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INTRODUCTION |
Calcitonin gene-related peptide
(CGRP)1 is a neuropeptide
found in the central and peripheral nervous systems. CGRP mediates sensory neurotransmission, decreases vascular tone, gastrointestinal motility and secretion, and inhibits the action of insulin on carbohydrate metabolism (1). CGRP is a member of a family of neurotransmitters and hormones including calcitonin, adrenomedullin, and amylin that have 24-46% sequence homology.
The receptors for CGRP, adrenomedullin, calcitonin, and amylin also
belong to a subfamily of seven transmembrane G protein-coupled receptors (GPCR) that also includes the receptors for secretin, vasoactive intestinal peptide and parathyroid hormone (PTH), among others in the "B family" of GPCRs. The receptor for CGRP is
one of very few GPCR that requires an accessory protein other
than a G protein for its function (2). The receptor for CGRP, the calcitonin receptor-like receptor (CRLR) requires interaction with the
accessory protein, receptor activity-modifying protein 1 (RAMP 1), to
form a functional CGRP receptor. RAMP 1 belongs to a three-member
family of integral membrane proteins that share ~30% sequence
homology. The RAMPs are 150-177 amino acids in size with a cleavable
signal peptide, relatively large N-terminal extracellular domain, one
transmembrane-spanning domain, and a nine-amino acid intracellular
C-terminal domain (see Fig. 1). CRLR associated with RAMP 1 has high
affinity for CGRP, whereas association with RAMP 2 and RAMP 3 results
in higher affinity for adrenomedullin (2).
The association of CRLR and RAMP 1 occurs early after translation. The
first report of the discovery of the RAMPs (2) and subsequent studies
show that RAMP 1 influences the glycosylation and trafficking of CRLR
to the cell surface (3). Glycosylation of RAMP 2 and 3 is required for
their cell surface expression in the absence of CRLR (4). RAMP 1, unlike RAMP 2 and 3, is not glycosylated and therefore requires
association with CRLR for cell surface expression. This functional
interaction continues at the cell surface where RAMP 1 enables CGRP
binding and signaling, and the heterodimer can be cross-linked and
immunoprecipitated with CGRP (5).
The extracellular domain (ECD) of RAMP is largely responsible for
determining CRLR ligand specificity for either CGRP or adrenomedullin although domain swapping between the ECD and TM/tail of RAMPs 1 and 2 suggests that the TM and/or tail may play a minor role (6).
Similar to the RAMPs, the extracellular N terminus of several members
of the B class of receptors also plays an important role in
receptor-ligand association as demonstrated by cross-linking (7),
chimera (8), and site-directed mutagenesis studies (9). In fact, the
exogenously expressed N-terminal domain of the PTH receptor is able to
associate with PTH (10).
Another accessory protein known to assist a GPCR in its function is
LRP6. LRP6 is also a single transmembrane-spanning protein that
associates with the GPCR, frizzled, and promotes its activity. The ECD
of LRP6 can associate with frizzled, and an LRP6 mutant lacking its
cytosolic tail is a dominant negative for frizzled function (11).
To determine the function of each of the three domains (ECD, TM, and
tail) of human RAMP 1, we made the following three mutants: the ECD and
TM of RAMP 1 without the tail, a chimera of the ECD of RAMP 1 with the
TM and tail from the platelet-derived growth factor (PDGF) receptor,
and the ECD of RAMP 1 alone. These RAMP 1 mutants were examined for
their ability to functionally couple with CRLR to effect
CGRP-stimulated intracellular cAMP accumulation, CGRP binding, CRLR
trafficking, and cell surface expression. All RAMP 1 mutants were able
to associate with CRLR with full efficacy for CGRP-stimulated cAMP
accumulation. However, the RAMP 1-ECD/PDGFR TM/tail mutant demonstrated
a moderate decrease in potency for CGRP signaling and binding, whereas
the RAMP-ECD mutant was more severely affected. CRLR cell surface
expression was retained with all RAMP 1 mutants, although the RAMP 1 mutant consisting of the ECD alone functioned less efficiently and was
secreted into the medium. The ECD of RAMP 1 was also shown to interact
specifically with the N terminus of CRLR.
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EXPERIMENTAL PROCEDURES |
Construction of Wild Type and Mutant RAMP 1--
The full-length
human RAMP 1 was PCR-amplified from human brain cDNA
(Clontech, Palo Alto, CA) and cloned into the TOPO
TA cloning vector (Invitrogen). An oligonucleotide primer
encoding a 5' HindIII restriction site, followed by the
Haemophilus influenza hemagglutinin cleavable signal
peptide sequence (MKTILALSTYIFCLVFA) (12) and the c-myc antigen
epitope sequence (EQKLISEEDL) was used to replace the native signal
peptide in-frame with the remaining RAMP 1 sequence using PCR.
The RAMP 1 C-terminal tail truncation was made by PCR amplification of
wild type RAMP 1 cDNA using an antisense primer that inserted a
stop codon after Trp-139, the last predicted amino acid of the
transmembrane domain (see Fig. 1). The RAMP 1 chimera consisting of the
RAMP 1-ECD and the PDGF receptor TM and tail domains was constructed by
PCR amplification of wild type RAMP 1 cDNA using a long antisense
primer coding for the TM and first nine amino acids of the C-terminal
tail of the human PDGFR (see Fig. 1). The RAMP 1-ECD was made by PCR
amplification of wild type RAMP 1 cDNA using an antisense primer
that replaced the codon for the first amino acid in the transmembrane
domain with a stop codon (see Fig. 1).
