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Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, 8008 Zurich, Switzerland
Address all correspondence and requests for reprints to: Dr. Walter Born, Klinik Balgrist, Forchstrasse 340, 8008 Zurich, Switzerland. E-mail: wborn{at}balgrist.unizh.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-calcitonin (CT) gene-related
peptide (-, ßCGRP) are structurally related vasodilatory peptides
with homology to CT and amylin. An originally orphan human CT
receptor-like receptor (hCRLR) is a Gs
protein-coupled CGRP or ADM receptor when coexpressed with recently
identified human single transmembrane domain receptor activity
modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the
function of the rat CRLR homologue (rCRLR) has been investigated in rat
osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and
presence of hRAMP1 and -2 and combinations thereof. Transient
expression of rCRLR in UMR-106 cells revealed an ADM receptor, and
[125I]rat (r) ADM binding was enhanced with hRAMP2 and
inhibited by 50% when hRAMP1 was coexpressed. Detectable
[125I]hCGRP binding required the presence of hRAMP1,
and the expression of CGRP binding sites was unaffected by coexpressed
hRAMP2. Specificity of ADM binding sites in
[125I]rADM binding inhibition experiments was
reflected by an over 1000-fold higher potency of rADM [half-maximal
effective concentration = 0.19 ± 0.05 nM
(mean ± SEM, n = 4)], compared with rCGRP
and rßCGRP, to induce a cAMP-responsive luciferase reporting gene
(CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing
predominantly CGRP binding sites, rßCGRP, rCGRP, and rADM induced
CRE-luc with half-maximal effective concentration of 0.27 ± 0.17
nM, 0.37 ± 0.27 nM, and 1.4 ± 0.9
nM, respectively. In COS-7 cells, the results were
comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor
function. This is consistent with higher levels of endogenous RAMP2
encoding messenger RNA in UMR-106, compared with COS-7 cells. In
conclusion, the recognition of RAMP1 and -2 as mediators of CRLR
expression as a CGRP or ADM receptor has been extended, with evidence
that endogenous RAMP2 is sufficient to reveal an ADM receptor in
UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by
RAMP1 and generation of a CGRP receptor is consistent with competitive
interactions of the different RAMPs with rCRLR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human (h) and rat (r) ADM are 52- and 50-amino acid polypeptides
belonging to the calcitonin (CT) family of peptides. hCT
gene-related peptide (C1–37) (hCGRP) and rCGRP are neuropeptides
encoded by alternative splicing products of corresponding CT genes (10, 11). ßCGRP(1–37) (ßCGRP), in man and rat, are products of a second
closely related gene differing in 3 and 1 amino acids from h- and
rCGRP, respectively (12, 13). - and ßCGRP are widely expressed
in the central and peripheral nervous system (14). Rat ADM (rADM)
shares 18% amino acid sequence homology with rCGRP and rat amylin
that also consists of 37 amino acids. Six amino acid ring structures,
linked by a disulfide bridge between cysteine residues in positions 14
and 19 in rADM and 2 and 7 in rCGRP and rßCGRP, as well as
amidated C-termini, are required for the comparable vasodilatory
activity of CGRP and ADM. The C-terminal fragments hCGRP(8–37) and
hADM(22–52), both lacking the ring structures, are antagonists of CGRP
and ADM (15, 16, 17).
ADM receptors, coupled to cAMP formation and phospholipase C activation, have been described in bovine aortic endothelial cells (18). In rat vascular smooth muscle cells, cAMP stimulation was inhibited by the CGRP antagonist CGRP(8–37) (16, 19). The receptors have the properties of ADM/CGRP receptors. A more potent stimulation of cAMP production by ADM than CGRP was observed in human vascular endothelial and in rat mesangial cells (20, 21). In mesangial cells, it was inhibited at lower concentrations of hADM(22–52) than of CGRP(8–37); and in endothelial cells, CGRP(8–37) did not antagonize ADM. These ADM receptors can be distinguished from ADM/CGRP receptors.
