Full text of DOI:10.1038/sj.bjp.0704761

British Journal of Pharmacology (2002) 136, 784 ± 792
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Comparison of the expression of calcitonin receptor-like receptor
(CRLR) and receptor activity modifying proteins (RAMPs) with
CGRP and adrenomedullin binding in cell lines
1
2
3
1,4Tejal Choksi, 1,4Debbie L. Hay, Stephen Legon, David R. Poyner, Stefanie Hagner,
1Stephen R. Bloom & *,1David M. Smith
1Department of Metabolic Medicine, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London
2
3
W12 0NN; School of Life Sciences, Aston University, Aston Triangle, Birmingham B4 7ET and Institute of Physiology,
Philipps-University, Deutschhausstrasse 2, D35037, Marburg, Germany
1
The calcitonin receptor-like receptor (CRLR) and speci®c receptor activity modifying proteins
(RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenome-
dullin in transfected cells.
2
There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/
RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding
correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express
these receptors. Speci®c human or rat CRLR antibodies were used to examine the presence of
CRLR in these cells.
3
We con®rmed CGRP subtype 1 receptor (CGRP1) pharmacology in SK-N-MC neuroblastoma
cells. L6 myoblast cells expressed both CGRP1 and adrenomedullin receptors whereas Rat-2
®broblasts expressed only adrenomedullin receptors. In contrast we could not con®rm CGRP2
receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP1-like in this
study.
4
L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 ®broblasts
had only RAMP2. No cell line had detectable RAMP3 mRNA.
SK-N-MC, Col-29 and Rat-2 ®broblast cells expressed CRLR mRNA. By contrast, CRLR
5
mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a di€erent
source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus,
circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the
presence of this receptor.
6 These data strongly support CRLR, together with appropriate RAMPs as binding sites for
CGRP and adrenomedullin in cultured cells.
British Journal of Pharmacology (2002) 136, 784 ± 792
Keywords: CGRP; adrenomedullin; CRLR; RAMP; SK-N-MC neuroblastoma cells; Col-29 cells; L6 myoblasts; Rat-2
®broblasts
Abbreviations: a-CGRP, calcitonin gene-related peptide; CGRP1 and CGRP2, CGRP receptor subtypes; CRLR, calcitonin
receptor-like receptor; [Cys(ACM)2,7]a-CGRP, [acetimidomethyl-Cys2,7]a-CGRP; [Cys(Et)2,7]a-CGRP, [ethyla-
mide-Cys2,7]a-CGRP; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IBMX, isobutylmethylxanthine;
RAMP, receptor activity modifying protein
Introduction
Adrenomedullin and calcitonin gene-related peptide (CGRP)
are structurally related members of the calcitonin family of
regulatory peptides, which share many overlapping biological
functions such as extremely potent vasodilatory behaviour
(Brain & Cambridge, 1996; Hinson et al., 2000). CGRP
receptors are of particular interest as anti-migraine targets
since CGRP release and the onset of head pain have been
clearly correlated (Edvinsson, 2001; Doods, 2001). Studies
with adrenomedullin gene knock-out mice demonstrate that
this peptide and its receptors are essential for the formation
of a functional cardiovascular system (Hay & Smith, 2001;
Caron & Smithies, 2001).
High anity binding sites, speci®c for either CGRP or
adrenomedullin, have been demonstrated in various tissues
and cell lines (Henke et al., 1987; Sexton et al., 1986; Poyner
et al., 1992; Owji et al., 1995; Coppock et al., 1999). CGRP
receptors have been divided into two subtypes (CGRP1 and
CGRP2) based, predominantly, on the ability of the
antagonist CGRP8-37 to inhibit the e€ects of CGRP in
functional studies; binding studies are not able to distinguish
these putative subtypes (Dennis et al., 1989; 1990; Rorabaugh
et al., 2001). At CGRP1 receptors this CGRP fragment is a
potent antagonist whereas CGRP2 receptors are more
resistant to its e€ects (Dennis et al., 1989; 1990; Chiba et
al., 1989). Two non-cyclic analogues of CGRP, a-[acetimi-
*Author for correspondence at: AstraZeneca, Mereside, Alderley
Park, Maccles®eld, Cheshire, SK10 4TG;
E-mail: dave.smith@astrazeneca.com
4The ®rst two authors contributed equally to this study

T. Choksi et al
CRLR, RAMPs and receptor binding in cell lines
785
domethyl-Cys2,7]hCGRP ([Cys(ACM)2,7]a-CGRP) and a-
[ethylamide-Cys2,7]hCGRP ([Cys(Et)2,7]a-CGRP) have been
proposed as CGRP2-speci®c agonists (Dennis et al., 1989;
Dumont et al., 1997), however their speci®city has recently
been questioned (Wu et al., 2000; Wisskirchen et al., 1998).
