(Received for publication, February 20, 1996)
From the
Calcitonin gene-related peptide (CGRP) is a neuropeptide with
diverse biological effects including potent vasodilator activity. We
report here the cloning of a complementary DNA (cDNA) encoding a human
CGRP receptor, which shares significant peptide sequence
homology with the human calcitonin receptor, a member of the
G-protein-coupled receptor superfamily. Northern blot analysis revealed
that the messenger RNA for this receptor is predominantly expressed in
the lung and heart. In situ studies showed specific
localization of the receptor mRNA to alveolar cells in the lung and to
cardiac myocytes in the heart. Stable expression of the cDNA in human
embryonic kidney 293 (HEK 293) cells produced specific, high affinity
binding sites for CGRP that displayed pharmacological and functional
properties very similar to native human CGRP
receptor.
Exposure of these cells to CGRP resulted in a 60-fold increase in cAMP
production, which was inhibited in a competitive manner by the
CGRP
receptor antagonist, CGRP-(8-37).
Calcitonin gene-related peptide (CGRP) ()is 37-amino
acid peptide that exists as highly homologous
or
isoforms
in both human and rat (1, 2) .
- and
-CGRP
display very similar biological activities, including peripheral and
cerebral vasodilation(3) , cardiac acceleration(4) ,
regulation of calcium metabolism(5) , reduction of intestinal
motility(6) , regulation of glucose metabolism (reduction of
insulin secretion and insulin sensitivity)(7) , diminution of
appetite (8) , and reduction of growth hormone
release(9) . The two CGRP peptides differ by 3 amino acids in
humans and 1 amino acid in rats. The amino acid sequences of CGRP
peptides are well conserved among species and can be considered as
members of a family of peptides including the related peptides amylin
(46% homology), salmon calcitonin (32% homology), and adrenomedullin
(24% homology). These peptides in general have N-terminal ring
structures of 6-7 amino acids involving a disulfide bridge and an
amidated C-terminal end(10, 11, 12) .
CGRP
peptides are localized predominantly in sensory afferent nerves and
central neurons(11, 12) . When released from the cell,
the peptides initiate their biological responses by binding to specific
cell surface receptors which are predominantly coupled to the
activation of adenylyl cyclase(3, 13) . CGRP receptors
have been identified and pharmacologically evaluated in several
tissues, including brain, cardiovascular, endothelial, and smooth
muscle tissues(12) . Multiple CGRP receptors have been
observed: based on pharmacological properties they are divided into at
least two subtypes and denoted as CGRP and
CGRP
, according to the classification of Dennis et
al.(14) . CGRP-(8-37), which lacks 7 N-terminal
amino acid residues, is a selective antagonist of CGRP
receptors, whereas the linear analog of CGRP, diacetoamidomethyl
cysteine CGRP (Cys(ACM2,7)CGRP), is a selective agonist of CGRP
receptors(14) .
A deeper understanding of the
physiological and pathophysiological effects mediated by the CGRP
peptides has been constrained for lack of a cloned cognate receptor.
Here we describe the isolation by expressed sequence tag (EST) analysis (15, 16, 17) of a human CGRP receptor that
exhibits ligand binding and functional properties of the CGRP receptor. Curiously, this receptor had been cloned previously as
an orphan calcitonin-like receptor receptor (rat (18) and,
recently, human(19) ), but the authors had been unable to
demonstrate an interaction with CGRP or any other ligands.
We hypothesized that the CGRP and recently cloned calcitonin
(CT) receptors might display close homology since (i) like the
G-protein-coupled CT receptor(26) , CGRP signaling appears to
be mediated through the activation of adenylyl cyclase, (ii) at high
concentrations CT interacts with the CGRP binding site(27) ,
and (iii) CGRP (the , not
, isopeptide) and CT share the same
mRNA transcript and are produced through alternate
splicing(1) , as are the substance P/K ligands that
cross-interact with tachykinin receptors (28) . Thus, when
expressed sequence tag (EST) analysis (15, 16, 17) of cDNA clones derived from a
human synovial tissue cDNA library identified a clone demonstrating
significant homology to the human CT receptor, this cDNA was selected
for further evaluation. Since the cDNA insert was incomplete (i.e. about 800 bp), it was used as a probe to screen a human lung cDNA
library to isolate a clone with a complete open reading frame.