Rat and human proteins, with seven transmembrane domains and approximately 55% amino acid sequence identity to cloned CT receptors, have initially been reported as orphan CT receptor-like receptors (CRLR) (22, 23). Subsequently, the hCRLR was identified as a CGRP receptor when expressed in a human embryonic kidney cell line (24, 25). Transfection of the same CRLR into other cells (including COS-7 cells) did not reveal a receptor ligand. In situ hybridization analysis of mRNA encoding rCRLR during rat fetal and early postnatal development showed predominant expression in the lung (26), known to express an ADM receptor (27), but not in the cerebellum and the spleen, which present a high density of CGRP binding sites (28).
Recently, complementary DNA (cDNA), encoding a human receptor activity modifying protein-1 (hRAMP1) that enhanced the activity of endogenous CGRP receptors, has been cloned through the expression of corresponding complementary RNA in Xenopus oocytes (29). The sequence of 148 amino acids includes an N-terminal signal sequence and putative single extracellular transmembrane and small intracellular domains. Limited nucleotide sequence homology of hRAMP1 encoding cDNA to expressed sequence tags in public databases has been used for the identification of structurally related hRAMP2 and -3 of 175 and 148 amino acids, respectively. The hCRLR became a functional CGRP or ADM receptor when cotransported with hRAMP1 or hRAMP2, respectively, to the cell surface (29).
In the present study, the rCRLR has been transfected into rat osteoblast-like UMR-106 cells that express endogenous RAMP2 to reveal an ADM receptor. COS-7 cells with presumably lower endogenous RAMP2 expression, based on Northern blot analysis, required transfection of rCRLR together with RAMP2 to reveal an ADM receptor. Inhibition of RAMP2-evoked rCRLR, as ADM receptor, and its conversion into a CGRP receptor by RAMP1 indicates competition between different RAMPs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP and rat amylin were purchased from Bachem AG (Bubendorf, Switzerland); rßCGRP, rCGRP(8–37), and rCT
from Peninsula Laboratories, Inc. (Belmont, CA);
rADM(1–50) from Peptide Institute (Osaka, Japan); and
rADM(20–50) from Phoenix Pharmaceuticals, Inc. (Mountain
View, CA). Culture media and FCS were obtained from Biological
Industries (Kibbutz Beit Haemek, Israel). Geneticin 418, OptiMEM
medium, and LipofectAMINE were from Life Technologies
(Gaithersburg, MD). Na[125I] was supplied by
Amersham International (Little Chalfont, UK). Chemicals
and other reagents were purchased from Sigma Chemical Co.
(St. Louis, MO) and E. Merck (Darmstadt, Germany) at the
highest grade available.
Plasmids
The cDNA encoding rCRLR (22), cloned between AflII
and EcoRI restriction sites in the mammalian expression
vector pcDNA1 (Invitrogen, Carlsbad, CA), was
obtained from M. G. Rosenfeld (University of California, San
Diego, CA). hRAMP1 and -2 encoding cDNA, cloned into the mammalian
expression vector pcDNA3 (Invitrogen), was provided
by S. Foord (GlaxoWellcome, UK).
Cell culture and DNA transfection
COS-7 cells were cultured in Ham’s F-12 and DMEM, containing
4.5 g/liter glucose (1:1) supplemented with 10% FCS, at 37 C, in a
humidified atmosphere of 95% air-5% CO2. The same medium,
supplemented with 400 µg/ml Geneticin 418, was used to culture a
subclone of rat osteoblast-like UMR-106 cells stably transfected with a
cAMP-responsive luciferase reporter gene (CRE-luc) (30). The cells were
subcultured by treatment with 0.1% trypsin and 0.5 mM EDTA
in PBS. For transfection and subsequent experiments, the cells were
grown in 24-well plates. COS-7 cells at 40–50% confluence and UMR-106
cells seeded at 1–1.5 x 105 cells/well and grown for
24 h were transfected with mammalian expression vector pcDNA1
(control transfection) and with expression constructs of rCRLR encoding
cDNA in pcDNA1 and of RAMP1 or -2 encoding cDNA in pcDNA3 and with
combinations thereof at equal amounts, as indicated in the figure
legends. pcDNA1 was added, in amounts required to keep the total
quantity of DNA in individual transfection mixtures constant. COS-7
cells were incubated at 37 C for 3 h in 300 µl/well OptiMEM
medium containing 300 ng plasmid DNA and 0.9 µl LipofectAMINE.
