From the Department of Pharmacology and the
§ Howard Florey Institute, The University of Melbourne,
Victoria 3010, Australia, the
Department of
Neuroendocrinology and Cell Biology, INSERM U410, Faculté de
Médecine Xavier Bichat, 75018, Paris, France, and the
** First Department of Internal Medicine, Miyazaki
Medical College, Miyazaki 889-1692, Japan
Received for publication, November 8, 2002
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ABSTRACT |
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The receptor activity-modifying proteins (RAMPs)
comprise a family of three accessory proteins that heterodimerize with
the calcitonin receptor-like receptor (CL receptor) or with the
calcitonin receptor (CTR) to generate different receptor phenotypes.
However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in
cellular processes. We have investigated the potential for RAMP
interaction with a number of Class II G protein-coupled receptors
(GPCRs) in addition to the CL receptor and the CTR. Using
immunofluorescence confocal microscopy, we demonstrate, for the first
time, that RAMPs interact with at least four additional receptors, the
VPAC1 vasoactive intestinal polypeptide/pituitary adenylate
cyclase-activating peptide receptor with all three RAMPs; the
glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the
PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL
receptor or the CTR, VPAC1R-RAMP complexes do not show altered
phenotypic behavior compared with the VPAC1R alone, as determined using
radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2
heterodimer displays a significant enhancement of agonist-mediated
phosphoinositide hydrolysis with no change in cAMP stimulation
compared with the VPAC1R alone. Our findings identify a new
functional consequence of RAMP-receptor interaction, suggesting that
RAMPs play a more general role in modulating cell signaling through
other GPCRs than is currently appreciated.
The discovery of receptor activity-modifying proteins
(RAMPs)1 has led to a
re-evaluation of what defines G protein-coupled receptor (GPCR)
phenotypic behavior toward agonists and/or G proteins (1). The RAMP
family comprises three accessory proteins (designated RAMP1, RAMP2, and
RAMP3) that were originally identified during attempts to clone the
receptor for calcitonin gene-related peptide (CGRP). Phenotypic
receptor behavior corresponding to that of the native CGRP receptor
could be demonstrated only in recombinant expression systems when
another seven-transmembrane receptor, the calcitonin receptor-like
receptor (CL receptor) was co-expressed with RAMP1 (1). Additional
studies extended these observations to identify a general role of RAMPs
in modifying the expression and the pharmacology of receptors related
to the calcitonin family of peptides (1-5).
To date, studies of RAMP-GPCR interactions have focused predominantly
on the receptors for calcitonin and its related peptides (i.e. CGRP, adrenomedullin, and amylin). However, these
receptors belong to the Class II family of GPCRs, members of which
share a number of structural features and are all activated by peptide ligands (6). Given these similarities, it is possible that other Class
II GPCRs may also interact with RAMP proteins to yield novel receptor
phenotypes or receptors with altered pharmacological profiles.
Importantly, RAMPs display a ubiquitous tissue distribution (1, 7, 8),
and cellular background can have a significant impact on the
effects of RAMP (5, 8). Therefore, it is conceivable that RAMPs play a
more generalized role in cellular signaling mediated by Class II GPCRs
than is currently appreciated.
Materials--
All peptides were from Auspep (Parkville,
Victoria, Australia) except for human GHRH, human secretin, and
helodermin, which were from Bachem (Bubendorf, Switzerland).
125I-VIP was prepared using IODO-BEADS (Pierce),
with the mono-iodinated peptide purified by reverse-phase high pressure
liquid chromatography. All other chemicals were reagent grade or better
and were purchased from Sigma (St Louis). Plasmid DNA for the hGHRH
receptor was a gift from Dr. Bruce Gaylinn (9). Plasmid DNA for the
hPTH1 receptor was a gift from Dr. Michael Chorev (10). Plasmid DNA for
the hPTH2 receptor was a gift from Dr. Ted Usdin (11). Plasmid DNA for
wild-type hRAMPs, c-Myc RAMP1, and hCL receptor was a gift from
Dr. Steve Foord (1). Plasmid DNA for c-Myc RAMPs 2 and 3 was prepared
as described previously (12). Plasmid DNA for the hVPAC1, hVPAC2, and
FLAG-hVPAC1 receptors was prepared as described previously (13, 14).
Plasmid DNA for the human glucagon, GLP-1, and GLP-2 receptors was
provided by Dr. Anette Sams Nielsen (Novo Nordisk, Copenhagen, Denmark).