Critical to the development of speci®c ligands for these
receptors and to fully understand the biology of these
peptides is a determination of the molecular nature of all
CGRP and adrenomedullin binding sites.
Methods
Materials
Rat adrenomedullin and rat [Tyr0]a-CGRP were obtained
from Peninsula Laboratories Inc (St Helens, Merseyside,
U.K.). Rat a-CGRP and human a-CGRP8-37 were custom
synthesized by ASG University (Szedgel, Hungary) and Dr
P.G.H. By®eld (Haemostasis Research Unit, Clinical Sciences
Centre, Hammersmith Hospital, London, U.K.) respectively.
[Cys(ACM2,7]a-CGRP was from Peninsula and [Cys(Et)2,7]a-
CGRP was from Phoenix Pharmaceuticals Inc (Mountain
View, CA, U.S.A.). All peptides were checked for correct
molecular weight by mass spectroscopy. Na[125I], polyvinyli-
dene di¯uoride (PVDF) membrane, ECL Plus reagent and
Hyper®lm ECL were supplied by Amersham Pharmacia
Biotech (Little Chalfont, Bucks, U.K.). Iodogen reagent was
supplied by Pierce (Rockford, Illinois, U.S.A.). Unless
otherwise speci®ed chemicals were from Sigma Chemical Co
(Sigma-Aldrich, Gillingham, Dorset, U.K.) and cell culture
materials from Gibco BRL (Life Technologies, Paisley,
Renfrewshire, U.K.).
Though the calcitonin receptor was cloned in 1991 (Lin et
al., 1991), CGRP and adrenomedullin receptors have proven
dicult to identify. In 1995 an orphan receptor known as
L1 was reported to be an adrenomedullin receptor and a
related receptor, RDC-1, to be a CGRP1 receptor (Kapas et
al., 1995; Kapas & Clark, 1995). However this work has
been dicult to reproduce (Kennedy et al., 1998). Recent
evidence suggests that both adrenomedullin and CGRP bind
to the same seven transmembrane domain receptor, the
calcitonin receptor-like receptor (CRLR, Njuki et al., 1993;
Fluhmann et al., 1995). Interestingly, the speci®city of
CRLR for these peptides is conferred by a novel family of
single transmembrane proteins, termed receptor-activity
modifying proteins (RAMPs, McLatchie et al., 1998).
Concurrent CRLR and RAMP expression is essential for
the formation of functional receptors. Furthermore, RAMPs
appear to be closely associated with CRLR at the cell
surface and may contribute part of the peptide binding site
(Hilairet et al., 2001). CRLR functions as a CGRP1 receptor
when co-expressed with RAMP1 but as an adrenomedullin-
preferring receptor in the presence of either RAMP2 or
RAMP3 in transfection studies using HEK 293T cells
(McLatchie et al., 1998). This basic hypothesis has been
supported by further transfection studies from a number of
groups (Mu€ et al., 1998; Kamitani et al., 1999;
Chakravarty et al., 2000), however more information is
required concerning the relevance of these receptor combina-
tions to the physiological actions of CGRP and adrenome-
dullin. Whether CGRP2 receptors are also formed by CRLR
and RAMP combinations is not yet known. IUPHAR
guidelines are that the complexes formed by CRLR/RAMP2
and CRLR/RAMP3 should be known as AM1 and AM2
receptors; however these entities show similar pharmacolo-
gical properties with respect to their anities for CGRP8-37
(Poyner et al., 2002).
Peptide iodination
Rat adrenomedullin and rat [Tyr0]a-CGRP were iodinated by
the Iodogen method as previously described (Owji et al., 1995;
Bhogal et al., 1993). Adrenomedullin (12.5 mg, 2 nmoles) in
10 ml of phosphate bu€er 0.2 M, pH 7.2 was incubated with
37 MBq of 125NaI and 10 mg of Iodogen reagent for 4 min at
228C. The 125I-labelled peptide was puri®ed by reverse phase
HPLC (Waters C18Novopak, Millipore, Milford MA, U.S.A.)
using a 15 ± 40% acetonitrile/water/0.05% tri¯uoroacetic acid
gradient. The speci®c activity of the label was estimated as
10 Bq fmol71 by comparison of simultaneous saturation and
competition binding data. Rat [Tyr0]a-CGRP was iodinated
by
a similar procedure except 12 mg (2.4 nmoles) were
incubated on ice for 5 min together with the 125NaI and the
products separated on a 20 ± 50% acetonitrile/water/0.05%
tri¯uoroacetic acid gradient. The speci®c activity of the label
was estimated as 36 Bq fmol71 by radio-immunoassay
(Upton et al., 1997). Fractions showing binding were
aliquoted, freeze-dried and stored at 7808C.
