with 5 µg of the pECE vector, with 5 µg of the pcDNA1
vector (the plasmid encoding wild-type pCTR cDNA), or with 5 µg of
the wild-type pCTR cDNA in the absence (-) or presence (+) of 100
nM sCT. The 100% activity is the activity stimulated by
100 µM isoproterenol in parental COS cells, 42.3 ±
6.9 pmol/min/mg protein. Values in all figures in this study represent
the mean ± SE of triplicate samples from at least two
independent transfections.
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The AC activity in membranes expressing either P3/IGF-IIR or P4/IGF-IIR
was comparable to or even higher than that measured in membranes
expressing the wild-type pCTR treated with 100 nM, a
saturating concentration of salmon CT (sCT) (Fig. 2
, A and B). As shown
in Fig. 2A
, as higher amounts of P3/IGF-IIR cDNA were transfected, more
increase was observed in AC activity. In contrast, the profile of the
relation between the DNA amount and the AC response was different for
P4/IGFIIR. Stimulation of AC activity was observed for lower cDNA
amounts. Activity peaked for 2.5 µg P4/IGF-IIR cDNA and then
decreased for higher amounts of this hybrid receptor. However, the
maximal AC activities attained by these two hybrid receptors were
comparable.
We also measured the reporter activity of the cAMP response element
(CRE) in the cell homogenates after transfection of either hybrid
receptor cDNA with the CRE-driven chloramphenicol acetyltransferase
(CAT) plasmid. CRE is typified by the consensus palindromic sequence
TGACGTCA, which is present in the promoters of many genes. Our CRE-CAT
reporter has multiple CRE sequences located in the promoter of the
somatostatin gene, which is highly and selectively responsive to cAMP
stimulation. Transient expression requires a prolonged period of time
and the gene transiently transfected is expressed randomly during this
period. This would make it technically difficult to quantitate the
cAMP-stimulating function of constitutive receptors by measuring cAMP
concentrations, which could rapidly fluctuate in the cells after the
receptor cDNA transfection. In contrast, with the CRE-CAT reporter
method, the accumulation of cAMP activity stimulated by the expression
of transfected activators could be more properly assessed.
Cells expressing P3/IGF-IIR or of P4/IGF-IIR exhibited significant
increases in CAT activity (Fig. 3
, A and
B). These increases were dependent on the amount of transfected
plasmids. Concerning each hybrid receptor, the profile of the relation
between the DNA amount and the CRE-CAT response was proportional to
that observed in the cell-free AC assay, suggesting that the
hybrid-induced CRE activation is tightly linked with the hybrid
receptor-induced AC activation. IGF-II treatment again exerted small
additional effects on the transcriptional activity of CRE in either
transfection of P3/IGF-IIR or P4/IGF-IIR (Fig. 3C
). In contrast, the
CRE activities in cells transfected with empty vectors or with either
of the two control hybrid receptors having reverse-oriented sequences
(revP3/IGF-IIR and revP4/IGF-IIR) were low (Fig. 3C
). Likewise, the
wild-type IGF-IIR had no significantly positive effect on CRE activity
with or without IGF-II (Fig. 3C
).

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Figure 3. CAT Activity in Cells Transfected with Hybrid
Receptor cDNAs and the CRE-CAT Reporter Plasmid
A and B, COS cells were cotransfected with various amounts of hybrid
IGF-IIR cDNAs and 2.5 µg of the CRE-CAT plasmid. After 48 h,
cell homogenates were prepared and CAT activity was assayed. As
controls, COS cells were transfected with pECE (5 µg), or wild-type
pCTR cDNA (5 µg) in the presence (+) or absence (-) of 100
nM sCT. The maximal activity elicited by
(Bu)2cAMP in mock-transfected cells was 400 ± 20
cpm/h (means ± SE, n = 3). C, COS cells
were transfected with 10 µg of P3/IGF-IIR, 2.5 µg of P4/IGF-IIR, or
wild-type IGF-IIR cDNA in the presence of 2.5 µg of the CRE-CAT
plasmid. During the last 12 h, cells were incubated with 3
nM IGF-II (+) or vehicle (-) (10 nM IGF-II was
used in case of IGF-IIR transfection). After 48 h, cell
homogenates were prepared and CAT activity was assayed. As controls,
COS cells were transfected with 5 µg of pECE, 5 µg of pcDNA1, 5
µg of wild-type pCTR cDNA in the presence (+) or absence (-) of 100
nM sCT, or with the hybrid receptors bearing
reverse-oriented sequences: revP3/IGF-IIR (10 µg cDNA) or
revP4/IGF-IIR (2.5 µg cDNA). The values indicate CAT activity
relative to that of pECE-transfected cells. The maximal activity
elicited by (Bu)2cAMP in mock-transfected cells was
approximately 12-fold of the control activity.