Transfection of UMR-106 cells was carried out at 37 C for 5 h in
300 µl/well OptiMEM medium containing 600 ng plasmid DNA and 3 µl
LipofectAMINE. After transfection, the cells were washed twice with
tissue culture medium and cultured for 48 h and assayed for ligand
binding and cAMP accumulation or luciferase induction.
Radioligand binding studies
[125I]-labeled ([125I])rADM and
[125I]hCGRP were prepared by a modified chloramine-T
method (31). The reactions were quenched with 1 mg tyrosine methylester
after 1 min for [125I]rADM and after 2 min for
[125I]hCGRP. Radioiodinated rADM and hCGRP were
separated from noniodinated peptides, detected by UV absorption at 215
nM, and from [125I]tyrosine methylester by
reversed phase HPLC, as described (32, 33). UMR-106 and COS-7 cells
were incubated for 3 h at 15 C with 1670 Bq
[125I]rADM or 1670 becquerels
[125I]hCGRP, in the absence and presence of nonlabeled
peptides in 0.2 ml/well Ham’s F-12 and DMEM (1:1), supplemented with
0.1% BSA (binding medium). Incubations were stopped by washing the
cells with ligand-free binding medium (15 C) and cell lysis in 0.5 ml
0.5% SDS. Radioactivity was measured in a 
-counter (MR252,
Kontron, Zurich, Switzerland). Binding in the presence of
0.1 µM nonlabeled rADM or hCGRP was considered to be
nonspecific.
cAMP accumulation and luciferase activation
cAMP was measured in extracts of cells incubated at 37 C for 15
min in 0.2 ml medium containing 136 mM NaCl, 5.4
mM KCl, 1 mM Na2 HPO4,
5.5 mM glucose, 1 mM CaCl2, 1
mM MgSO4, 1 mM
isobutylmethylxanthine, 20 mM HEPES (pH 7.45), and 0.1%
BSA, in the absence and presence of the indicated peptides. cAMP was
extracted with 500 µl 95% ethanol (pH 3) at 4 C for 1 h. The
extracts were lyophilized, and cAMP was determined by RIA, as described
(34).
cAMP-responsive element-driven luciferase expression was estimated in UMR-106 cell extracts as a measure of cAMP production. Before stimulation, the cells were kept serum-free for 24 h in Ham’s F-12 and DMEM (1:1) supplemented with 0.1% BSA. The indicated peptides were then added at 10-12–10-6 M in 0.2 ml/well fresh medium, and incubations were carried out in duplicate for 4 h at 37 C in a humidified atmosphere of 95% air-5% CO2. Luciferase extraction and activity measurements were carried out as described (30).