We recently hypothesized that CRLR/RAMP combina-
tions account for the majority of observed CGRP and
adrenomedullin binding in rat tissues. We found a signi®cant
correlation between CGRP binding and RAMP1 mRNA
expression but the correlation between adrenomedullin
binding and RAMP2 expression was not signi®cant
(Chakravarty et al., 2000). As an extension to this study
we have evaluated the relationship between CGRP and
adrenomedullin binding and CRLR/RAMP transcript levels
in immortalized cell lines. These cell lines have previously
been reported to express various combinations of CGRP
and/or adrenomedullin receptors and should represent a less
complex system than tissues for correlating receptor type
with CRLR and RAMP mRNA (Longmore et al., 1994;
Coppock et al., 1996; 1999; Kirkland, 1985). We have also
established the presence of CRLR protein in each of these
cell lines using speci®c CRLR antibodies (Hagner et al.,
2001).
Cell culture
SK-N-MC human neuroblastoma (a gift from Prof S.
Nahorski, University of Leicester, widely considered to
express the `standard' CGRP1 receptor (Longmore et al.,
1994)), Rat-2 ®broblast (European Collection of Animal Cell
Cultures (ECACC), previously reported to only express
adrenomedullin receptors (Coppock et al., 1999)), L6G8C5
rat myoblasts (L6-1 cells from Aston University stock and
L6-2 from ECACC, shown to express both CGRP and
adrenomedullin receptors (Coppock et al., 1996)) and Col-29
human colon epithelium cells (previously reported to express
CGRP2 receptors, kindly provided by Dr S.C. Kirkland,
Department of Histopathology, ICSM, Hammersmith Hos-
pital, London, U.K. (Kirkland, 1985; Cox & Tough, 1994;
Poyner et al., 1998)) were cultured as previously described
(Morgan et al., 1998; Coppock et al., 1996; 1999; Poyner et
al., 1998). The cells were grown to con¯uence and were used
British Journal of Pharmacology vol 136 (5)

786
T. Choksi et al
CRLR, RAMPs and receptor binding in cell lines
for whole cell binding, cAMP assay, RNA and protein
extraction.
1.561079 mg71) and equivalent amounts of radiolabelled
probe were used for hybridizations. Northern analysis was
quanti®ed using a PhosphorImager with ImageQuant soft-
ware (Molecular Dynamics Inc, Sunnyvale, CA, U.S.A.). The
blots were then probed successively for glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) and b-actin mRNA and
®nally for the total amount of polyA+ RNA in each track
using a 45mer oligo-dT probe labelled by tailing with a single
32P-dCTP residue. For each standard, hybridization in each
tissue was expressed as a percentage of the total and the
mean ®gures for the three standards were used to normalize
the values for the test probes.
Whole cell adrenomedullin and CGRP ligand binding
assays
Con¯uent cell monolayers (approximately 26105 cells per
well), grown on poly-D-lysine coated 24-well plates, were
washed once with binding bu€er (mM): HEPES 20, pH 7.4,
MgCl2 5, NaCl 10, KCl 5, EDTA 1; phosphoramidon 1 mM
and 0.1% w v71 BSA, and incubated for 60 min at 48C for
adrenomedullin or 228C for CGRP in binding bu€er contain-
ing 1000 Bq of 125I-adrenomedullin or 125I-[Tyr0]a-CGRP.
Unbound label was removed by aspiration of binding bu€er
and cells were washed three times with ice-cold binding bu€er.
Cells were solubilized in 500 ml of NaOH 0.1 M and counted in
a gamma counter. Non-speci®c binding was determined in the
presence of excess unlabelled rat adrenomedullin (100 nM) or
a-CGRP (1 mM). These concentrations are sucient to occupy
499% of the receptors. All binding was performed under
equilibrium conditions (Bhogal et al., 1993; Owji et al., 1995).
Anti-CRLR antibodies and Western blot analysis
Two anity-puri®ed rabbit polyclonal antibodies were
employed in these investigations. One (MR567) was raised to
the carboxy-terminus of human CRLR and has been
characterized elsewhere (Hagner et al., 2001). The other
antibody (MR003) was raised in an identical manner to
MR567 but to the carboxy-terminus of rat CRLR. This
antibody has been shown to detect the rat sequence following
its transfection into HEK293 cells by both Western blot and
immunohistochemical analysis (D.L. Hay et al., unpublished
observations). MR567 was used at a dilution of 1 : 200 and
MR003 at a dilution of 1 : 500. Total protein was prepared in
the following manner; cells were harvested and centrifuged at
5006g for 5 min. The resulting pellets were incubated in cell
lysis bu€er (1% v v71 Triton X-100, 10% v v71 glycerol, (mM):
NaCl 150, NaF 50, HEPES 20, pH 7.5) containing protease
inhibitors, on ice for 30 min. The resulting cell lysate was
centrifuged at 10,000 r.p.m. in a bench-top centrifuge for
20 min at 48C. The supernatant was collected and the protein
concentration determined. The supernatants were subjected to
10% SDS ± PAGE, then transferred to PVDF membranes
using a wet transfer method (25 V, overnight, 48C). Non-
speci®c binding was blocked by incubating the PVDF
membrane in phosphate-bu€ered saline (PBS) containing 5%
non-fat milk for 1 h. Membranes were then incubated at room
temperature for 1 h with primary antibody in PBS containing
0.25% non-fat milk and 0.02% Tween 20 and then with a
horseradish peroxidase-linked secondary antibody (in PBS,
5% milk, 0.02% Tween 20) for 1 h at room temperature.