Construction of HA-CRLR-GFP--
The full-length human CRLR was
PCR-amplified from human brain cDNA (Clontech,
Palo Alto, CA) and cloned into the TOPO TA cloning vector (Invitrogen).
An oligonucleotide primer encoding a 5' HindIII restriction
site, followed by the H. influenza hemagglutinin cleavable signal peptide sequence (MKTILALSTYIFCLVFA) (12) and the
influenza virus hemagglutinin antigen epitope (HA1) sequence
(YPYDVPVYA), was used to replace the native signal peptide in-frame
with the remaining CRLR sequence using PCR. This construct of CRLR and a similar construct containing an enhanced GFP
(Clontech, Palo Alto, CA) C-terminal fusion
(CRLR-GFP) were cloned into PEAK 12 vector (Edge Biosystems,
Gaithersburg, MD) at the HindIII and NotI
restriction sites.
Cell Culture and cDNA Transfection--
All studies were
performed using human embryonic kidney 293 tsA 201 cells (tsA 201) (a
kind gift from Ronald Li, Institute of Molecular Cardiobiology, The
Johns Hopkins University School of Medicine, Baltimore, MD). tsA 201 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (BioFluids Inc., Rockville,
MD) and 1% penicillin/streptomycin and incubated in humidified air
supplemented with 5% CO2 at 37 °C.
Transfections of tsA 201 cells were performed in either six-well
culture plates (5 × 105 cells per well) or 10-cm
round culture plates (5 × 106 cells) using Polyfect
(Qiagen, Santa Clarita, CA) according to the manufacturer's protocol.
For the cAMP and binding studies, cells were split 8 h after
transfection and cultured in the above medium until assayed at 24 h post-transfection.
Radioligand Binding to Transfected tsA 201 Cells--
Eight h
after transfection, cells were trypsinized and reseeded in 24-well
plates at a density of 100,000 cells per well. The cells were washed
once with cold phosphate-buffered saline (PBS), pH 7.4, containing
0.1% bovine serum albumin (BSA) and incubated in DMEM containing 0.1%
BSA for 60 min at 37 °C, 50 pM [125I]CGRP
(human 8-37) (2200 Ci/mmol) (PerkinElmer Life Sciences) for 30 min with and without increasing concentrations of unlabelled human
CGRP. The cells were washed one time with PBS containing 1% BSA and
were collected with 0.5 ml of 0.1 N NaOH added to each well. Radioactivity was detected in a
-counter (Packard
Instruments, Downers Grove, IL). Nonspecific binding (determined
in the presence of 1 µM CGRP) was always less than 10%
of total binding. Binding assays were performed in triplicate in at
least three separate experiments. IC50 values were
determined using a nonlinear regression curve-fitting computer program
within PRISM, version 2.0 (Graph-Pad Software, Inc., San Diego, CA).
cAMP Assays--
Intracellular cAMP levels were assayed using a
modification of the procedure described by Salomon et al.
(13). Briefly, 8 h after transfection, tsA 201 cells were
trypsinized and reseeded in 24-well plates at a density of 100,000 cells per well in DMEM containing 10% calf serum and
[3H]adenine (2 mCi/ml). The cells were washed with DMEM
alone for 10 min and incubated in DMEM with or without the indicated
concentrations of human CGRP, BSA (1%), and
isobutylmethylxanthine for 30 min. Following the aspiration of the
incubation medium, 100 µl of 5% SDS/1 mM cAMP solution
was used to lyse the cells. Intracellular [3H]cAMP
released into the lysate was measured by successive column chromatography using a Dowex AG-50W-X4 resin (Bio-Rad) followed by
aluminum oxide (Sigma). Eluates were collected in scintillation vials,
and radioactivity was counted using a liquid scintillation counter
(Beckman). cAMP assays were performed in triplicate in at least six
separate experiments. Each assay included an experimental positive
control group of cells expressing native human HA-CRLR-GFP and native
RAMP 1. Positive control cells stimulated 20-fold over basal, whereas
mock (vector alone) and untransfected cells were unresponsive to CGRP.
Results were expressed as a percent of the maximal response observed
for the positive control group of cells, and EC50 values
were determined using a nonlinear regression curve-fitting computer
program within PRISM, version 2.0 (Graph-Pad Software, Inc., San Diego, CA).
Immunoblotting--
tsA 201 cells were grown in six-well culture
plates (5 × 105 cells/well) and processed 24 h
post-transfection. The cells were solubilized after washing away the
growth medium and incubating with 1 ml of PBS containing 1% Triton
X-100, 0.2% SDS, and a protease inhibitor mixture (Roche
Molecular Biochemicals) for 1 h at 4 °C. Following
centrifugation (20,000 × g at 4 °C for 15 min), a
fraction of the supernatant was mixed with sample buffer (final concentration: 1% SDS, 4 M urea, and 50 mM
dithiothreitol), separated on a 4-12% NuPAGE gel (Invitrogen) run in
MOPS buffer, and elector-transferred onto nitrocellulose membranes.