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As a positive control in this assay, we measured the CRE activity in
cells transfected with wild-type pCTR cDNA with or without CT
treatment. Cells were transfected and after 28 h were treated with
saturating concentrations of sCT for 20 h of culture. The cells
treated with 100 nM sCT showed a few hundreds % of
increase in CRE activity compared with untreated cells (Fig. 3
, A, B, and C). Therefore, both P3/IGF-IIR and P4/IGF-IIR stimulated the
transcriptional activity of CRE to levels comparable to or even higher
than the maximal CT-induced stimulation of the wild-type pCTR. These
findings indicate that AC stimulation by both P3- and P4-containing
hybrid receptors does trigger in vivo activation of
intracellular responses leading to nuclear gene expression.
Although the observed CRE stimulation most likely resulted from
in situ stimulation of AC by the expressed hybrid receptors
within a single cell, it was also possible that the hybrid receptor
expression resulted in an extracellular secretion of factors that could
trigger the CRE activation cascade in the neighboring cells. If so, the
CRE-CAT assay might not reflect direct stimulation of AC activity by
the hybrid receptors. To clarify this issue, we initially transfected
COS cells with each DNA (hybrid receptors and CRE-CAT), respectively,
later mixed them, and measured whether CRE-CAT activity was elevated.
As shown in Fig. 4
, the mixture of the
separately transfected cells resulted in no significant increase in
CRE-CAT activity, as compared with the elevated CRE-CAT activity in
cotransfected cells. This observation provides evidence that
stimulation of CRE-CAT should be a direct result from the activation of
intracellular cAMP pathways triggered by hybrid receptors. In support,
the coincidence of immunoreactivities was observed when the cells
cotransfected with hybrid receptor and CRE-CAT plasmids were doubly
stained with anti-IGF-IIR antibody and anti-CAT antibody (data not
shown). Therefore, both P3- and P4-containing hybrid receptors
activated the AC and consequent CRE cascade in situ within a
single cell.

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Figure 4. Comparison of the Effect of the Mixed Transfection
of a Hybrid Receptor and CRE-CAT Plasmid with That of Independent
Transfection/Subsequent Mixture
In the right two columns, 0.9 µg of P3/IGF-IIR or
P4/IGF-IIR was cotransfected with 0.9 µg of CRE-CAT into COS cells
(3 x 105). Twenty-four hours after transfection,
those cells were replated into one dish at 4 x 104.
CAT activity was measured 36 h after replating. In the left
three columns, 0.9 µg of P3/IGF-IIR or P4/IGF-IIR was
transfected into COS cells (3 x105) in one dish and 0.9
µg of CRE-CAT into COS cells (3 x105) in the other dish.
Twenty-four hours after transfection, those cells (2 x
104 each) were mixed into one dish, and CAT activity was
measured 36 h after mixture. The values indicate the CAT activity
relative to that of cells transfected separately with 0.9 µg of pECE
and 0.9 µg of CRE-CAT. In this setting, 0.9 µg of a plasmid was
equivalent to 3 µg in other experiments in this study. The values
represent the mean ± SE of eight independent
experiments.
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Finally, we applied a conventional deletion approach to the P3 and P4
domains. We constructed mutant pCTRs lacking either the P3 or P4 region
from the wild-type pCTR (
P3-CTR or
P4-CTR; Fig. 5A
). To construct the
P3-CTR, we first
prepared pCTR possessing XhoI sites immediately before and
after the P3 region (CTR-XhoI). As the XhoI site
represents Leu-Glu (LE) in amino acids, the CTR-XhoI
contains two LEs flanking the P3 region.