Following each incubation with antibody, the membrane was
washed four times (at 15 min intervals) with PBS containing
0.02% Tween 20. Bound antibodies were detected using the
ECL Plus Western blotting detection kit and autoradiography
was performed with Hyper®lm ECL.
Assay of cAMP production
Cells were grown to con¯uence and prior to experimentation
were incubated with serum free medium for 2 h 30 min to
minimize unstimulated cAMP levels. Cells were then
incubated in medium containing isobutylmethylxanthine
(IBMX) 0.5 mM for 30 min and all media thereafter also
contained IBMX. For agonist studies, cells were incubated
with the required agonist for 15 min and cAMP extracted in
ethanol for 1 h at 48C. Cyclic AMP was then measured by
radio-immunoassay using Amersham SPA kits (Amersham
Pharmacia Biotech) or NEN ¯ashplate kits (NEN life Science
Products, Hounslow, Middlesex, U.K.) (Abbott et al., 2000).
In experiments where the antagonist CGRP8-37 was used,
following serum starvation (as above) cells were incubated
with antagonist for 5 min prior to addition of agonist (plus
antagonist) for 15 min. Cyclic AMP was then extracted and
measured as above. Concentration e€ect curves were
analysed using Prism 3 (GraphPad Software, Inc., San Diego,
CA, U.S.A.). From these, dose-ratios were calculated (using
the curves obtained in the presence of 30, 100 and 300 nM
antagonist). These were used to produce a Schild plot; the
slopes were calculated by linear regression and found not to
be di€erent from unity. They were then constrained to 1 to
calculate pKb values.
Northern blot analysis
Statistical analysis
Total RNA was prepared and analysed on formaldehyde
agarose gels as previously described (Sharma et al., 1992).
The rat probes have also been described in a previous
publication (Chakravarty et al., 2000). For human probes,
full length myc-tagged CRLR (1467 bp), and full length
RAMP1 (501 bp), RAMP2 (588 bp) and RAMP3 (508 bp)
cDNAs were obtained as a gift from Dr S.M. Foord, (Glaxo
Wellcome Research and Development, Stevenage, Herts,
U.K. (McLatchie et al., 1998)). Northern analysis was
performed as described previously (Chakravarty et al.,
2000). Brie¯y, the insert of each clone was labelled using
random primer synthesis (32P incorporation eciency
Statistical analysis was undertaken using the Students t-test
(two-tailed).
Results
Analysis of receptor type in SK-N-MC, Rat-2, L6 and
Col-29 cells
Since we have found that receptor expression in cell lines can
vary, it was essential to verify the receptor types present in
British Journal of Pharmacology vol 136 (5)

T. Choksi et al
CRLR, RAMPs and receptor binding in cell lines
787
the exact cells used in this study thus allowing accurate
comparisons of pharmacology, mRNA and protein expres-
sion to be made.
agonist treated wells (not shown). [Cys(Et)2,7]a-CGRP was a
more potent agonist in both cell types but there was only a 2
fold di€erence between the two cell lines with an EC50 of
67 nM in SK-N-MC cells and 152 nM in Col-29 cells (Figure
1d). The similarity of responses with these agonists also
suggests that the Col-29 cells used in this study have a CGRP1
rather than CGRP2 phenotype.
Analysis of receptor type by whole cell binding assay Whole
cell binding for 125I-[Tyr0]a-CGRP and 125I-adrenomedullin
was performed for all cell lines (Table 1). For most of the cell
lines, CGRP and adrenomedullin binding was as previously
reported. SK-N-MC cells had clear CGRP binding but no
adrenomedullin binding. Rat-2 cells bound adrenomedullin
but not CGRP. Col-29 cells showed CGRP binding but no
adrenomedullin binding. The amount of adrenomedullin
binding in L6-1 cells was similar to what we have previously
reported but CGRP binding in this study was less than
previously shown (Coppock et al., 1996). Similar results were
obtained with L6-2 cells. There was a considerable di€erence
in the non-speci®c binding of the radioligands between these
two populations of L6 cells; the reason for this is unknown.