Blots were blocked in 5% non-fat milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, incubated with primary
antibody, either monoclonal HA.11 (anti-HA ascites; 1:3000) or 9E10
(anti-c-myc; 1:3000) (BabCo, Berkeley, CA), in PBST/1% milk for 1 h at room temperature. The blots were then washed in three changes of
PBST for 15 min, incubated with secondary antibody, goat anti-mouse
IgG, horseradish peroxidase-conjugated (Kirkegaard and Perry
Laboratories, Gaithersburg, MD), in PBST/1% milk for 1 h at room
temperature, and washed in three changes of PBST for 15 min at room
temperature. Bands were visualized using enhanced chemiluminescence
(ECL plus kit; Amersham Biosciences).
Immunoprecipitation--
tsA 201 cells were grown in six-well
culture plates (5 × 105 cells/well) and processed
24 h post-transfection. The cells were solubilized after washing
away the growth medium and incubating with 1 ml of PBS containing 1%
Triton X-100, 0.2% SDS, and a protease inhibitor mixture (Roche
Molecular Biochemicals) for 1 h at 4 °C. Following
centrifugation (20,000 × g at 4 °C for 15 min), the
supernatant was mixed on a rocker with either HA.11 (1:200) or 9E10
(1:200) antibody overnight at 4 °C. The immune complexes were bound
to protein G-Sepharose beads (Amersham Biosciences) after
rocking for 1 h at 4 °C. The beads were washed with PBS/0.1% Triton X-100 and eluted with sample buffer (final concentration: 1%
SDS, 4 M urea, and 50 mM dithiothreitol).
Samples were analyzed by gel electrophoresis and Western blotting as
described above.
Surface Immunoprecipitation--
tsA 201 cells, grown in
six-well culture plates (5 × 105 cells), were
incubated 24 h post-transfection by washing three times with PBS
and incubated with blocking buffer (PBS/0.2% bovine serum albumin) for 1 h on ice (4). HA.11 anti-HA antibody was added (1:200) for 2 h on ice. The cells were washed once with 4 °C
blocking buffer and twice with 4 °C PBS and then lysed, centrifuged,
bound to protein G beads, and Western blotted as described above.
Flow Cytometry--
tsA 201 cells transiently expressing either
wild type or one of the mutant RAMP 1 constructs and N-terminal HA
epitope-tagged human CRLR-GFP (HA-CRLR-GFP) were harvested 24 h
post-transfection using Versene (Invitrogen). The cells were
washed with DMEM, 10% fetal bovine serum at 4 °C and resuspended
at 1 × 106 cells/ml in the same medium
containing a 1:100 dilution of anti-HA antibody (HA.11) (BabCo,
Berkeley, CA) for 60 min at 4 °C. The cells were subsequently washed
twice with DMEM/10% fetal bovine serum at 4 °C and resuspended at
1 × 106 cells/ml in the same medium containing
a 1:100 dilution of phycoerythrin-conjugated goat anti-mouse IgG
(Molecular Probes, Eugene, OR) for 60 min at 4 °C. The cells were
washed twice with DMEM/10% fetal bovine serum and resuspended in the
same media at 1 × 106 cells/ml at 4 °C. Flow
cytometric analyses were performed on 1 × 104 cells
using an EPICS ELITE ESP flow cytometer (Coulter, Hialeah, FL). Argon
laser excitation and either a 525 or 575 nM bandpass filtering was used to detect GFP or phycoerythrin. Cells that fluoresced at least two standard deviations above the mean
autofluorescence of unmanipulated tsA 201 cells were defined as positive.
Statistical Analysis--
Differences between mean values were
analyzed by Student's t test. Differences were considered
statistically significant for p values <0.05.
EC50 and IC50 values and their 95% confidence intervals (CI), were determined using a nonlinear regression
curve-fitting computer program within PRISM, version 2.0 (Graph-Pad
Software, Inc., San Diego, CA).
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RESULTS |
Role of RAMP 1 Domains in CGRP-stimulated Signal
Transduction--
To determine the role of the ECD, TM, and tail
domains of human RAMP 1, three mutants were constructed. The role of
the cytoplasmic tail was evaluated using a mutant consisting of the ECD
and TM (ECD/TM). The importance of the specific sequence of the TM and tail was determined using a chimera composed of the ECD of RAMP 1 and
the TM and tail of the PDGF receptor (R1-ECD/PDGF-TM/tail). A mutant
consisting of the ECD alone (ECD) was used to further evaluate the role
of the TM domain, as well as the role of the ECD (see "Experimental
Procedures" and Fig. 1). tsA 201 cells were transiently transfected with human HA-CRLR-GFP and either wild
type human RAMP 1 or one of the RAMP 1 mutants. Twenty-four h
post-transfection, dose response assays for CGRP stimulation of
intracellular cAMP accumulation were performed. CGRP stimulated cAMP
accumulation with an EC50 of 36 pM (95%
CI, 24-55 pM) (see Fig. 2
and Table I). Truncation of the
nine-amino acid intracellular C-tail of RAMP 1 to form the ECD/TM
mutant resulted in an ~4-fold increase in EC50
(EC50 = 125 pM, 95% CI, 52-299
pM) that was not significantly different from wild type
RAMP 1 (Table I). Maximal stimulation (efficacy) was unaffected by the
tail truncation, and therefore the ECD/TM mutant was statistically
indistinguishable from wild type RAMP 1 in its biological function as
measured by cAMP stimulation.

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Fig. 1.