P3-CTR, which was
constructed by digesting CTR-XhoI with XhoI, has
LE instead of the P3 region. In contrast,
P4-CTR has no additional
LE in the third intracellular loop or the C-terminal tail, as it was
constructed from the authentic CTR cDNA. We also constructed a
revP3-addback
P3-CTR mutant (rP3-
P3-CTR), a pCTR mutant
which, instead of the P3 region, retains the reverse (at a nucleotide
level) oriented sequence revP3 flanked by two LEs in the third
cytoplasmic loop. In this regard, CTR-XhoI is identical to
the P3-addback
P3-CTR. We initially found that the expression level
of CTR-XhoI decreased severalfold relative to that of
authentic CTR (Fig. 5C
, lower inset), although both the CT
affinity and the molecular capacity to produce a cAMP response were
similar between authentic CTR and CTR-XhoI (see below). We
thus used CTR-XhoI as a positive control in the following
experiments (only for
P4-CTR, authentic CTR provided a control). It
should also be noted that as we used the pCTR containing a
hemagglutinin (HA) tag between residues 66 and 67, CTR-XhoI,
P3-CTR, rP3-
P3-CTR, and
P4-CTR were all tagged with an HA
epitope.

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Figure 5. Examination Of COS Cells Transfected with Mutant
CTR cDNAs
A, Illustrations of the mutant CTRs. B, cAMP production in cells
transfected with mutant CTR cDNAs. COS cells were transfected with
mutant CTR cDNAs at 48 h before cAMP assay. To test for
hormone-induced cAMP responses, cells in triplicate wells were
incubated with either test buffer alone or with increasing
concentrations of sCT. Similar experiments were performed three times,
each of which resulted in similar results. Inset,
dose-response curves of the sCT effect in COS cells transfected with
authentic pCTR cDNA (open circles) or
CTR-XhoI cDNA (closed circles). COS cells
were transfected with CTR cDNAs at 48 h before cAMP assay, in
which cells in triplicate wells were incubated with either test buffer
alone or with increasing concentrations of sCT. The cAMP response was
indicated as a fold of the basal cAMP content, which was similar to
that shown in panel B. C, CT binding to cells transfected
with mutant CTR cDNAs. Cells were transfected with mutant CTR cDNAs at
48 h before the binding assay. The binding of radioactive CT to
transfected cells in duplicate wells was measured in the presence of
increasing concentrations of nonradioactive sCT. The values represent
the mean of the specific binding (total binding - nonspecific
binding; nonspecific binding is the binding in the presence of 100
nM nonradioactive CT). Each value indicates the mean of
results obtained from two independent dishes. The data indicate a
representative result of four independent experiments. Lower
inset, CT binding to cells transfected with authentic pCTR cDNA
(open circles) is shown with the CT binding to
CTR-XhoI-transfected cells (closed circles) shown
in panel C. Experiments were performed similarly to those in
panel C. Upper inset, Immunoblot
analysis of transfected CTR mutants (1: pcDNA1, 2: CTR-XhoI,
3: rP3- P3-CTR, 4: P4-CTR, 5: P3-CTR). Forty-eight hours after
transfection of mutant CTR cDNAs, cell lysates (30 µg protein/lane)
were submitted to immunoblot analysis with anti-HA antibody (1.6
µg/ml; Roche Molecular Biochemicals). Similar
experiments were performed four times, resulting in similar data.
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sCT treatment of cells transfected with CTR-XhoI resulted in
a dose-dependent increase in cAMP production (Fig. 5B
). In clear
contrast, sCT treatment of transfected cells elicited little cAMP
response, when cells were transfected with
P3-CTR or rP3-
P3-CTR.
Cells transfected with
P4-CTR exhibited a significant CT-induced
cAMP response. We then examined the surface binding of radiolabeled sCT
to transfected cells. As shown in Fig. 5C
, CTR-XhoI and
P4-CTR were similarly expressed on the surface of the transfected
cells with similar affinity for CT. In contrast, there was minimal CT
binding to cells transfected with either
P3-CTR or rP3-
P3-CTR.
Immunoblot analysis using anti-HA antibody revealed that
CTR-XhoI, rP3-
P3-CTR, and
P4-CTR were comparably
expressed in transfected cells, whereas
P3-CTR was poorly expressed
(Fig. 5C
, upper inset).
As rP3-
P3-CTR, in which the same length peptide was replaced for the
P3 region, was significantly expressed, the poor expression of
P3-CTR was probably due to rapid degradation based upon an improper
folding induced by the replacement of the P3 region, which occupies a
major portion in the third intracellular loop, with the amino acids LE.