Detection of CRLR and RAMP mRNA
Total RNA was extracted from all cells in parallel with the
receptor characterization for that cell type to ensure that the
binding or cAMP results observed could be correlated with
the mRNA data. The RNA was analysed on four parallel
Northern blots and probed with CRLR, RAMP1, RAMP2
and RAMP3 cDNA probes. The signals were normalized by
probing successfully with GAPDH, b-actin and oligo dT45 as
controls for RNA loading. The ®gures for the target RNAs
have been normalized using the mean of these three controls
(Table 2). Rat lung total RNA was used as a positive control
on all blots as this tissue expresses CRLR, RAMP1 and
RAMP2 and both adrenomedullin and CGRP binding
(Chakravarty et al., 2000).
SK-N-MC cells at passage 5 showed the highest levels of
CRLR and RAMP1 mRNA and low levels of RAMP2
(Figure 2 and Table 2). Note the characteristic multiple bands
seen in human CRLR mRNA compared with the single band
seen in rat CRLR mRNA (Chakravarty et al., 2000) (Figures
2 and 3). This may be due to either alternative promoters or
splicing of the mRNA but this needs further investigation.
Col-29 cells showed expression of CRLR and RAMP1
mRNA but only low levels of RAMP2 mRNA (Figure 2).
Rat-2 cells showed some CRLR mRNA expression, no
RAMP1 mRNA but an abundance of RAMP2 mRNA
(Figure 3, Table 2). In contrast to these three cell lines we
could not detect CRLR in L6-1 cells. However RAMP1 and
RAMP2 mRNA were present (Figure 3, Table 2). We chose
to investigate a second source of L6 cells for CRLR
expression to evaluate whether this apparent lack of CRLR
was speci®c to this batch of cells. These cells, designated L6-
2, showed a di€erent pattern of expression to L6-1 cells with
CRLR, RAMP1 and 2 all present at moderate levels (Figure
3, Table 2). We found no expression of RAMP3 mRNA in
any of the cell lines we examined in this study (results not
shown).
Analysis of receptor type by cAMP assay The EC50 values
for stimulation of cAMP by CGRP in SK-N-MC and Col-29
cells were 1.5 and 2.1 nM respectively (Figure 1a,b). Maximal
cAMP stimulation achieved by CGRP in these cell lines was
42.8+1.0 and 8.6+0.3 pmol per well for SK-N-MC and Col-
29 cells respectively. This approximately 5 fold di€erence
between the cell lines correlates with the density of CGRP
receptors (Table 1). Analysis of inhibition of CGRP
stimulated cAMP production by CGRP8-37 showed similar
characteristics for SK-N-MC and Col-29 cells (Figure 1a,b).
When these data were used to calculate a Schild plot (Figure
1c), the slopes of the lines were not signi®cantly di€erent from
unity (SK-N-MC, 1.01+0.06; Col 29, 1.27+0.03). When the
lines were constrained to 1, the resulting pKb values were not
signi®cantly di€erent (SK-N-MC, 8.14+0.02; Col 29,
8.19+0.08). Since we would expect the pKb value for
CGRP8-37 in Col-29 cells to be at least one log unit lower
than in SK-N-MC cells (Poyner et al., 1998), in this
circumstance the CGRP receptor in these Col-29 cells could
not be con®rmed as CGRP2. This result is contrary to
published data (Cox & Tough, 1994) and indeed our own data
(Poyner et al., 1998) so we further investigated these cells using
the putative CGRP2 selective agonists [Cys(ACM)2,7]a-CGRP
and [Cys(Et)2,7]a-CGRP. [Cys(ACM)2,7]a-CGRP weakly sti-
mulated cAMP in both cell lines. In SK-N-MC cells, 100 nM
[Cys(ACM)2,7]a-CGRP produced weak but signi®cant cAMP
stimulation from
a
basal level of 1.41+0.18 to
5.91+0.96 pmol per well (P40.05, n=3) in agonist treated
wells (not shown). In Col-29 cells, 100 nM [Cys(ACM)2,7]a-
CGRP produced a non-signi®cant increase in cAMP from a
basal level of 2.47+0.35 to 2.94+0.55 pmol per well (n=3) in
Detection of CRLR protein
In order to con®rm the presence or absence of CRLR in our
cell lines we used speci®c human or rat anti-CRLR antibodies
Table 1 125I-CGRP and 125I-adrenomedullin binding in SK-N-MC, Col-29, L6-1, L6-2 and Rat-2 cell lines
125I-adrenomedullin 125I-CGRP
Total binding+s.e.mean
NSB+s.e.mean
Total binding+s.e.mean
NSB+s.e.mean
(fmols per 106 cells)
(fmols per 106 cells)
(fmols per 106 cells)
(fmols per 106 cells)
SK-N-MC
Col-29
L6-1
L6-2
Rat-2
2.3+0.3
2.6+0.2
29.7+0.8
35.0+2.0
10.8+0.5
2.7+0.2
2.2+0.2
5.6+0.3
21.0+1.0
3.6+1.2
5.2+0.3
1.5+0.1
2.4+0.1
3.5+0.3
0.13+0.03
1.3+0.1
0.7+0.03
0.9+0.03
2.6+0.3
0.13+0.01
NSB, non-speci®c binding. Results are expressed as s.e.mean of quadruplicate determinations.