Preparation of Epitope-tagged RAMPs--
Double (RAMP2) or
triple (RAMP3) hemagglutinin (HA) tags were inserted in the RAMP N
terminus, downstream of the signal peptide (between amino acids 55 and
56 of RAMP2 and amino acids 33 and 34 of RAMP3), by overlap extension
PCR and cloned directionally into the HindIII and
XbaI restrictions sites of pcDNA3.1/Zeo (Invitrogen). For incorporation of the V5 tag at the C terminus of RAMPs, coding regions were generated by PCR using the High Fidelity Taq
polymerase (Roche Molecular Biochemicals) and ligated in-frame into
pcDNA3.1V5His6 (Invitrogen) using TA-topoisomerase. All
constructs were confirmed by sequencing.
Cell Culture and Transfection--
HEK-293 and COS-7 cells were
maintained as previously described (3). HEK-293 cells were grown to
60% confluence in 6-well plates or 4-well chamber slides and were
transfected with the indicated plasmid using LipofectAMINE 2000 (Invitrogen). COS-7 cells were grown in 175-cm2 flasks
(membrane preparations) or in 24-well plates (whole cell binding and
signaling assays) to 90% confluence and transfected using
LipofectAMINE (3).
Immunofluorescence Microscopy--
Immunofluorescence
experiments were carried out 24 h post-transfection. HEK-293 or
COS-7 cells, transiently transfected with 150 ng of epitope-tagged RAMP
cDNA with/without 100 ng cDNA of an individual Class II GPCR
(see "Results"), were fixed with 3.4% paraformaldehyde in
phosphate-buffered saline with subsequent detection of the subcellular
distribution of the RAMPs using anti-c-Myc (9E10, gift from Dr.
Jörg Heierhorst, SVIMR, Fitzroy, Australia), anti-HA (12CA5,
Roche Molecular Biochemicals) or anti-V5 antibodies (Invitrogen)
performed as described previously (3).
Western Blot Analysis--
N-terminally c-Myc-tagged RAMP1 or
C-terminally V5-tagged RAMP1 was transfected into 6-well plates as
described above with or without increasing amounts of CL receptor or
VPAC1 receptor. Whole cell lysates were separated on 12% SDS-PAGE,
transferred to nitrocellulose, and probed using either anti-c-Myc
antibody (1:1000) or anti-V5 antibody (1:1000) with labeled proteins
visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotech).
Radioligand Binding--
COS-7 cells transfected with the VPAC1
receptor with or without RAMP were incubated in binding buffer
(Dulbecco's modified Eagle's medium containing 1% w/v bovine
serum albumin) with 50,000 cpm/well of 125I-VIP in the
absence (total binding) or presence of increasing concentrations of
unlabeled receptor ligands at 48 h post-transfection. Following
incubation for 60 min at 37 °C (competition binding) or 90 min at
22 °C (saturation binding), the cells were washed with ice-cold
phosphate-buffered saline and solubilized with 0.5 M NaOH.
Cell lysates were collected and counted in a Packard Phosphoinositide (PI) Hydrolysis--
COS-7 cells were
transfected overnight with 100 ng of VPAC1 receptor or CTR cDNA
together with 150 ng of pcDNA3 (vector control), RAMP1, RAMP2, or
RAMP3 cDNA. The DNA-lipid complex was removed from the wells after
16 h. Cells were loaded with myo-[3H]inositol (1 µCi/ml) at 24 h post-transfection, and agonist-mediated phosphoinositide hydrolysis was determined using ion-exchange chromatography as described previously (17).
cAMP Accumulation--
COS-7 cells were transfected as described
for the PI assay. Cells were then loaded with [3H]adenine
(2 µCi/ml) overnight at 37 °C, and agonist- or forskolin (100 µM)-mediated cAMP accumulation was determined using
ion-exchange chromatography as described previously (17). PI
hydrolysis, cAMP accumulation, and saturation binding assays were
always performed in parallel for each transfection.
Data Analysis--
Saturation and competition binding data were
analyzed via nonlinear regression using PRISM 4 beta (GraphPad
Software, San Diego, CA). In all instances, data shown are the
mean ± S.E. Comparisons between means were performed by one-way
analysis of variance. Unless otherwise stated, values of
p < 0.05 were taken as significant.
