(Received for publication, August 22, 1995)
From the
Adrenomedullin is a potent vasodilator peptide that exerts major
effects on cardiovascular function. Its actions are mediated through an
abundant class of specific binding sites that activate adenylyl cyclase
through a G protein-coupled mechanism. We report here the
identification of a cDNA clone for the adrenomedullin receptor that was
originally isolated as an orphan receptor from rat lung. The cDNA
encodes a polypeptide of 395 residues that contains seven transmembrane
domains and has a general structural resemblance to other members of
the G protein-linked receptor superfamily. When expressed in COS-7
cells, this receptor mediates a cAMP response to adrenomedullin with an
EC of 7
10
M, and
binds
I-adrenomedullin with a K
of 8.2
10
M, properties
that are consistent with those observed in cardiovascular and other
target tissues. The receptor gene is expressed as several mRNA species
of which the most prominent is a 1.8-kilobase transcript found in the
lung, adrenal, heart, spleen, cerebellum, and other sites.
Identification of this receptor cDNA should facilitate further
investigation of the cellular actions of adrenomedullin and its
regulatory effects in normal and disordered states of cardiovascular
function.
The potent hypotensive peptide, adrenomedullin (AM), was originally isolated from a human adrenal phaeochromocytoma
using a detection system based on its ability to elevate platelet cAMP
levels (1) . Subsequent work revealed that this peptide is
produced by a wide variety of tissues, most notably the adrenal, lung,
kidney, and heart (2, 3, 4, 5) . It
is also synthesized and secreted in abundance by vascular endothelial
cells (6) and is detectable in the
circulation(1, 4, 5) . The principal
physiological action of AM appears to be as a potent vasodilator, and
its systemic administration causes a rapid and profound fall in blood
pressure and an increase in pulmonary blood flow (1, 7, 8) . Other actions as a
bronchodilator(9) , an inhibitor of drinking
behavior(10) , and an inhibitor of angiotensin-induced
aldosterone secretion(11) , have been described.
There has
been some difficulty in defining the receptor sites of AM, and several
studies have suggested that its actions are mediated through calcitonin
gene-related peptide (CGRP) receptors. There is modest sequence
homology between these peptides, in particular the presence of a six
residue intramolecular disulfide-linked ring structure that is also
present in islet amyloid polypeptide. CGRP has similar vasodilator
properties to AM, and this action of AM can be inhibited by the CGRP
antagonist CGRP[8-37](12) . However, while the
specific AM binding sites in rat vascular smooth muscle cells exhibit a K of 1.3
10
M, the K
for CGRP at these sites
(3
10
M) was significantly greater
than that for binding to the CGRP receptor(13) , suggesting
that specific receptors for AM do exist and may have low affinity for
CGRP. Identification of a cDNA clone for such an AM receptor (AM-R)
would enhance our understanding of the physiology of this peptide, and
facilitate the development of therapeutically useful drugs that could
stimulate or block these receptors. In this report we describe the
cloning and expression of a rat lung cDNA encoding a functional AM-R.
The DNA products derived from low stringency PCR with rat adrenal cDNA were excised from an agarose gel and ligated into pCR1000. After transformation, individual colonies were picked, plasmid DNA was prepared and subjected to DNA sequencing. Several of these contained inserts that bore no homology to the equivalent region of other seven transmembrane domain receptors. However one of these, denoted pAD6, contained an insert that appeared to encode a novel member of the seven transmembrane domain receptor family. When this was used to probe a Northern blot prepared from various rat tissues, high levels of expression were evident in the lung.
A gt11 rat lung cDNA
library was therefore screened with pAD6 and this led to the isolation
of two plaques, one of which contained a 1.8-kbp cDNA insert. This was
sequenced on both strands and was found to contain a single long open
reading frame of 1185 bp with a polyadenylation signal and a poly(A)
tail at its 3` end. The cDNA sequence and its predicted translation
product are shown in Fig. 1. The polypeptide chain is 395
residues long and has a molecular mass of 45,196 Da. This protein has
seven putative
-helical transmembrane domains, two predicted sites
of N-linked glycosylation in the N terminus, and four
potential protein kinase C phosphorylation sites: one in the first
cytoplasmic loop, one in the third cytoplasmic loop, and two in the
C-terminal tail (Fig. 1). Sequence alignment using the BLAST
program (20) indicates that this cDNA sequence is essentially
identical to that of an orphan G protein-linked receptor that has been
previously reported by two groups(21, 22) . The next
most closely related cDNA sequence is that of RDC1, an orphan
seven-transmembrane domain receptor cloned from the canine thyroid
gland(23) .