As anti-HA antibody nonspecifically recognized an approximately 80-kDa
protein as strongly as the transfected CTR proteins in the
immunoblotting (data not shown), we were unable to further clarify, by
means of the immunostaining analysis, whether rP3-
P3-CTR was
expressed on the cell surface. While it remained unclear whether the
poor CT binding to the rP3-
P3-CTR-transfected cells was due to a
loss of the affinity for CT and/or due to inefficient trafficking from
the cytoplasm to the surface, it became difficult, with the
conventional deletion approach, to further address the in
vivo function of the P3 region in CTR, because CT failed to bind
to the cells transfected with the P3-deleted mutants, one with a simple
deletion and the other with a replacement of the same length
polypeptide. In this regard, the hybrid receptor approach, which
detects a positive function of a receptor domain without any necessity
for a receptor ligand (CT in this case), points to a role for the P3
region in the signal transduction mechanism of CTR.
In contrast to the P3 region study, the function of the P4 region could
be examined by our mutagenesis approach. The data suggest that the P4
region was dispensable for AC activation per se. However,
comparison of the maximal CT-induced cAMP responses standardized by
Bmax between authentic CTR and
P4-CTR
indicated a decrease in the molecular capacity to produce cAMP response
by the deletion of P4 domain from CTR. The Bmax
data indicated that transfection of authentic CTR, CTR-XhoI,
and
P4-CTR cDNAs resulted in expression of the cognate receptors at
260 fmol/well, 51 fmol/well, and 62 fmol/well, respectively,
corresponding to 3.9 x 106, 7.7 x
105, 9.3 x 105
(number per cell). Cells transfected with authentic CTR,
CTR-XhoI, and
P4-CTR cDNAs and incubated with sCT
increased cAMP content to a mean maximum of 21, 17, and 9.5
(pmol/receptor x 10-10), respectively. Not
only the expression level of transfected authentic CTR (number per
cell) but also the calculated value of the transfected CTR capacity to
produce maximal cAMP response (pmol/receptor) was comparable to those
previously reported (27 ). These data indicate that the maximal capacity
of CTR to produce cAMP in response to CT was impaired by the deletion
of P4 domain. It is thus highly likely that P4 domain regulates the CTR
function in transducing the CT signal to its effector by modulating the
maximal response of Gs activation by this
receptor.
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DISCUSSION
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We have herein shown novel domain-specific
Gs-activating functions in the third
intracellular loop and the C-tail of CTR, using the cassette IGF-IIR
method. The IGF-IIR containing two independent CTR sequences, but
neither wild-type IGF-IIR nor the IGF-IIR containing irrelevant
sequences of the same length, augmented both membrane AC activity and
nuclear CRE transcriptional activity. In addition, CRE activity was
stimulated only when the hybrid IGF-IIR cDNAs were cotransfected with
the reporter plasmid, indicating that CRE stimulation was the direct
result of the activation of intracellular cAMP pathways triggered by
hybrid receptors. In support of this notion, the AC stimulation and the
CRE response caused by each hybrid receptor were highly proportional.
These results indicate that hybrid IGF-IIRs containing either the P3 or
P4 domain of CTR stimulated the ACCRE cascade in the hybrid
receptor-expressing cells. Multiple controls ensured that the inserted
CTR sequences conferred cAMP-stimulating ability on the hybrid
receptors. The conventional mutagenesis study supported a significant
role of the P4 domain in the maximal capacity of CTR to activate
Gs. However, in this case, the mutagenesis study
did not allow for further investigation of the P3 function in CTR,
suggesting that it would have been difficult to specify the P3 function
in vivo without using the hybrid receptor approach.
The data measuring membrane AC activity in cells transfected with CTR
or hybrid IGF-IIRs were quantitatively consistent among experiments,
when standardized by maximal response to isoproterenol: the basal
activity was 3040%, and the activities stimulated by 5 µg CTR cDNA
and 100 nM sCT, by 10 µg P3/IGF-IIR cDNA, and by 2.5 µg
P4/IGF-IIR cDNA were similarly 7080% of the maximal AC activity
stimulated by isoproterenol. In contrast, the data measuring CRE
activity were more variable among experiments. For the CRE assay, we
were unable to employ isoproterenol as an interexperimental standard,
since an increase in CRE-CAT by high concentrations of isoproterenol
was marginal in this system, as reported previously (22 ), although this
observation suggested that ß-adrenergic receptors were rapidly
down-regulated after isoproterenol stimulation. While
(Bu)2cAMP yielded the standard maximal response
of CRE-CAT stimulation in this assay, normalization among CRE-CAT
experiments was not simple, even with (Bu)2cAMP
as a standard, particularly for CTR. This was so because 1) basal
CRE-CAT production fluctuates within a range of approximately 1/12 to
1/4 of the maximal production by (Bu)2cAMP; and
2) the quantity as well as the timing of down-regulation of CTR
occurring after CT stimulation should vary, depending on each
transfected cell, whereas (Bu)2cAMP stimulation
may not be down-regulated as it is with receptors. The variability in
the CRE response to CTR was thus, at least in part, attributable to the
receptor down-regulation. Furthermore, CRE activation by CTR might be
conditionally influenced by cell cycle, whereas the cell cycle
condition of transfected cells could not be reproduced precisely. It
has been reported that Gi is activated by CTR in
a cell-cycle dependent manner (2 ). Such a cell cycle-dependent action
of CTR could be responsible for interexperimental variability in the
CRE response to CTR.