Because the influence of RAMPs on the pharmacology of receptors
was unpredictable, we utilized the capacity of receptors to modulate
the subcellular distribution of RAMPs as a marker of receptor-RAMP
interaction. The distribution of HA or c-Myc N-terminally epitope-tagged RAMPs was monitored by immunofluorescence confocal microscopy in the absence or presence of co-transfection with the
following Class II GPCRs: PTH1 and PTH2 receptors, glucagon, glucagon-like peptide-1 (GLP-1) and GLP-2 receptors, growth
hormone-releasing hormone (GHRH) receptor, and VPAC1 and VPAC2
receptors. The CTR and CL receptors were used as positive controls.
Each RAMP displayed different degrees of cell surface expression when
transfected alone in HEK-293 cells. In contrast to the c-Myc RAMP1,
high levels of cell surface expression were observed when c-Myc RAMP 2 or 3 were transfected alone into cells. A similar pattern of behavior
has been observed for FLAG-tagged murine RAMPs 2 and 3 in oocytes,
where receptor-independent cell surface expression requires
glycosylation of the RAMPs (18). Nonetheless, as our c-Myc constructs
contained an artificial signal peptide sequence (12), we also performed
experiments with RAMPs 2 and 3 that incorporated HA tags after the
natural, predicted signal peptide sequence of the RAMPs. Using these
constructs, a much lower level of cell surface RAMP2 or -3 expression
was seen, and indeed the total level of RAMP expressed was also lower
than that seen with the c-Myc-tagged RAMPs, particularly for RAMP2.
Given the divergence in subcellular distribution with the different
tags, we also looked at the localization of RAMP3 incorporating a V5
tag at the C terminus. Like the HA-tagged RAMP3, this construct was
principally retained inside the cell in the absence of co-transfection
of an interacting receptor, suggesting that either the c-Myc tag or the
artificial signal sequence of the c-Myc-tagged construct contributed to
the high levels of receptor-independent cell surface expression of RAMPs 2 and 3. It has been suggested that the presence of either the
c-Myc or HA tags can differentially affect protein transit and terminal
glycosylation in the Golgi (19), which would be consistent with our
findings. We have principally used the HA-tagged RAMPs 2 and 3 in our
primary assay for RAMP-receptor interaction.
Co-transfection of the GHRH (Fig. 1,
D-F), VPAC2, GLP-1, or GLP-2 (not shown)
receptors with each of the RAMPs failed to alter the level of cell
surface-expressed RAMP1, -2, or -3. In contrast, co-transfection of the
CL receptor (Fig. 1, G-I), CTR (not shown), or VPAC1
receptor (Fig. 1, J-L) led to a significant translocation of each of the RAMPs to the cell surface. Furthermore, co-expression of
the PTH2 receptor with RAMP3 (Fig. 1O), but not the other
RAMPs (not shown), led to increased cell surface expression of RAMP3, whereas expression of the PTH1 (Fig. 1N) or glucagon (Fig.
1M) receptors with RAMP2, but not other RAMPs (not shown),
led to translocation of RAMP2 to the cell surface as well as an
increase in total RAMP2 levels. This latter property is likely to be a result of stabilization of the RAMP2 protein when complexed with its
receptor partner and is also seen with the classic RAMP partners CTR
and CL receptor (not shown). The specificity of RAMP-receptor interactions was also maintained when the c-Myc-tagged RAMPs were used,
although the capacity to observe modulation of RAMP distribution required titration of the level of RAMP co-transfected, because of the
high level of receptor-independent cell surface expression (not shown).
Our findings with the VPAC1, glucagon, PTH1, and PTH2 receptors are the
first to demonstrate an interaction between these receptors and RAMPs.
Previous study of "unglycosylated" murine RAMPs and PTH1 and
glucagon receptors in oocytes have failed to reveal the association
between these receptors and RAMP2 (18). Although this
discrepancy in results may represent a species difference in
RAMP-receptor interaction or an effect of removal of the consensus glycosylation sites, we believe it is more likely a consequence of the
highly divergent cellular backgrounds. We have shown previously that
interaction of the CTR with RAMPs, particularly RAMP2, is sensitive to
the cellular background in which it is expressed, indicating that other
cellular components, such as G proteins, are likely to contribute to
RAMP-receptor interactions (5). The specific pairing of the PTH1, PTH2,
and glucagon receptors with only one of the RAMPs highlights the
specificity of RAMP-receptor interactions and the diversity of the
impact that RAMPs may have on receptor function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-counter to
determine bound radioactivity. Nonspecific binding was defined using 1 or 10 µM unlabeled VIP. For membrane binding, aliquots of
crude membrane were prepared (15) and incubated as described above for
competition binding for 60 min at 37 °C with bound and free
radiolabel separated by filtration. Cell surface FLAG-VPAC1 receptor
expression was determined in 24-well plates at 72 h
post-transfection using 4 µg/well anti-FLAG antibody (AMRAD,
Boronia, Australia) and 50,000 cpm/well 125I-IgG
with 125I-VIP binding determined in adjacent wells
(16).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (83K):
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Fig. 1.