British Journal of Pharmacology vol 136 (5)

788
T. Choksi et al
CRLR, RAMPs and receptor binding in cell lines
Figure 1 Stimulation of cAMP production in SK-N-MC (a) and Col-29 (b) cells by CGRP in the absence or presence of di€erent
CGRP8-37 concentrations. Cyclic AMP production is shown as pmol cAMP per well. Points represent the mean and s.e.mean of
three determinations. (c) Schild plot showing the e€ects of CGRP8-37 on SK-N-MC and Col-29 cells. This illustrates the case where
the slope of the plot has not been constrained to unity. (d) E€ect of [Cys(Et)2,7]a-CGRP on cAMP production in SK-N-MC and
Col-29 cells. Cyclic AMP production is shown as a per cent of the maximal stimulation level in those cells. Points represent the
mean and s.e.mean of three determinations.
Table 2 Northern analysis of SK-N-MC (passage numbers
5 or 25), L6-1, L6-2 or Rat-2 cells
discrepancy in molecular weights relates to di€erences in
the preparation of the protein samples or is another example
of the inherent variability of the SK-N-MC cells. The rat
band at approximately 75 kDa corresponds well to the band
found in our SK-N-MC cells. Furthermore, the size of this
rat band agrees with published cross-linking data (Coppock
et al., 1996; 1999).
CRLR
(arbitrary units) (arbitrary units) (arbitrary units)
RAMP1
RAMP2
Cell type
SK-N-MC (5)
SK-N-MC (25)
L6-1
L6-2
Rat-2
Rat lung
2652
0
8260
1392
2956
318
34
1405
384
192
4792
916
6134
48238
0
963
250
22978
Effect of continuous passage on SK-N-MC binding,
cAMP signal and receptor mRNA level
Values are arbitrary PhosphorImager units after normal-
ization. Values of zero are indicative of a normalized mRNA
value equal to or less than background levels for the CRLR
probe. Total RNA (25 mg) was probed in all cases.
During this and other studies using SK-N-MC cells we
noticed that binding and cAMP activity in response to
agonist stimulation are lost as these cells undergo continuous
passage. The di€erence in CGRP binding and cAMP activity
in response to CGRP over a period of 20 passages is shown
in Figure 5a,b. At passage 25 SK-N-MC cells have negligible
CGRP binding compared to passage 5 cells (Figure 5a; the
di€erence in total binding between the two passages of cells
was signi®cant (P50.001) whereas there was no signi®cant
di€erence between non-speci®c binding). Figure 5b shows
dose response analysis of cAMP production in response to
CGRP in low and high passages of SK-N-MC cells. The
EC50 of cAMP production in low passage SK-N-MC cells
was 0.2 nM whereas in high passage cells the EC50 value was
in excess of 1 mM. Both populations of cells showed a
signi®cant elevation of cAMP (P50.05) when challenged
with 10 mM forskolin. mRNA was prepared from both
raised against the C-terminal portion of the receptor. In
contrast to our mRNA data we found that all of the cell
lines, including L6-1 cells were positive for CRLR protein
expression (Figure 4a,b). Whereas the human antibody
detected only a single band at approximately 80 kD, the rat
antibody detected multiple bands. In the initial characteriza-
tion of the human CRLR antibody on SK-N-MC cells a
single band of 59 kDa was detected (Hagner et al., 2001).
Subsequent studies in human tissues have shown that this
antibody predominantly detects a band of approximately
70 kDa (S. Hagner, unpublished observations), broadly in
line with our determination. It is possible that the
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CRLR, RAMPs and receptor binding in cell lines
789
Figure 2 Northern analysis of Col-29 and SK-N-MC cells showing
(from left to right) CRLR, RAMP1, and RAMP2 mRNA. Note that
human CRLR mRNA appears as multiple bands, as discussed in the
text. The blots were also probed with GAPDH and the resulting
autoradiogrphs are aligned under the original blots. The position of
the 18S rRNA band, which was used as a marker, is shown on each
blot.