A model illustrating the sequence
modifications of human RAMP 1 used to construct myc-RAMP 1 and
the truncation and chimeric mutants. In all constructs the native
signal peptide was replaced with the hemagglutinin signal peptide and
the myc epitope. RAMP 1 was truncated after Trp-139 to form the ECD/TM
mutant and after Ile-118 to form the ECD mutant. The RAMP
1-ECD/PDGFR-TM/tail chimera was constructed by replacing the TM and
tail of RAMP 1 with the TM and tail of PDGFR at the position denoted by
the line separating the extracellular and transmembrane
domains.
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Fig. 2.
CGRP-stimulated intracellular cAMP
accumulation in tsA 201 cells expressing HA-CRLR-GFP with each RAMP 1 mutant. Eight h after transfection with human HA-CRLR-GFP and RAMP
1, or one of the RAMP 1 mutants, tsA 201 cells were trypsinized and
reseeded in 24-well plates at a density of 100,000 cells per well.
Twenty-four h post transfection, the cells were stimulated with the
indicated concentrations of human CGRP for 30 min at 37 °C and
subsequently assayed for intracellular cAMP accumulation. Results are
expressed as a percent of the maximal stimulation (mean, 95% CI;
n > 5) observed for positive control cells
expressing wild type RAMP 1 and HA-CRLR-GFP.
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Table I
EC50 and IC50 values for CGRP-stimulated increases in
intracellular cAMP accumulation and [125I]CGRP binding for
wild type RAMP 1 and each of the RAMP 1 mutants derived from Figs. 2
and 6, respectively
Values were determined using a nonlinear regression curve-fitting
computer program within PRISM, version 2.0. ND, not detected.
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The importance of the TM domain of RAMP 1 was examined by replacing the
TM and tail with the TM and first nine amino acids of the C-terminal
tail of the PDGF receptor (Fig. 1). This chimera allows the exploration
of the contribution of the specific sequence of the RAMP 1 TM domain
versus a nonspecific membrane-tethering role it may serve
for the ECD. The EC50 for the CGRP dose response curve for
cAMP stimulation in cells expressing the RAMP 1-ECD/PDGFR-TM/tail chimera and HA-CRLR-GFP was increased ~12-fold compared with wild type RAMP 1 (470 pM, 95% CI, 201-1099 pM)
(see Fig. 2 and Table I), and the maximal response (efficacy) was
unaffected. These data indicate that the specific sequence of the TM of
RAMP 1 contributes to CGRP sensitivity, although the remote possibility
that the PDGFR tail interferes with HA-CRLR-GFP action cannot be ruled out.
This RAMP 1/PDGFR chimera shows that although the TM domain is
important for CGRP sensitivity, it is still unclear whether the TM
domain is necessary for full efficacy or whether the ECD of RAMP 1 alone is sufficient for full biologic function. Therefore, the ECD
mutant of RAMP 1 was transiently expressed with HA-CRLR-GFP in tsA 201 cells, and the cAMP response to increasing doses of CGRP was
determined. Although the EC50 for the ECD of RAMP 1 was increased ~4000-fold (145 nM, 95% CI, 78-272
nM) compared with wild type RAMP 1, surprisingly, the
maximal response was increased nearly 2-fold. CGRP stimulation of
HA-CRLR-GFP expressed alone was the same as non-transfected tsA 201 cells (data not shown). These data indicate that the ECD alone is
sufficient for association with HA-CRLR-GFP and full biological
efficacy as determined by the cAMP response to CGRP and that the
specific amino acid sequence and tethering function of the TM domain
are required for full receptor sensitivity to CGRP.
Role of RAMP 1 Domains in CRLR Trafficking--
Previous studies
have shown that RAMP 1 associates early with CRLR to form a heterodimer
and functions to promote their trafficking through the Golgi to the
cell surface. As a result of passing through the Golgi, CRLR increases
in size as it becomes terminally glycosylated. This increase in the
size of CRLR can be used to assess the functional association and
trafficking of CRLR and RAMP 1. To determine whether the decrease in
CGRP potency for stimulation of cAMP observed for the various mutants
of RAMP 1 was because of impaired association and trafficking with
CRLR, we examined the effect of each mutant of RAMP 1 on HA-CRLR-GFP glycosylation.
Total lysates of tsA 201 cells expressing HA-CRLR-GFP alone or with
RAMP 1 were analyzed by SDS-PAGE (4-12%) and immunoblotting with
mouse monoclonal anti-HA antibody. In the absence of RAMP 1, HA-CRLR-GFP runs as two bands of ~70 and 82 kDa (Fig.
3A) that have been shown
previously to represent intracellular core-glycosylated and cell
surface terminally glycosylated forms, respectively (4). HA-CRLR-GFP
co-expressed with wild type RAMP 1 ran at an intermediate size of 75 kDa.

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Fig. 3.
Immunoblot analysis of HA-CRLR-GFP alone and
with RAMP 1 and RAMP 1 mutants. Twenty-four h after transient
transfection of tsA 201 cells, total lysates from solubilized cells
were analyzed by SDS-PAGE (4-12%) and immunoblotted with mouse
monoclonal anti-HA antibody. Comparison of the size of HA-CRLR-GFP
expressed either alone or with wild type (WT), tail
truncation (ECD/TM), chimeric
(R1-ECD/PDGFR-TM/Tail) (A)
or ECD truncation (B) mutants of RAMP 1 is shown.