Northern blot analysis
PolyA+RNA was isolated from UMR-106 and COS-7 cells
with the FastTrack 2.0 mRNA isolation kit from Invitrogen
(Leek, The Netherlands). The RNA (10 µg per lane) was
size-fractionated on a formaldehyde/agarose (1% wt/vol) gel,
electroblotted to 
-Probe membranes (Bio-Rad Laboratories, Inc., Glattbrugg, Switzerland), and UV-cross-linked with a
Stratalinker 2400 (Stratagene, Basel, Switzerland). Size
markers in parallel lanes were stained, after UV-cross-linking, with
methylene blue (Molecular Research Center, Inc.,
Cincinnati, OH), marked with a waterproof pen, and superimposed to
autoradiograms for size estimation of hybridizing RNA. Human and mouse
RAMP1 and -2 specific cDNA hybridization probes were amplified by PCR
from cloned cDNA and labeled with [32P]deoxy-ATP
(110·1012/mmol, Amersham Life Science,
Little Chalfont, UK) using the Prime-It II random primer labeling kit
from Stratagene. The incorporation of
[32P]deoxy-ATP was between 41% and 78%. Filters were
hybridized overnight at 50 C in 5 x saline-sodium phosphate/EDTA
according to a CLONTECH Laboratories, Inc. (Palo Alto, CA)
protocol. Subsequently, the filters were washed 4 times in 2 x
saline-sodium citrate, 1% SDS, and twice in 0.1% saline-sodium
citrate, 0.5% SDS at 50 C. Then, 1.8-kb human ß-actin and 1.3-kb
glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA fragments were
labeled with the same kit and used for control
hybridizations, under the conditions recommended by CLONTECH Laboratories, Inc.. The filters were exposed to x-ray films at
-80 C with intensifying screens.
Statistical analyses
Results presented in Figs. 1
and 2 (also see
Figs. 4–6) are mean
values ± SEM of triplicate determinations; and in
Fig. 3, of duplicate determinations of a
representative experiment carried out at least three times. The values
for half-maximal inhibitory concentration (IC50) and for
half-maximal effective concentration (EC50) were calculated
by nonlinear regression analysis using Fig. P 6.0 software
(Biosoft, Cambridge, UK) and are means ±
SEM of at least three independent experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP was investigated 48 h after
transfection of UMR-106 cells with cDNA encoding rCRLR and hRAMP1 and
-2, and combinations thereof (Fig. 1
). In control-transfected UMR-106
cells and in cells transfected with individual hRAMP expression
constructs alone, total binding of [125I]rADM and
[125I]hCGRP was below 3% of added tracer, and
specific binding was not detected. In cells transfected with rCRLR
encoding cDNA alone, specific binding of [125I]rADM
varied between 1.3% and 2.6% of added tracer, but specific
[125I]hCGRP binding was not observed. Specific
[125I]rADM binding was increased to between 9.2% and
24% of added radioligand when rCRLR and hRAMP2 were coexpressed.
Expression of rCRLR, together with hRAMP1 alone or with hRAMP1 and
hRAMP2 together, lowered [125I]rADM binding to
approximately 50% of that observed in the absence of hRAMP1 and
revealed specific binding of [125I]hCGRP, varying
between 21% and 35% of added tracer. [125I]hCGRP
binding was not affected by the presence of hRAMP2. Taken together,
transfection of cDNA encoding rCRLR in UMR-106 cells brings about
expression of [125I]rADM binding sites that is
enhanced by coexpressed hRAMP2. The same rCRLR reveals
[125I]hCGRP binding sites in the presence of hRAMP1,
unaltered by hRAMP2.
Radioligand displacement experiments were carried out in UMR-106 cells
transfected with rCRLR encoding cDNA together with hRAMP2 or hRAMP1
alone or together with hRAMP1 and -2 (Fig. 2). In UMR-106 cells,
coexpressing rCRLR and hRAMP2 (Fig. 2A) [125I]rADM
binding was inhibited by rADM and rADM (20–50) with IC50
of 0.35 ± 0.08 (mean ± SEM, n = 3)
nM and 2.6 ± 1.2 nM, respectively. Rat
ßCGRP and rCGRP (8–37) were 270- and 110-fold less potent than
rADM. Up to 10-6 M rCGRP and rat amylin
inhibited [125I]rADM binding by less than 50%. In cells
coexpressing rCRLR and hRAMP1 (Fig. 2C), [125I]hCGRP
binding was inhibited by rCGRP (8–37), rßCGRP, rCGRP, and rADM
with IC50 of 1.6 ± 0.7 nM, 4.6 ±
2.3 nM, 11.5 ± 6.0 nM, and 141 ± 57
nM, respectively. Inhibition of
[125I]hCGRP binding, by up to 10-6
M rat amylin and rADM (20–50), was less than 50%. Up to
10-6 M rCT did not interfere with
[125I]rADM and [125I]hCGRP binding. The
IC50 of rADM and rCGRP, with respect to
[125I]rADM, and of [125I]hCGRP binding
was the same in UMR-106 cells subjected to the combined transfection of
hRAMP1 and -2 and rCRLR encoding cDNA, and in cells with rCRLR and
hRAMP2 or hRAMP1 alone (Fig. 2, B and D). As a result, the observed
50% reduction of maximal specific [125I]rADM binding in
cells coexpressing hRAMP1 and -2 together with rCRLR (compared with
cells expressing rCRLR with hRAMP2 alone) reflects a reduction in the
number of [125I]rADM binding sites by hRAMP1 in the
presence of hRAMP2. hRAMP2, on the other hand, was unable to reduce
hRAMP1-dependent [125I]hCGRP binding in UMR-106 cells.