Figure 4 Western blots of rat (a) or human (b) cell line total
protein. (a) Twenty mg of total protein analysed on a 10% SDS ±
PAGE gel was exposed to an anti-rat CRLR antibody. A band of
75 kDa, representing rat CRLR was detected in L6-1, L6-2 and Rat-
2 cells. (b) Total protein (20 mg) from human cell lysates was
analysed in the same way as for the rat cells. A single band of
80 kDa representing human CRLR was detected in SK-N-MC and
Col-29 cells. Molecular weight markers, shown on the left of the blots
are: b-galactosidase, 131 kDa; bovine serum albumin, 75 kDa and
carbonic anhydrase, 42 kDa.
Figure 3 Northern analysis of L6-2, L6-1 and Rat-2 cells showing
(from left to right) CRLR, RAMP1 and RAMP2 mRNA. These
blots were also probed with GAPDH and the resulting autoradio-
graphs are aligned under the original blots. The position of the 18S
rRNA band, which was used as a marker, is shown on each blot.
to determine whether CRLR and RAMPs are responsible for
generating CGRP and/or adrenomedullin binding sites in cell
lines commonly used as models for these receptors.
passages of cells as described above. We found that CRLR
mRNA was reduced to below our limits of detection between
passages 5 and 25 and that RAMP1 was reduced by 83.1%
(Figure 5c and Table 2). RAMP2 mRNA also decreased but
to a lesser extent (50%).
Although it is often considered that cultured cells represent
dependable systems for analysis of receptors there can be
considerable variability of apparently the same cell line
between laboratories. For example, in our hands HEK293
cells express calcitonin receptors (Han et al., 1997) but in
others this is not the case (Findlay et al., 1994). In the
RAMP ®eld, the background of a particular cell type can
contribute greatly to the ®nal observed receptor phenotype
following transfection and this background can vary between
batches of cells (Tilakaratne et al., 2000). As shown clearly
with SK-N-MC cells in this study, receptors can also be lost
or gained over time (Sulistiyani & St Clair, 1991; Makino et
al., 2001). Hence, we assessed the receptor type and extracted
RNA and protein from the same cells, on the same day. Our
data cannot be used to make general assumptions on the
characteristics of a cell line, only on these particular batches
of cells at de®ned passage.
Here we show that, in accordance with the proposed
hypothesis, CGRP1 receptor binding sites in SK-N-MC cells
are likely to be formed by CRLR and RAMP1. Low levels of
RAMP2 in these cells and higher levels of RAMP1 are
associated with no adrenomedullin binding but good CGRP
binding. This is also supported by RAMP1 being dominant
in binding phenotype (Buhlmann et al., 1999). Interestingly,
McLatchie et al. (1998) found equivalent levels of RAMP1
and 2 in SK-N-MC cells, another illustration of the intrinsic
variability of cell lines. In our study CRLR mRNA is
abundant and CRLR protein is present. To add extra weight
Discussion
There is increasing evidence to support the original
hypothesis of Foord and co-workers that CRLR with
RAMP1 forms a CGRP1 receptor and CRLR with RAMP2
or RAMP3 forms an adrenomedullin preferring receptor in
transfected cell lines (McLatchie et al., 1998; Mu€ et al.,
1998; Kamitani et al., 1999; Chakravarty et al., 2000).
Speci®c antibodies to CRLR and RAMPs and mRNA
studies are now being used to evaluate the role of these
receptor components in tissues and cell lines (Hagner et al.,
2001; Nikitenko et al., 2001; Oliver et al., 2001; Ueda et al.,
2001). Recently, we presented evidence showing a strong
correlation between CRLR and RAMP1 mRNA expression
and CGRP binding in a selection of rat tissues (Chakravarty
et al., 2000). However, the association of CRLR with
RAMP2 or 3 and adrenomedullin binding was less convin-
cing (Chakravarty et al., 2000). A potential explanation of
these results is that a proportion of adrenomedullin binding is
not associated with CRLR/RAMP2 or 3. Receptor hetero-
geneity within tissues also adds complication. In this study we
have used homogenous cell lines to circumvent this issue and
British Journal of Pharmacology vol 136 (5)

790
T. Choksi et al
CRLR, RAMPs and receptor binding in cell lines
Figure 5 The e€ect of passage number on 125I-CGRP binding (a), cAMP stimulation (b) and CRLR, RAMP1 and RAMP2
mRNA levels (c) in SK-N-MC cells. (a) Total and non-speci®c binding (NSB) were measured in the cells as described in Methods.