Arrows indicate the molecular mass of the receptors
extrapolated from linear regression analysis of four protein standards
(ranging from 39 to 97 kDa). Data were derived from at least three
separate experiments.
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Similar to the wild type RAMP 1, all three RAMP 1 mutants, the ECD/TM
mutant lacking the nine-amino acid intracellular tail and the RAMP
1-ECD/PDGFR-TM/tail chimera (Fig. 3A), as well as the RAMP
1-ECD alone (Fig. 3B), were able to completely convert the
70- and 82-kDa bands to the 75-kDa form of HA-CRLR-GFP. The significance of the differences in the intensity of the bands of
HA-CRLR-GFP co-expressed with the various mutants is difficult to
interpret because of the variability in transfection efficiency and
mutant expression. Nonetheless, this size shift functional assay
demonstrates that only the ECD of RAMP 1 is necessary for the early
association and trafficking with HA-CRLR-GFP. Therefore the decreased
potency for CGRP stimulation of cAMP observed for the RAMP 1 mutants is
not because of impaired early association and trafficking with
HA-CRLR-GFP.
Role of RAMP 1 Domains in CRLR Surface Expression--
Several
expression studies in a variety of heterologous cells have shown that
RAMP 1 association with CRLR is necessary for full expression of CRLR
at the cell surface (2). Although one study has questioned this
requirement for RAMP 1, the data still suggest that RAMP 1 is important
for surface expression of the functional CRLR/RAMP 1 heterodimer (5).
Therefore, to determine whether the absence or mutation of one domain
within RAMP 1 that resulted in the decreased potency for cAMP
production reported above could be because of impaired surface
expression of CRLR, each of the RAMP 1 mutants was compared with wild
type RAMP 1 for its ability to promote HA-CRLR-GFP cell surface expression.
Cell surface expression of HA-CRLR-GFP was detected with anti-HA
monoclonal antibody and phycoerythrin-labeled, goat anti-mouse secondary antibody. The GFP fused to the C terminus of CRLR-GFP was
used to measure the level of whole cell expression of HA-CRLR-GFP. Fluorescent flow cytometric analysis was applied for the simultaneous measurement of both the phycoerythrin and whole cell GFP (HA-CRLR-GFP) fluorescent signals. Fluorescent measurements for 10,000 cells are
depicted on scatter plots (Fig.
4A). HA-CRLR-GFP surface
expression could be then be normalized for the level of total cell
receptor expression of GFP and expressed as a ratio that could be
meaningfully compared among cells co-expressing wild type
versus mutant or absent domains of RAMP 1 (Fig.
4B). Among all the mutant RAMPs, both the RAMP
1-ECD/PDGFR-TM-tail chimera and the RAMP 1-ECD mutant lacking the TM
and tail domains were impaired with statistical significance (70.3 ± 11.2%) and (56 ± 1.5%, respectively) compared with wild type
RAMP 1 in their ability to promote HA-CRLR-GFP surface expression.

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Fig. 4.
Fluorescent flow cytometric analysis of the
effect of RAMP 1 mutants on the cell surface expression of
HA-CRLR-GFP. A, tsA 201 cells transiently
co-transfected with wild type or mutant RAMP 1 and HA-CRLR-GFP were
incubated with monoclonal anti-HA antibody for 60 min at 4 °C,
washed, incubated with phycoerythrin-conjugated goat anti-mouse IgG for
60 min at 4 °C, and washed again prior to analysis. Flow cytometric
analyses were performed on 1 × 104 cells per
experimental group of transfected cells. 525- or 527-nm bandpass
filtering was used to detect GFP (abscissa) and
phycoerythrin (ordinate) fluorescence for each cell,
respectively. Cells that fluoresced at least two standard deviations
above the mean autofluorescence of unmanipulated tsA 201 cells were
defined as positive. B, the relative mean fluorescent
intensity at 575 nm (phycoerythrin) relative to 525 nm (GFP) for each
group of transfected cells shown in A was plotted as a
percent of the control wild type HA-CRLR-GFP/RAMP 1-transfected cells.
Results are expressed as the mean ± S.E. from three separate
experiments. R1, RAMP 1. Differences between mean values
were analyzed by Student's t test and considered
statistically significant for p values < 0.05.
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Role of Transmembrane Domain of RAMP 1 in RAMP 1 CRLR Heterodimer
Stability--
Although the impaired function of the mutant that
resulted in a 50% reduction in HA-CRLR-GFP surface expression may
contribute to the observed 4000-fold decrease in CGRP potency for cAMP
stimulation, it cannot account for all of the reduction. We
demonstrated that the ECD of RAMP 1 could associate and traffic with
HA-CRLR-GFP similar to wild type RAMP 1 using an N-linked
glycosylation maturation assay (see "Experimental Procedures" and
Fig. 3B). To determine whether the heterodimer consisting of
the ECD of RAMP 1 and HA-CRLR-GFP was unstable, perhaps because of the
absence of the TM and tail of RAMP 1, the media of cells either
expressing myc-ECD of RAMP 1 alone or co-expressed with HA-CRLR-GFP was
examined for the presence of the ECD of RAMP 1. Myc-ECD of RAMP 1 was
immunoprecipitated from the medium using mouse monoclonal anti-myc
antibody and analyzed by PAGE and immunoblotting with the same anti-myc
antibody. The presence of RAMP 1-myc-ECD in the media of cells
expressing RAMP 1-Myc-ECD alone was nearly undetectable (Fig
5, lane 1) whereas there was a
dramatic increase of the 11-kDa RAMP 1-Myc-ECD in the media of cells
co-expressing HA-CRLR-GFP (Fig. 5, lane 2). These data
confirm that the ECD of RAMP 1 alone is capable of associating with
HA-CRLR-GFP and trafficking through the Golgi to the cell surface as
shown above. In addition, the presence of the ECD of RAMP 1 in the
media indicates that the ECD has dissociated from HA-CRLR-GFP
presumably as a result of a decrease in the stability of its
interaction with HA-CRLR-GFP at the surface. Although it is difficult
to quantitate the degree of instability between the ECD and
HA-CRLR-GFP, because there are so many unknown variables, the
interaction is stable enough to allow full efficacy of CGRP stimulation
of intracellular cAMP accumulation.