This points to a dominant action of hRAMP1 over hRAMP2 for the
functional expression of rCRLR as a CGRP receptor.
cAMP accumulation, measured after 15 min in UMR-106 cells (not shown),
paralleled the induction after 4 h of a stably transfected CRE-luc
by ADM, CGRP, amylin, and CT (Fig. 3). UMR-106 cells express endogenous
CT receptors and rCT induced luciferase expression in control-, rCRLR-,
and rCRLR and hRAMP1-transfected cells with EC50 of 10–30
nM. In control-transfected cells (Fig. 3A), luciferase
induction by rCGRP, rßCGRP, and rat amylin was only observed at
over 100 nM, probably because of cross-reaction with an
endogenous CT receptor; and rADM was inactive. In cells transfected
with rCRLR alone (Fig. 3B) or together with hRAMP2 (not shown), rADM
maximally induced luciferase expression 10 ± 4- and 8 ±
3-fold, with EC50 of 0.19 ± 0.05 nM and
0.12 ± 0.04, respectively. Increased luciferase responses in
rCRLR expressing cells by rCGRP and rßCGRP, compared with
control-transfected cells, is attributed to cross-reaction of CGRP with
the transiently expressed ADM receptor. This was not observed with rat
amylin. In UMR-106 cells expressing rCRLR together with hRAMP1 (Fig. 3C), rCGRP (EC50 = 0.37 ± 0.27 nM) and
rßCGRP (EC50 = 0.27 ± 0.17 nM) induced
luciferase expression at over 1000-fold higher potency than in cells
expressing rCRLR alone; but the potency of rADM decreased 7-fold. These
findings are consistent with the function of rCRLR as a CGRP receptor
in the presence of hRAMP1. Luciferase induction in these cells, by high
nanomolar concentrations of rat amylin, is most likely brought about
through its interaction with a hRAMP1-dependent CGRP receptor. Taken
together, the results parallel those of radioligand binding
experiments, demonstrating ADM receptor expression in UMR-106 cells
transfected with a rCRLR encoding cDNA; and a CGRP receptor is revealed
through coexpression with hRAMP1.
Rat CRLR function in COS-7 cells in the absence and presence of
RAMPs
COS-7 cells were transfected with cDNA encoding rCRLR and hRAMP1
and -2, and combinations thereof, as indicated in Fig. 4. In cells transfected with pcDNA1
control vector or rCRLR, hRAMP1, or -2 expression constructs, total
binding of [125I]rADM or [125I]hCGRP was
below 2% of added tracers, and specific binding was undetectable.