In contrast, CRE activity stimulated by hybrid receptors was
consistently 6070% of the maximal response by (Bu)2cAMP; the maximal
CRE responses to both hybrid receptors were comparable. This suggests
that hybrid receptors were more resistant in receptor down-regulation
than CTR and that the variability in the CRE response by hybrid
receptors was thus mainly due to the fluctuation of the basal CRE-CAT
production. In support of this idea, Wada et al. (28 )
reported a cAMP-dependent protein kinase A (PKA)-mediated
down-regulation of CTR as a major mechanism underlying homologous
desensitization; in contrast, the hybrid IGF-IIRs would not be affected
by CTR-mediated down-regulation, as IGF-IIR is highly resistant to both
homologous and heterologous down-regulation (29 ), and no potential PKA
phosphorylation site is included in either the P3 or P4 domain. The
observed discrepancy between the hybrid receptors and liganded CTR
could thus be attributable, at least in part, to the difference in the
receptor sensitivity to CTR- or CTR sequence-mediated
down-regulation.
As compared with P3/IGF-IIR, the P4 hybrid receptor caused both cAMP
and CRE responses by transfection with lower DNA concentrations.
As both receptors were expressed similarly at each DNA
concentration, this finding suggests that the P4 sequence may be
more effective in activating the cAMP system. However, it remained
unclear why impaired actions of P4/IGF-IIR plasmid were observed at
higher DNA doses. A simple interpretation was that AC activation by
P4/IGF-IIR was attenuated by a negative feedback mechanism acting on
P4/IGF-IIR: the mechanism was only triggered by highly expressed
P4/IGF-IIR. Such a feedback mechanism should be other than through PKA-
or PKC-mediated regulation, because the P4 sequence contains no
potential phosphorylation sites for these protein kinases.
Alternatively, the feedback mechanism could act on
Gs. For instance, it has been reported that
phosphorylation of the Tyr residue at the C terminus of the G protein
subunits (G
s and
G
q/11) alters the efficiency of receptor-G
protein interactions (30 31 ), and that CT stimulation of CTR activates
Shc tyrosine phosphorylation and downstream pathways (32 ). It is thus
conceivable that Gs may change its response to
P4/IGF-IIR through phosphorylation, potentially resulting in altered
actions of highly expressed P4/IGF-IIR to act on AC. Obviously, more
detailed investigation is necessary to address this issue.
The magnitude of the maximal stimulation by the P3 and P4 hybrid
receptors was comparable to or even higher than that observed with
maximally liganded CTR, suggesting that those two short domains in the
CTR can quantitatively mimic the action of the entire receptor.
Activation of the intracellular cAMP system appears to be the major
signaling pathway involved in regulation of the physiological effects
of CT in target cells (1 4 5 7 8 9 10 11 ). Therefore, it is likely that
the dissected AC-activating domains in CTR are of physiological
significance, and it is tempting to examine whether the active hybrid
receptors bearing the CTR domains can mimic the biological functions of
CTR in primary cultured target cells or in transgenic mice.
Although the P3 and P4 regions were selected based upon the structural
characteristics of the regions shared by various GPCRs as well as those
of a small number of the regions that have been shown to activate G
proteins in vitro, the universality may not necessarily be
given to this procedure. It has become evident that a complex inner
surface defined by multiple intracellular loops and the C-tail is
necessary for receptors to interact with G proteins, which has limited
the usefulness of sequence-based approaches toward defining the
function of GPCRs. However, this notion does not deny the
domain-specific functions in GPCRs or the sequence-based approach to
determine the functions of G protein-linked molecules other than GPCRs.