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Fig. 5.
Immunoblot analysis of the stability of the
interaction between the ECD of RAMP 1 and HA-CRLR-GFP. Forty-eight
h post-transfection of tsA 201 cells with Myc-ECD of RAMP 1 alone
(lane 1) or with HA-CRLR-GFP (lane 2), the medium
was examined for the presence of the ECD of RAMP 1. The Myc-ECD was
detected in the medium by immunoprecipitation with mouse monoclonal
anti-myc antibody. Immunoprecipitates were analyzed by PAGE (4-12%)
and immunoblotting with the same anti-myc antibody. The
arrow indicates the position of the Myc-ECD determined to be
11 kDa on the basis of linear regression analysis of three protein
standards (ranging from 6-17 kDa). The position of two dark
upper bands in both lanes is consistent with
nonspecific immunostaining of the heavy and light chains of the
immunoprecipitating antibody.
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Role of RAMP 1 Domains in CGRP Affinity--
RAMP 1 association
with CRLR at the cell surface is necessary for CGRP high affinity
binding and activation of signal transduction. Having demonstrated that
the RAMP 1-ECD is sufficient for association, trafficking, and cell
surface expression of HA-CRLR-GFP, the effect of the mutations of each
of the RAMP 1 domains on CGRP affinity was examined as a possible basis
for the observed decrease in potency for stimulating cAMP accumulation.
Radioligand binding dose inhibition studies were performed in tsA 201 cells co-expressing HA-CRLR-GFP and each of the RAMP 1 mutants using 50 pM [125I]CGRP and increasing concentrations
of unlabeled CGRP. The results of the competitive binding studies were
consistent with the results from the cAMP studies. Similar to the
results of the cAMP studies, CGRP affinity for HA-CRLR-GFP co-expressed
with the RAMP 1-ECD-TM (tail truncation) mutant (IC50 = 4.5 nM, 95% CI, 3.5-5.9 nM) was not significantly
different from co-expression with wild type RAMP 1 (IC50 = 2.8 nM, 95% CI, 2.1-3.7 nM) (see Fig.
6 and Table I). Also similar to the cAMP
studies, the RAMP 1-ECD/PDGFR-TM-tail chimera had a nearly 10-fold
decrease in CGRP affinity compared with wild type RAMP 1 (IC50 = 2.8 nM, 95% CI 2.1-3.7
versus 23 nM, 95% CI 7.3-74 nM,
respectively). As might be expected from the 4000-fold increase in
EC50 for cAMP stimulation, the IC50 for CGRP
binding to the RAMP 1-ECD mutant/HA-CRLR-GFP heterodimer was too great
to be determined using radioligand binding dose inhibition studies. The
parallel decrease in potency for CGRP stimulation of cAMP with the
decrease in CGRP binding affinity suggests that the majority of the
loss in biologic function for both the RAMP 1-ECD/PDGFR-TM/tail chimera
and the RAMP 1-ECD mutant is attributable to loss in CGRP affinity.
Taken together, the data indicate that the stability conferred by the
presence of the TM of RAMP 1 is important for high affinity binding of
CGRP to the RAMP 1/CRLR heterodimer.

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Fig. 6.
Displacement of [125I]CGRP
binding to tsA 201 cells expressing wild type RAMP 1 or RAMP 1 mutants
with HA-CRLR-GFP. Eight h after transfection with human
HA-CRLR-GFP and RAMP 1 or RAMP 1 mutants, tsA 201 cells were
trypsinized and reseeded in 24-well plates at a density of 100,000 cells per well. Twenty-four h post-transfection, the cells were
incubated with [125I]CGRP (50 pM) either
alone or with the indicated concentrations of unlabeled CGRP for 60 min
at 37 °C. Results are expressed as a percent of the saturable
binding in the absence of unlabeled ligand. Each value represents the
mean and 95% CI (n > 5).
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Interaction of the ECD of RAMP 1 with the N Terminus of
CRLR--
Having demonstrated the ability of the RAMP 1-ECD to
associate with HA-CRLR-GFP early in protein processing and traffic
through the Golgi to the cell surface, we sought to identify the region of CRLR that was interacting with the ECD of RAMP 1. The N terminus of
several B class GPCRs have been shown to be major determinants for
ligand binding (7-10). In addition, the N-terminal ECD of RAMP 1 has
been shown to determine CGRP versus adrenomedullin specificity when co-expressed with CRLR (6). Therefore, a chimera was
constructed in which the N-terminal extracellular domain of CRLR
replaced the N-terminal extracellular domain of the secretin receptor.