Figure 1:
DNA and deduced amino acid
sequences of the pGEM.L1 cDNA clone. The nucleotide sequence is 1719 bp
in length and contains a single long open reading frame of 1185 bp,
terminating in a TAG termination codon (underlined). Two
overlapping poly(A) addition signals are found at the 3` end of the
gene followed by a poly(A) tract (double underlined). The
amino acid sequence, denoted in the single-letter amino acid
code, includes seven predicted transmembrane domains, which are underlined. There are two putative sites of N-linked
glycosylation () and four consensus sites for phosphorylation by
protein kinase C (
) (see (21) and (22) ).
The full-length cDNA was used to probe Northern blots derived from a variety of rat tissues (Fig. 2). The predominant mRNA is approximately 1.8 kb in size, with lesser species of 2.2, 3.0, and 5.0 kb. The lung is clearly the most abundant site of this mRNA, followed by the adrenal, ovary, heart, spleen, cerebellum, and cerebral cortex. It was recognized that this distribution was similar to the distribution of AM binding sites recently reported by Owji et al.(24) ; therefore, the hypothesis that this cDNA encoded the rat AM-R was tested. Although there is recent evidence to suggest that AM activates both cAMP and calcium mobilization pathways(25) , studies in many different cell types have described a cAMP-stimulating action that can be abrogated by pretreatment with cholera toxin(26) . Therefore, cAMP responses to AM were measured after transfection of this cDNA clone into heterologous cells.
Figure 2:
Northern blot analysis of AM-R transcripts
in various rat tissues. The principal transcripts of 1.8, 2.2, 3.0, and
5.0 kb can be seen in several lanes. The tissues examined include
placenta (Pl), psoas muscle (Mu), ovary (O),
testis (T), uterus (U), heart (H), lung (Lu), spleen (S), kidney (K), liver (Lv), adrenal capsule (Ad C), whole adrenal (Ad), cerebral medulla (Md), cerebellum (Cb), pituitary (Pit), hypothalamus (Hy),
thalamus and hippocampus (Th), and cerebral cortex (CC). All lanes contained 3 µg of poly(A) RNA except for kidney, adrenal, and adrenal capsule, which
contained only 1 µg of poly(A)
RNA. The blot was
exposed for 48 h with image intensifying screens at -70
°C.
The full-length cDNA was ligated into the
expression vector pcDNA1, and this was used to transiently transfect
COS-7 cells cultured in six-well plates. 48 h after transfection, cells
were washed with serum-free medium and stimulated with selected
concentrations of rAM or CGRP. As shown in Fig. 3a,
there was a specific cAMP response to AM in transfected cells with a
threshold of around 10M, a peak response
at 10
M, and an EC
of 7
10
M. No response was found in sham
transfected cells or in cells stimulated with 10
M CGRP. CGRP[8-37] is an antagonist for CGRP
receptors, and evidence suggests that it also antagonizes the actions
of AM(13) . When used at various concentrations in the presence
of 10
M AM, this peptide appeared to have a
weak antagonistic effect with a K
of
10
M.
Figure 3:
Panel a, dose-response curve of cAMP
production in AM-R transfected COS-7 cells. cAMP is expressed per mg of
protein, and each point represents the mean and S.E. of six
estimations. , rat AM;
, rat CGRP. Panel b, ligand
binding of rat AM to AM-R expressed in COS-7 cells. The main figure
shows the displacement curve obtained with rat AM. Each point is the
mean of three estimations. Scatchard analysis (inset)
indicates a K
= 8.2 nM and a B
= 680 fmol/mg
protein.
Ligand binding studies lend
further support to the identification of this cDNA clone as the AM-R (Fig. 3b). The calculated K of
this receptor when expressed in COS-7 cells was 8.2
10
M, a value similar to that reported for
the wild type receptor in other studies (13, 24) .
The technique of low stringency consensus primer PCR has been
successfully used in the cloning of a number of G protein-coupled
receptors, including the adenosine A1 and A2 receptors (23, 27, 28) and the MC1 (-MSH) and ACTH
receptors(29) . Moreover, the gene that we have now identified
as the rAM-R has been cloned in this way and recognized as an orphan
receptor by three independent groups. We have previously tested a large
number of potential agonists for activation of this receptor without
success, and it was the recent description of the prevalence and
distribution of the AM-R that led to its identification as reported in
this paper. It is not yet clear whether the widespread tissue
distribution of AM-R transcripts is mainly a reflection of its vascular
role, or whether it has other non-vascular functions in these tissues.
The latter seems highly likely in view of recent reports proposing a
role for AM as a bronchodilator(9) , an inhibitor of drinking
behavior(10) , an aldosterone release inhibitory
factor(11) , and an ACTH inhibitory factor(30) .
The AM-R cDNA seems to be a relatively unique member of the G protein receptor superfamily, being most homologous (30%) to another orphan receptor known as RDC1. It remains to be seen whether this gene will define a new subfamily among the G protein-linked receptors.