Cotransfection of COS-7 cells with rCRLR and hRAMP2 or hRAMP1 revealed
between 3.0% and 6.6% and between 7.7% and 11.4% specific
[125I]rADM or [125I]hCGRP binding,
respectively. Expression of rCRLR together with hRAMP1 and hRAMP2
reduced [125I]rADM-specific binding by about 60% but
left [125I]hCGRP binding unchanged. In COS-7 cells
cotransfected with rCRLR and hRAMP2 encoding cDNA (Fig. 5A), rADM, rADM (20–50), rCGRP
(8–37), and rßCGRP inhibited [125I]rADM binding with
IC50 of 1.6 ± 1.5 nM, 3.5 ± 0.1
nM, 72 ± 39 nM, and 146 ± 58
nM, respectively. Up to 10-6 M
rCGRP and rat amylin inhibited [125I]rADM binding by
less than 50%. In COS-7 cells cotransfected with rCRLR and hRAMP1
(Fig. 5B), [125I]hCGRP binding was inhibited by
rCGRP (8–37), rßCGRP, rCGRP, and rADM with IC50 of
1.2 ± 0.1 nM, 4.4 ± 1.9 nM,
5.3 ± 2.5 nM, and >100 nM, respectively.
Binding inhibition with up to 10-6 M rat
amylin and rADM (20–50) was below 50%. rCT, at up to
10-6 M, did not inhibit
[125I]rADM and [125I]hCGRP binding.
Similar to UMR-106 cells, rADM stimulated cAMP levels in COS-7 cells
coexpressing rCRLR and hRAMP2, with an EC50 of 0.66 ±
0.21 nM (Fig. 6A). cAMP
accumulation, in response to rCGRP and rßCGRP, was only observed
at over 10-7 M, as a result of cross-reaction
with an ADM receptor. Rat amylin and rCT were inactive in these cells.
In COS-7 cells coexpressing rCRLR and hRAMP1 (Fig. 6B), on the other
hand, rßCGRP, rCGRP, rADM, and rat amylin stimulated cAMP
accumulation, with EC50 of 0.05 ± 0.06
nM, 0.11 ± 0.03 nM, 10 ± 4
nM, and >100 nM, respectively. rCT was again
inactive.
Expression of endogenous RAMP mRNA in UMR-106 and COS-7 cells
Northern blot analysis of polyA+ RNA, from
nontransfected UMR-106 cells, recognized mRNA of approximately 1 kb
hybridizing to an hRAMP2-specific cDNA probe, but an hRAMP1-specific
probe revealed no signal (Fig. 7). A much
weaker signal with the hRAMP2 probe was obtained with
polyA+ RNA isolated from nontransfected COS-7 cells,
although apparently higher amounts of COS-7, compared with UMR-106
polyA+ RNA reflected by the more marked signals of
ß-actin and G3PDH mRNA, were recognized. mRNA hybridizing to
hRAMP1-specific cDNA remained again undetectable. The results were
similar when cDNA probes encoding functional mouse RAMP1 and -2
(manuscript in preparation) were used (not shown). A computer search in
the GenBank database revealed 91% and 89% nucleotide sequence
homology of mouse RAMP1 and -2 to rat expressed sequence tags AI 012424
and AI 012814, respectively, representing putative rat RAMP1 and -2
encoding sequences. Candidate monkey RAMP encoding sequences were not
found. Taken together, the results imply inactive RAMP1 genes in
UMR-106 and COS-7 cells and a higher abundance of RAMP2 encoding mRNA
in UMR-106, compared with COS-7 cells. But different relative intensity
of hybridization signals, caused by the use of heterologous probes,
cannot be ruled out.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat CRLR expressed in rat osteoblast-like UMR-106 cells is an ADM receptor recognizing CGRP at over 100-fold higher concentrations. Its functional expression in UMR-106 cells is brought about by a putative rRAMP2 encoded by a 1-kb mRNA hybridizing with an hRAMP2-specific cDNA probe. RAMP2-independent expression of rCRLR as an ADM receptor in UMR-106 cells cannot be excluded, but the enhanced expression of [125I]rADM binding sites upon coexpression of hRAMP2 suggests that an endogenous RAMP2-like protein was present in limited amounts in UMR-106 cells transiently overexpressing transfected rCRLR. Expression of a monkey hRAMP2 homologue at low levels in COS-7 cells is consistent with the observed much weaker RAMP2 hybridization signal of COS-7, compared with UMR-106 mRNA, on Northern blot analysis. This may explain the dependence of rCRLR on exogenous hRAMP2 for ADM receptor function in COS-7 (unlike UMR-106) cells.