The VPAC1, PTH1, PTH2, and glucagon receptors
translocate RAMPs to the cell-surface. HEK-293 cells were
transiently transfected with N-terminally epitope-tagged RAMP in the
absence or presence of co-transfection with receptor. A-C,
RAMP alone; D-F, RAMP plus GHRH receptor;
G-I, RAMP plus CL receptor; J-L, RAMP plus
VPAC1 receptor; M, RAMP2 plus glucagon receptor;
N, RAMP2 plus PTH1 receptor; O, RAMP3 plus PTH2
receptor. A, D, G, and J,
c-Myc-RAMP1; B, E, H,
K, M, and N, HA-RAMP2; C,
F, I, L, and O, HA-RAMP3. The
figure shown is of nonpermeabilized cells and is representative of at
least three independent experiments. Bar = 10 µm.
Similar results were obtained in transfected COS-7 cells (not
shown).
The most striking pharmacological consequence of RAMP association with receptors for the calcitonin family of peptides is the generation of unique receptor phenotypes that can readily be detected pharmacologically in both radioligand binding and cell signaling studies (1, 3, 5). Because we established a significant interaction between the VPAC1 receptor and each of the RAMPs, we chose to investigate the pharmacological consequences of this association in greater detail to determine whether the VPAC1 receptor phenotype was also altered. For the sake of comparison with our functional experiments (see below), binding assays were conducted on both membrane homogenates and whole cells. Saturation binding experiments using 125I-VIP as the radioligand revealed no significant effect of co-expression of RAMPs on the maximal density of cell surface VPAC1 receptor binding sites when compared with those determined in the absence of RAMP (not shown). Table I shows the results of subsequent competition binding assays between 125I-VIP and a variety of low and high affinity VPAC1 receptor ligands. Unlike their behavior with either the CTR or CL receptor, the association of RAMPs with the VPAC1 receptor did not cause a significant alteration in the binding properties of the receptor in either whole cells or membranes (Table I).
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The VPAC1 receptor is known to couple promiscuously to different
classes of G proteins, leading to the intracellular stimulation of cAMP
accumulation and PI hydrolysis (20). We chose to quantify the
generation of these second messengers in response to VPAC1 receptor
agonists in the absence or presence of co-transfection of RAMPs to
determine the effect of receptor-RAMP interaction on the signaling
properties of the VPAC1 receptor. Initial experiments utilized a single
concentration, at an approximate EC50 value, of a
variety of VPAC1 receptor agonists (Fig.
2, A and B,
left panels) and compared the responses of these
agonists with agonists acting via the CTR (Fig. 2, A and
B, right panels). As shown in Fig. 2A, the
co-expression of the VPAC1 receptor and RAMP2 led to a significant
enhancement of the PI response to the VPAC1 receptor agonists. Although
there was a slight enhancement in the PI response when the receptor was
co-expressed with RAMP3, it was not statistically significant. In
contrast, the cAMP response mediated by the VPAC1 receptor was not
significantly affected by co-expression of any of the RAMPs (Fig.