In addition, a method that can dissect the in vivo function
of a given region is useful. The present study provides such a method,
which is applicable to unlimited numbers of regions in G protein-linked
molecules.
For the purpose of examining the G protein-linked function of receptor
domains in vivo, the IGF-IIR vector provides several
advantages. A plasmid encoding a protein interactive with G proteins
would be appropriate as an expression vector for a sequence of interest
in testing its G protein-stimulating activity, as such a vector protein
is accessible to G proteins when expressed in intact cells. Also, a
receptor domain natively involved in the coupling to G proteins would
be appropriate as a site for the substitution of the test sequences.
Accumulated evidence indicates that most GPCRs contain multiple
nonoverlapping G-protein-interacting domains in various cytoplasmic
loops or in the C-tail (8 33 34 ). Therefore, it is quite difficult to
assign a specific G protein-linked function to specific receptor
domains by conventional mutational or deletional approaches (35 ). Also,
this redundancy makes it hard to specify a G protein-coupling function
for a short sequence derived from other receptors by expressing these
sequences in a recombinant GPCR. In contrast, IGF-IIR contains only one
site for interaction with G-proteins (6 ), allowing for a simple
interpretation of the data obtained from hybrid IGF-IIRs expressing
putative G protein-interacting domains of other receptors. Furthermore,
IGF-IIR is highly resistant to down-regulation (29 ), as also suggested
by the experiments with the hybrid receptors. When receptor
down-regulation occurs after cellular expression of the receptor cDNA
used for expressing test sequences, it becomes difficult to examine the
sequence function. Even if receptor down-regulation does not occur
completely, when it occurs randomly after expression of the
constitutive receptors, the data examining the signal of the expressed
receptors should accompany large quantitative variability. For these
reasons, the present system would be suitable for the examination of
the domain-specific function to activate Gs
in vivo.
There are advantages and disadvantages in both the conventional
mutagenesis approaches and the developed hybrid receptor approach. For
instance, there is an obvious limitation in assessing the physiological
role of a selected domain in the entire function of the relevant
receptor, using the cassette IGF-IIR method. We must also emphasize
that the successfulness of the hybrid receptor approach may still be
very dependent on the sequence of GPCRs chosen and that the usefulness
of this method may therefore be limited to a narrow spectrum of GPCRs.
However, once it works, this method will be able to dissect a
domain-specific signaling function, whereas the mutagenesis approach
cannot usually exclude interference from other domains in the same
receptor. Furthermore, as observed in this study, when
domain-mutagenized receptors lose proper surface expression or ligand
binding, the mutagenesis approach does not allow for further
investigation of the domain-specific signal transduction. Even in such
a case, the hybrid receptor approach is able to investigate it in
vivo. Mutagenesis study primarily assumes a region-specific
function by deleting or substituting the region of interest to observe
negative changes or disappearance of cellular outputs, whereas the
hybrid receptor approach provides an in vivo method to
detect domain-specific function positively. Combining these
supplementary methods, researchers will be able to deepen the
understanding of the signal transduction mechanism for transmembrane
receptors.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
The IGF-IIR-XhoI cDNA was described previously (22 ).
This cDNA has two unique XhoI sites, corresponding to LE in
amino acids, before and after the R2410-K2423 region of the human
IGF-IIR. These additional residues do not alter the capacity of IGF-IIR
to couple to Gi, as described (22 ).
Oligonucleotides corresponding to the P3 domain
(TCGAGAA-ACTTAAAGAATCTCAAGAAGCTGAATCTCATATGTATCTT-AAAGCTGTTAGAC
and CGAGTCTAACAGCTTTAAGATA-CATATGAGATTCAGCTTCTTGAGATTCTTTAAGTTTC)
or to the P4 domain
(TCGAGAAAAGACAATGGAATCAGTACCAA-GCTCAAAGATGGGCTGGTAGAC and
CGAGTCTACCGCCCATCTTTGAGCTTGGTACTGATTCCATTGTCTTTTC) were hybridized and
ligated between the XhoI sites. Double-stranded plasmids
were sequenced to confirm the correct orientation of the P3 and P4
inserts in the IGF-IIR-XhoI cDNA sequence. The porcine CTR
cDNA (the 1a version) (1 ) was inserted into pcDNA1 with the HA tag
PYDVPDYA between residues 66 and 67, as described previously (36 ). A
PCR strategy was designed to delete the P3 sequence at nucleotide
position 12341287 (the nucleotide numbering is by GenBank M74420),
while inserting a XhoI site at nucleotide position 1234,
using the following couples of primers CCAACTACACTATGTGCAATGC and
AGTGGCCTCGAGCTTCACGAGCACGCGGAGG for the 5'-side,
CCCTGGAAGTGGATCAGAGAGTGCACCACGT and
TCGTGAAGCTCGAGGCCACTCTGATCTTGGTGCC for the 3'-side.