mRNA, hybridizing to human and mouse RAMP1-specific hybridization probes, was undetectable in UMR-106 and COS-7 cells, even under low stringency hybridization conditions. In view of nucleotide sequence homology of over 75% among human, mouse, and putative rat RAMP1-encoding cDNA, it seems unlikely that the monkey RAMP1 encoding sequence is more distantly related and undetectable by human and mouse probes. But low-level expression of RAMP1-encoding mRNA may have been missed in UMR-106 and COS-7 cells because of the use of heterologous hybridization probes. The results from the Northern blot analysis imply that RAMP1 mRNA, if expressed in UMR-106 and COS-7 cells, is present at much lower levels than RAMP2 mRNA. Undetectable RAMP1 mRNA expression in UMR-106 and COS-7 cells is also consistent with the inability of these cells to functionally express rCRLR as a CGRP receptor without added exogenous hRAMP1.
Expression of hRAMP1 together with rCRLR, in the absence and presence of hRAMP2 in UMR-106 cells and in its presence in COS-7 cells, inhibited ADM receptor expression. hRAMP2, on the other hand, did not interfere with hRAMP-1-dependent CGRP receptor expression in both cell lines. This suggests a dominant functional interaction of hRAMP1 over r- and hRAMP2 with rCRLR. In cells coexpressing hRAMP1 and -2 together with rCRLR, higher hRAMP1 than -2 expression levels, caused by shorter half-lives of hRAMP2 mRNA and/or protein (compared with hRAMP1), cannot be excluded. Detailed regulatory mechanisms remain to be elucidated.
Residual [125I]rADM binding in UMR-106 cells transiently
cotransfected with rCRLR and hRAMP1 encoding cDNA has been observed. It
may result from a subpopulation of cells expressing rCRLR and
endogenous rRAMP2 alone and/or incomplete suppression of rCRLR–rRAMP2
interaction by hRAMP1 in cells expressing all three proteins. Residual
ADM receptor expression in this mixed cell population also explains an
only 3.8-fold lower potency of rADM, compared with rCGRP, to induce
the stably expressed CRE-luc reporter gene. As indicated by the
relative potencies of ADM and CGRP to stimulate cAMP accumulation in
COS-7 cells, cross-reaction between ADM and CGRP receptors was more
pronounced than between CGRP and ADM receptors (36).
McLatchie et al. (29) were the first to demonstrate that hCRLR requires coexpression of hRAMP1 or hRAMP2 for transport into the plasma membrane of human embryonic kidney 293T cells and functional expression as a CGRP- or an ADM receptor, respectively. The present report, extending these results, demonstrates RAMP2-independent ADM receptor expression of the rat homologue of hCRLR in an osteoblast-like cell line of the same species expressing endogenous RAMP2 encoding mRNA. Furthermore, competition of hRAMP1 with cotransfected hRAMP2 and presumably also with endogenous rat RAMP2, causing inhibition of ADM receptor expression, has been revealed for the first time. Vasorelaxant and hypotensive actions of CGRP, comparable with those of ADM, are likely mediated in vivo by rCRLR when coexpressed with RAMP1, but the existence of nonidentified CGRP receptors different from RAMP1-dependent CRLR and similar to CGRP binding sites in the cerebellum cannot be excluded. Conversion of rCRLR from an ADM into a CGRP receptor, upon cotransfection of RAMP1, points to important functions of RAMPs determining ligand specificity in vivo and suggests a wider role for these and similar proteins in G protein receptor-coupled signaling.
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Footnotes |
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2 N. Bühlmann and K. Leuthäuser made equal contributions
to this work.
Received November 19, 1998.
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),
r
), rCT(
),
rADM(20–50)(
), and r
). cDNA expression constructs
were those of Fig. 1