The secretin receptor was chosen for chimerization, because it does not
associate with RAMP 1, and a variety of secretin/PTH receptor chimeras
have maintained biologic function (9, 10). Because the N terminus of
CRLR contains all of the N-linked glycosylation sites, we
were able to use the previously demonstrated RAMP 1 associated change
in glycosylation as a measurement of the functional interaction between
RAMP 1 and the N terminus of CRLR (Fig.
7A). As expected, RAMP 1 had
no effect on the mobility of the HA epitope-tagged secretin receptor as
determined by immunoblot (Fig. 7B). However, the HA
epitope-tagged CRLR/secretin receptor chimera demonstrated the familiar
shift in size when co-expressed with RAMP 1 (Fig. 7C). The
total and surface immunoprecipitation of the HA-CRLR/secretin receptor
chimera showed a pattern similar to the wild type HA-CRLR-GFP (Fig.
7A), namely two bands when expressed alone and one
intermediate band when co-expressed with RAMP 1. The surface
immunoprecipitation shows that in the absence of RAMP 1, the lower band
does not make it to the cell surface unlike the upper band and the
solitary intermediate size band formed in the presence of RAMP 1 (Fig. 7, B and C). The remarkable similarity in the
size shift of the CRLR/secretin chimera and the wild type CRLR because
of RAMP 1-associated changes in glycosylation suggests that RAMP 1 interacts with the N-terminal extracellular domain of CRLR.

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Fig. 7.
Immunoblot analysis of the interaction of
RAMP 1 with HA-CRLR, HA-secretin receptor, and chimeric
HA-CRLR-secretin receptor. All receptors were expressed 24 h
following transient transfection of tsA 201 cells and analyzed by
SDS-PAGE (4-12%) and immunoblotted with mouse monoclonal anti-HA
antibody. Immunoprecipitation of HA-CRLR (A), HA-secretin
receptor (B), and chimeric HA-CRLR-secretin receptor
(C) expressed alone or with RAMP 1 from lysates of whole
cells (total IP) or whole cells that were incubated with
mouse monoclonal anti-HA antibody and washed prior to lysis
(surface IP) is shown. Arrows indicate the
molecular mass of the receptors extrapolated from linear regression
analysis of four protein standards (ranging from 39 to 97 kDa). Data
were derived from at least three separate experiments.
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DISCUSSION |
The human RAMP 1 protein can be divided into at least three
structural domains, the N-terminal ECD, a transmembrane domain, and a
nine-amino acid intracellular C-terminal tail domain. Three mutants of
RAMP 1 were constructed to help ascribe a potential biological function
to each of these domains: the ECD and TM of RAMP 1 without the tail, a
chimera of the ECD of RAMP 1 with the TM and tail from the PDGFR, and
the ECD of RAMP 1 alone. Each of the mutants was compared with wild
type RAMP 1 for their ability to associate and traffic with HA-CRLR-GFP
from the endoplasmic reticulum, through Golgi to the cell surface,
where they were assessed for their ability to bind CGRP and transduce a
signal leading to the measured accumulation of intracellular cAMP. On the basis of these functional criteria, it appears that the C-terminal tail of RAMP 1 is unnecessary. The specific sequence and presence of
the TM domain of RAMP 1 appears to contribute to CGRP affinity and
thereby also the potency for stimulation of receptor-mediated signal
transduction. The ECD of RAMP 1 is sufficient for normal association
and trafficking of HA-CRLR-GFP. However, the surface expression of
HA-CRLR-GFP, as well as CGRP affinity and potency for signal
transduction, were all diminished. This observed decrease in the
function of the ECD is possibly a result of the unstable association of
the RAMP 1-ECD with HA-CRLR-GFP as evidenced by the secretion of the
ECD in the media. Finally, the functional interaction of RAMP 1 with
the N-terminal CRLR/secretin receptor chimera indicates that the
N-terminal ECD of RAMP 1 interacts with the N terminus of CRLR to form
a functional heterodimer capable of trafficking to the cell surface.
The RAMP 1-ECD/TM mutant lacking the nine-amino acid tail domain was
functionally indistinguishable from wild type RAMP 1. Loss of the
C-terminal tail had no affect on the association and trafficking with
HA-CRLR-GFP. Similarly, surface expression and CGRP affinity and
potency for signal transduction were unimpaired. During the preparation
of this manuscript, a report was published (14) confirming that this
same RAMP 1 tail truncation mutant interacted and trafficked with
HA-CRLR-GFP to the cell surface normally. However, unlike the results
presented here, this report found a 10-fold decrease in potency and a
35% decrease in efficacy for CGRP-stimulated cAMP response in a
similar HEK 293 cell line transiently expressing the tail-truncated
mutant (14). The reason for this discrepancy is unclear.