Underlined nucleotides indicate the sequence for the
XhoI restriction site to be added exactly at the site of the
deletion. Two separate PCRs were performed using each set of primers
and the HA-tagged pCTR cDNA in pcDNA1 as template, with 25 cycles (1
min at 95 C/2 min at 55 C/3 min at 72 C). Subsequently, a second-round
PCR was performed, using a three-step procedure. In the first step, 20
µmol of each PCR product from the first reactions
were mixed together with deoxynucleoside triphosphates and
MgCl2, and heated at 80 C for 10 min under a
layer of wax. Then, 0.5 µl of Taq polymerase was added on
top of the wax and one cycle was performed (1 min at 94 C/2 min at 50
C/3 min at 72 C). Finally, a preheated mix of both outside primers was
added and the final PCR was run for 20 cycles (1 min at 94 C/2 min at
50 C/3 min at 72 C). The final PCR product, encoding a cDNA fragment
exhibiting the deletion of the P3 sequence and the insertion of the
XhoI site, was digested with appropriate restriction enzymes
and ligated to the HA-tagged pCTR cDNA digested with the same
combination of restriction enzymes. Positive clones were selected and
sequenced to confirm the deletion of the P3 domain and the presence of
the XhoI site. The same PCR strategy was used to delete the
P4 sequence at nucleotide position 14651509, except that no
XhoI site was inserted at the site of the deletion. The
5'-side primers were CCAACTACACTATGTGCAATGC and
GGTGGAGCGCAGGGCTCCCTGAAC-CTCG, and the 3'-side primers were
CAACACCCTCCACCTCC and GGAGCCCTGC-GCTCCACCCGGGCCGC. All plasmid DNAs
were prepared by maxipreps of overnight Escherichia coli
cultures, using the alkaline lysis procedure, followed by two cycles of
CsCl equilibrium density gradient centrifugation, and repeated
isopropanol and phenol/chloroform extractions. Purified plasmid cDNAs
were stored at -80 C until use for cell transfection.
Cell Transfection
Plasmids were transiently expressed in COS cells by transfection
using LipofectAMINE (2 µl/µg DNA, Life Technologies, Inc., Gaithersburg, MD), as described previously (24 ). COS cells
were plated at 106 cells in a 100-mm dish 24
h before transfection and cultured in DMEM supplemented with 10% calf
serum. After transfection, cells were cultured for 24 h in a
humidified atmosphere of 5% CO2-95% air at 37
C. After removing transfection media, prewarmed DMEM plus 10% calf
serum was added to the cultures. Cells were cultured for another
24 h.
AC Assay
The AC assay was performed, as described previously (22 ).
Membranes were prepared from cells 48 h after transfection. Cells
were incubated for 30 min at room temperature with PBS containing 5
mM mannose 6-phosphate, washed three times with PBS,
scraped, suspended in ice-cold PBS, and centrifuged at 1500 rpm for 5
min. The pellet was suspended in ice-cold buffer A [20 mM
HEPES/NaOH (pH 7.4), 1 mM EDTA, 1 mM
dithiothreitol, 20 µM leupeptin, and 20 µg/ml
aprotinin], homogenized, and centrifuged at 1,500 rpm for 5 min. The
pellet was again suspended in buffer A, homogenized, and centrifuged.
The first and second supernatants were mixed and centrifuged at 15,000
rpm for 60 min at 4 C. The final pellet suspended in buffer A was
subjected to AC assay. AC activity of prepared membranes (60 µg of
membrane protein) was measured for 20 min at 30 C in 100 µl of 20
mM HEPES/NaOH (pH 8.0) buffer containing 0.37
µM [
-32P]-ATP (NEN Life Science Products ; specific activity, 30 Ci/mmol), 1
mM ATP, 1 mM cAMP, 10 µM GTP, 1
mM EDTA, 2 mM MgCl2, 5
mM phosphoenol pyruvate, 25 µg/ml pyruvate kinase, 0.1
mM isobutyl methylxanthine, 0.1 mg/ml BSA. Recombinant
IGF-II (Roche Molecular Biochemicals), sCT
(Peninsula Laboratories, Inc., San Carlos, CA), or a
respective vehicle was added to the appropriate reaction solutions,
just before mixing them with the aliquots of membrane preparations.