Passage number is shown in parentheses. Each bar represents the mean and s.e.mean of quadruplicate determinations. (b) Cyclic
stimulation by CGRP in SK-N-MC cells at passage 5 or 25 expressed as a percentage of the response to 10 mM forskolin (assay
points represent the mean of 2 to 3 separate, duplicate experiments, vertical lines are s.e.mean). The cAMP production in response
to forskolin was 30 pmol per well (passage 5) and 6.5 pmol per well (passage 25). (c) Northern analysis of low passage (5) or high
passage (25) SK-N-MC cells showing (from left to right) CRLR, RAMP1 and RAMP2 mRNA. These blots were also probed with
GAPDH and the resulting autoradiographs are aligned under the original blots. The position of the 18S rRNA band, which was
used as a marker, is shown on each blot.
to this ®nding, over 20 passages, CRLR mRNA is reduced to
below our detection limit in accordance with a dramatic loss
of RAMP1 mRNA and a large reduction of CGRP binding
and cAMP production.
manner to SK-N-MC cells and appear to express similar
molecular components. Given that the cells can express
CRLR and RAMPs, it is possible that an extra component
may be responsible for modulating the CGRP8-37 anity to
give a CGRP1 or CGRP2-like receptor. If this is the case, the
expression of this unknown factor appears to be erratic.
Rat-2 ®broblasts bound adrenomedullin but not CGRP, as
we have previously reported (Coppock et al., 1999). This
correlated well with RAMP2 mRNA being present and
RAMP1 mRNA being absent. CRLR mRNA levels were
very low but an anti-rat CRLR antibody detected CRLR
protein.
Despite the identical pharmacology of L6-1 and L6-2 cells
(both CGRP and adrenomedullin receptors present), CRLR
and RAMP mRNA composition between these cells was
heterogenous. Whereas L6-2 cells had moderate levels of
CRLR, RAMP1 and RAMP2 mRNA, we could not detect
CRLR mRNA in L6-1 cells. RAMP1 and RAMP2 mRNA
levels were much higher in L6-1 than in L6-2 cells. It is likely
that CRLR mRNA in L6-1 cells was below our detection
limit since CRLR was detected with the anti-rat CRLR
antibody. The stability of the CRLR protein is not known
and it is possible that a very small amount of CRLR mRNA
gives rise to a very long half-life protein. However, the
presence of both RAMP1 and RAMP2 mRNA corresponds
The behaviour of the Col-29 cells in this study stands in
contrast to the CGRP2-like phenotype that we and others
have previously found (Poyner et al., 1998; Cox & Tough,
1994). Perhaps signi®cantly there were di€erences between the
behaviour of the cells in the hands of Cox and Tough, who
found that 3 mM CGRP8-37 had no e€ect on the response to
CGRP and ourselves, who found that this concentration was
able to antagonize CGRP. Subsequent to the study presented
here we have examined the pharmacology of another batch of
these cells. These showed a signi®cant di€erence in anity for
CGRP8-37 compared to SK-N-MC cells (pKb values;
7.34+0.19, n=7 versus 8.35+0.18, n=6; Hay et al.,
unpublished observations), but the anity for CGRP8-37
was clearly high. Unfortunately it was not possible to
examine expression of CRLR or RAMPs in these cells. All
clones of the Col-29 cells have originated from the same
source but it seems that their phenotype with regard to
CGRP pharmacology is particularly unstable. We have not
been able to ®nd any explanation for this behaviour nor have
we succeeded in controlling it. It is clear that in the present
study the cells are behaving pharmacologically in an identical
British Journal of Pharmacology vol 136 (5)

T. Choksi et al
CRLR, RAMPs and receptor binding in cell lines
791
well with adrenomedullin and CGRP binding in both sources
of L6 cells.
and protein data for CRLR in the L6-1 cells. This is likely to
be due to very low levels of CRLR mRNA in these cells
being below the detection limits of our system. Thus,
circumstances where mRNA for CRLR is apparently absent
by Northern analysis do not exclude this protein as the
binding site for CGRP and/or adrenomedullin.
We did not detect RAMP3 mRNA in any cell line in this
study. Either RAMP3 was not present or its levels were
below our detection limits. Indeed we ®nd RAMP3 mRNA
to be expressed at very low levels in rat tissues (Chakravarty
et al., 2000). At this time the precise function of RAMP3 is
not known. It may be more important in forming amylin
receptors with the calcitonin receptor (Tilakaratne et al.,
2000; Mu€ et al., 1999) than adrenomedullin receptors in vivo
or may have entirely di€erent functions.
We thank the British Heart Foundation (Project grant PG/97091),
the Wellcome Trust and P®zer Ltd (Dr Claire Bihoreau, P®zer
Global Research and Development, Fresnes Laboratories, Fresnes,
France) for supporting this work. Thanks also to Sue Kirkland for
advice on the culture of Col-29 cells. D.L Hay is funded by a
Medical Research Council studentship.
In conclusion, these results are in accordance with the
CRLR/RAMP hypothesis proposed by Foord and co-work-
ers. However there is an apparent mismatch between mRNA
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(Received January 3, 2002
Revised March 25, 2002
Accepted April 15, 2002)
British Journal of Pharmacology vol 136 (5)