Considering the highly conserved (Q/R)(S/T)K(R/D)(S/T) sequence within
the nine-amino acid tail of all three members of the RAMP family, it
was somewhat unexpected to find that truncation of the tail was without
a functional consequence. However, also during the preparation of this
manuscript, it was reported that the QSKRT sequence in the tail of
human RAMP 1 functions as an intracellular retention motif that can be
overcome by the association of RAMP 1 with CRLR (14). Although
trafficking of RAMP 1 expressed alone was not addressed in this
study, this finding provides a suspected function for the conserved
sequence in the C-terminal tail. This reported retention function for
the C-terminal tail of RAMP 1 does not exclude other potential
functions such as interaction with intracellular proteins similar to
LRP6, another accessory protein for the GPCR, frizzled, that interacts
with axin and catinin (11).
A chimera replacing the TM and tail of RAMP 1 with the TM and first
nine amino acids in the C terminus of the PDGF receptor had no effect
on the efficacy of CGRP-stimulated cAMP accumulation; however, both the
affinity and potency of CGRP were decreased to a similar extent of
10-12-fold. These results, along with the data from the tail
truncation mutant, indicate that the TM of RAMP 1 can be substituted
without a loss in maximal function. However, the loss in CGRP affinity
and potency suggests that the specific sequence of the TM domain of
RAMP 1 is required for normal function. Although it is possible that
the PDGFR tail hinders the function of CRLR, the small size,
intracellular location, and irrelevance of the native C-tail suggest
that this is unlikely. The importance of the TM domain in ligand
sensitivity found here is not consistent with a previous study of RAMP
1 and RAMP 2 chimeras (6). In this study, the RAMP 1-ECD/RAMP 2-TM-tail
chimera function, as measured by the maximal CGRP-induced membrane
channel current in Xenopus laevis oocytes, was
40% of RAMP 1. In addition, this chimera exhibited increased affinity
for adrenomedullin compared with RAMP 1 when expressed in HEK 293 cells. Although the ECD is the major determinant for ligand affinity
and specificity, these results indicate that the TM domain of RAMP 1 contributes to ligand affinity, as well.
Co-expression of just the ECD of RAMP 1 with HA-CRLR-GFP resulted in
full HA-CRLR-GFP glycosylation and a maximal response to CGRP greater
than observed for full-length RAMP 1; however, surface expression and
affinity for CGRP were impaired. These data indicate that the ECD alone
is sufficient for association with HA-CRLR-GFP and full biological
efficacy and that the tethering function of the TM domain is only
required for full receptor sensitivity to CGRP. A recent study (14)
reported that removal of the C-terminal tail plus 2, 7, or 20 amino
acids from the 22-amino acid TM domain of RAMP 1 resulted in the
complete loss of CGRP-stimulated increase in cAMP in HEK 293 cells
transiently co-expressing CRLR despite normal CRLR glycosylation and
RAMP 1 truncation mutant cell surface expression for all but the
smallest mutant. From these non-functioning truncation mutants, they
conclude that the TM domain is essential for CGRP receptor function.
Our data using an ECD mutant of RAMP 1 that was truncated one amino
acid shorter than their shortest truncation mutant demonstrated
impairment only in affinity and potency. Not only was the TM domain of
RAMP 1 not essential, but the efficacy for CGRP-stimulated cAMP
accumulation in the absence of the TM domain was actually greater than
with the TM domain present. The discrepancy between their negative and
our positive functional data is presently unknown and may be on the
basis of single amino acid difference in length between their construct and ours.
The doubling of the biological efficacy of the RAMP 1-ECD mutant was
unexpected. The ability to recover the ECD from the culture medium by
immunoprecipitation indicates the importance of the TM domain for
anchoring RAMP 1. In the absence of the TM domain, the interaction of
the ECD of RAMP 1 with the N terminus of HA-CRLR-GFP is of low enough
affinity to allow it to dissociate into the medium. The dissociation of
the ECD of RAMP 1 should result in a diminution in functional cell
surface receptors and hence the paradox presented by the observed
increase in efficacy. Possible explanations for the paradox could
include loss of inhibition of signaling by the TM domain or increased
receptor turnover leading to more active receptors on the surface.
Although these hypotheses are not the focus of this work, they could be
interesting for future studies.
The ability of RAMP 1-ECD to functionally interact with HA-CRLR-GFP
suggests that the region of interaction is in the extracellular N
terminus and/or loops of CRLR. The fact that RAMP 1 was able to
interact with the N terminus of CRLR fused to the secretin receptor to
recapitulate the glycosylation pattern characteristic of the holo-CRLR,
combined with the fact that RAMP 1-ECD can change the glycosylation
pattern of HA-CRLR-GFP, indicates that the main interaction is between
the ECD of RAMP 1 and the N terminus of CRLR. Because the CRLR-secretin
receptor chimera doesn't have signaling capability with CGRP or
secretin (data not shown), it is premature to conclude more about what
portion of CRLR is sufficient for the effect of RAMP 1 on CRLR
specificity and functional signaling.
In conclusion, we find that the RAMP 1-ECD interacts with the
N-terminal domain of CRLR and that this interaction is sufficient for
trafficking from the endoplasmic reticulum through the Golgi to the
cell surface. Furthermore, the interaction of the ECD of RAMP 1 with
CRLR is sufficient for full biological efficacy. Although the ECD is
sufficient for full efficacy, the TM domain of RAMP 1 is important for
achieving full CGRP affinity and potency, as well as surface expression
of CRLR, perhaps as a result of tethering the ECD to the membrane and
specifically interacting with the TM domain of CRLR. In view of these
findings, future structural studies of the interaction of the ECD of
RAMP 1 with the N terminus of CRLR may be feasible.