Resultant radioactive cAMP was measured by two-step column
chromatography according to the method of Salomon. The maximal AC
activity was measured in untransfected COS cell membranes stimulated by
100 µM isoproterenol.
CRE-CAT Assay
The CAT plasmid fused with triple CRE of the somatostatin gene
(22 ) [provided by Dr. S. Ishii (Institute of Physical and Chemical
Research, Saitama, Japan)] was cotransfected with plasmids encoding
hybrid receptors. Unless otherwise specified, 106
cells were used for these assays. Transfected cells were treated with
IGF-II or vehicle for the last 20 h of day 2 after transfection;
cells transfected with wild-type pCTR were treated with 100
nM sCT or vehicle for the last 20 h of the
transfection, similarly. Forty-eight hours after transfection, cells
were washed twice with ice-cold PBS and scraped in 40 mM
Tris/HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA.
After centrifugation at 8,000 rpm for 2 min at 4 C, the pellet was
suspended in 250 mM Tris/HCl (pH 8.0), and samples were
homogenized by three cycles of freezing and thawing using liquid
nitrogen and 37 C water. After heating at 65 C for 15 min, samples were
centrifuged at 15,000 rpm for 10 min at 4 C. CAT activity of the
pellets (50 µg of protein) was measured in 262.5 µl 100
mM Tris/HCl (pH 8.0), 1.25 mM chloramphenicol,
1 µCi [14C]butyryl-CoA (finally 0.1
mM, NEN Life Science Products ; specific
activity, 4.0 mCi/mmol), gently overlaid with 5 ml Econofluor-2
(NEN Life Science Products). Capped vials were incubated
at room temperature for a few hours and the radioactivity was counted
in a liquid scintillator. The cAMP assay was performed in COS cells
transfected with the mutant CTR cDNAs, as previously described
(37 ).
CT Binding Assay
The binding of CT to cells transfected with mutant CTR cDNAs was
measured according to the method described previously (1 ), with
modification. In brief, 48 h after transfection, cells were
incubated with 43.052.0 pM
[125I]sCT in 40 mM HEPES buffer
containing 0.1% BSA in the presence of various concentrations of
nonradioactive sCT for 16 h at 4 C. After being washed, cells were
lyzed by 0.2 N NaOH, and the radioactivity of the sample
was measured by a
-counter.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. Byrne, A. Gorn, M. Flannery, T. Yamatsuji, and
T. B. Kinane for expert advice and helpful discussion; J. T.
Potts Jr. and Y. and Y Tamai for essential support; K. Takahashi, T.
Okamoto, and K. Tsuchiya for indispensable cooperation and technical
assistance; S. Ishii for CRE-CAT plasmid; and K. Nishihara, J. Hatomi,
L. Duda, and D. Wylie for expert assistance. We are also especially
indebted to T. Hiraki, T. Yoshida, and M. C. Fishman for
indispensable support of this study; and anonymous reviewers for
constructive criticism.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ikuo Nishimoto, Department of Pharmacology, KEIO University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
This work was supported in part by grants from the Naito Foundation,
the Brain Science Foundation, the Mitsubishi Foundation, the Takeda
Science Foundation, the Pathological Metabolism Foundation, the Mochida
Memorial Foundation for Medical and Pharmaceutical Research, the
Suzuken Memorial Foundation, Japan Brain Foundation, the Ministry of
Health and Welfare of Japan, the Ministry of Education, Science, and
Culture of Japan, and the Organization for Pharmaceutical Safety and
Research (I. N.) and a Massachusetts General Hospital Research
Fellow grant (P. O.). P. O. was also supported by grants from
the French Ministry of Foreign Affairs (Bourse Lavoisier), the
Société Française de Rhumatologie, and the European
League Against Rheumatism. This work was also supported by NIH Grant
RO1-DK-46772 and PD1AR-03564 to S.R.G. and S.M.C.
1 The first two authors equally contributed to this study. 
Received for publication March 25, 1998.
Revision received August 11, 1999.
Accepted for publication September 15, 1999.
 |
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