2B). When similar experiments were performed with the CTR
and its agonists, no selective augmentation of the PI response by any
of the RAMPs was noted (Fig. 2A). In subsequent experiments,
we established complete concentration-response curves to VIP for both
PI and cAMP accumulation. Fig. 2C shows that the potency of
VIP to mediate PI hydrolysis remained unaltered, whereas the maximal
response was significantly enhanced in the presence of RAMP2. When
VIP-mediated cAMP accumulation was investigated, there was no
significant effect of RAMP2-VPAC1 receptor co-expression on either the
maximal agonist response or agonist potency. The lack of change in
agonist potency is consistent with our competition binding data in
which no change in ligand affinity was observed. To ensure that the
augmented PI response seen with RAMP2 was not due to increased cell
surface receptor expression, additional experiments were performed in
which N-terminally FLAG-tagged VPAC1 receptors were co-transfected with
each of the RAMPs and both 125I-VIP and
125I-IgG binding (to detect anti-FLAG antibody as an
independent measure of cell surface-expressed receptor) were assessed
in parallel. No significant difference was observed in either
125I-VIP binding or anti-FLAG antibody binding, confirming
that co-expression with RAMPs did not modify the levels of cell
surface-expressed receptor. Taken together, these findings indicate
that the association of RAMP2 and the VPAC1 receptor differentially
affects the coupling efficiency of the receptor through different
signal transduction pathways. Importantly, the effect of RAMPs on
intracellular signal strength is receptor specific, with no parallel
modulation of CTR-mediated PI hydrolysis found. Thus, we have
identified a novel behavior of RAMPs that is distinct from their effect
on expression of receptor phenotype.
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The finding that PI signal strength is specifically augmented by RAMP2 is highly relevant because the classic coupling pathway associated with VPAC1 receptor activation in a number of cell and tissue types has always been the stimulation of cAMP accumulation via coupling to Gs proteins; receptor-mediated PI hydrolysis is either weakly stimulated or not observable (20). Our finding of a selective enhancement of the VPAC1 receptor PI response in the presence of RAMP2 is one possible mechanism that would explain some of the contradictory results observed in other studies of VPAC1 receptor signaling. Specifically, the endogenous RAMP complement may exert a hitherto unappreciated impact on the signaling efficiency of the VPAC1 receptor.
Important questions remain regarding the functional roles of the VPAC1-RAMP1, VPAC1-RAMP3, PTH1-RAMP2, glucagon receptor-RAMP2, and PTH2-RAMP3 complexes that we have identified. In the case of the VPAC1-RAMP1 heterodimers, despite very potent translocation of RAMP1 to the cell surface and reduction of RAMP1 homodimerization (not shown), we were unable to observe any obvious effects on receptor binding or classic signal pathways such as cAMP or PI accumulation. However, this does not mean that the complexes are pharmacologically indistinguishable from the VPAC1 receptor monomer. For example, the VPAC1-RAMP1 complexes may display altered trafficking properties, altered protein-protein interactions, or differential signaling through other intracellular pathways. Indeed, it is now clear that ligand efficacy is manifested in a variety of both classic and nonclassic modes (21), and thus RAMP-receptor complexes may impact upon these modes to differing extents.
In conclusion, we have provided evidence for the direct interaction of
RAMPs with four new receptor partners, thus illustrating for the first
time that RAMP-receptor complexing extends to receptors beyond those
activated by the calcitonin family of peptides. The ability of RAMP2 to
augment VPAC1 receptor signaling with no concurrent alteration in cAMP
identifies a new functional consequence of RAMP-receptor interactions
and suggests that RAMPs may play a more general role in modulating cell
signaling through other GPCRs than is currently appreciated.
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ACKNOWLEDGEMENTS |
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We thank Drs. Michael Chorev, Ted Usdin, Anette Sams Nielsen, and Gaylinn for generously donating receptor cDNA for this study.
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FOOTNOTES |
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* This work was funded by National Health and Medical Research Council (NHMRC) Grants 990024 and 145702.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ A C. R. Roper senior research fellow of the Faculty of Medicine, Dentistry, and Health Sciences, University of Melbourne.
A senior research fellow of the National Health and Medical
Research Council of Australia. To whom correspondence should be addressed. Tel.: 61-3-8344-7334; Fax: 61-3-9348-1707; E-mail: p.sexton@hfi.unimelb.edu.au.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.C200629200
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ABBREVIATIONS |
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The abbreviations used are: RAMP, receptor activity-modifying protein; GPCR, G protein-coupled receptor; CGRP, calcitonin gene-related peptide; CL receptor, calcitonin receptor-like receptor; CTR, calcitonin receptor; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating peptide; VPAC1R, VIP and PACAP 1 receptor; PTH, parathyroid hormone; PTH1R, parathyroid hormone 1 receptor; PTH2R, parathyroid hormone 2 receptor; PI, phosphoinositide; h, human; GHRH, growth hormone-releasing hormone; HA, hemagglutinin; GHRHR, growth hormone-releasing hormone receptor; GLP, glucagon-like peptide.
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