Fig. 1shows the 2995-bp nucleotide sequence and deduced protein of the cloned cDNA. Three potential in-frame ATG codons precede the open reading frame of the protein; however, translation from the second or third ATG codon will encode a protein with a size consistent with that predicted for the cloned rat CT-like ``orphan'' receptor, with which it shares 91% amino acid sequence homology and thus likely represents a receptor ortholog(18) . Also, since the third ATG most closely approximates a Kozak consensus translation initiation site(29) , it is probably the translation initiation codon. Consequently, the cDNA encodes a protein of 461 amino acids, sharing several features in common with the G-protein-linked receptors (30, 31) . Most prominent is the existence of seven hydrophobic regions of 16 to 28 amino acids each, which are likely to be membrane-spanning domains that form a seven-transmembrane motif found among G-protein-coupled receptors. In addition, 52 amino acid residues highly conserved among a recently described subfamily of G-protein-coupled receptors including calcitonin (CT), secretin, parathyroid, glucagon, and other receptors(20, 26, 32, 33, 34) , are also present, interspersed within the sequence. Among this subfamily, the cloned protein shares its greatest sequence identity, 55.5%, with the human CT receptor. Furthermore, within the N-terminal domain there are several sites for post-translational modification including three asparagine residues within consensus sites for glycosylation and a potential cleavage site of an N-terminal hydrophobic sequence that may be a signal peptide.
Figure 1:
Nucleotide and deduced amino acid
sequence of the human CGRP receptor. Amino acids
(represented by the one-letter code) are indicated below their
respective codons and numbered 1 at the left beginning with
the ATG initiation methionine codon (M). Other numbers on the right indicate nucleotide positions starting at position 1 of
the A nucleotide of the ATG codon. Underlined amino acid
sequences, putative seven transmembrane domains; potential sites for
post-translational glycosylation are marked above the sequence by closed circle; circled amino acids are those that are
highly conserved among the secretin/CT/PTH/PTH-RP/GLP1/GHRH/GLU
G-protein-coupled receptor
subfamily(20, 26, 30, 31, 32) ; arrow, a putative secretory signal sequence cleavage site; double-underlined ATG codons, in-frame potential initiation
codons; underlined AATAAA sequence, consensus polyadenylation
signal.
Northern blot
analysis using the full-length cDNA as a hybridization probe revealed
mRNA species of approximately 7.5, 5.5, and 3.5 kb predominantly in the
lung and to a lesser degree in the heart (Fig. 2A). RNA
dot-blot analysis of over 20 different human tissues indicated
widespread, but generally low levels, of receptor mRNA expression (data
not shown). In situ analysis of mouse lung and heart tissues
demonstrated specific expression of the CGRP receptor mRNA
in alveolar cells in the lung and cardiac myocytes in the heart (Fig. 2B).
Figure 2:
Tissue expression of CGRP
receptor mRNA. A, Northern blot analysis of CGRP
mRNA expression. RNA size markers are indicated on the left.
B, in situ gene amplification analysis of CGRP
mRNA expression. The adjacent sections of mouse lung (a, b, c, d,
100) and heart (e, f, g, h,
400) were used for these
studies. a and e, use of the reverse transcriptase. b and f, omission of the reverse transcriptase. c and g, omission of amplification primers. d and h, hematoxylin and eosin staining.
To determine the binding and functional
properties of the receptor, we used HEK 293 cells stably transfected
with the cDNA subcloned within an expression vector. Membranes prepared
from untransfected 293 cells had very little specific I-CGRP binding (data not shown). Membranes prepared from
transfected 293 cells displayed high-affinity, low-density binding
sites for
I-CGRP. The apparent dissociation constant (K
) and maximum binding (B
)
were 19 ± 3 pM and 86 ± 14 fmol/mg protein,
respectively (Fig. 3A). In competition binding studies,
the rank order potency for a series of related peptides to inhibit
I-CGRP binding to these membranes was CGRP >
CGRP-(8-37) > Cys(ACM2,7)CGRP > adrenomedullin (ADM) >
salmon CT-(8-32) >>> salmon CT > human CT >
vasoactive intestinal peptide > angiotensin II (Fig. 3B). The binding affinity of
I-CGRP
for the recombinant receptor as well as the pharmacological profile of
the competing ligands were very similar to that observed for endogenous
CGRP receptors present in membranes prepared from human neuroblastoma
SK-N-MC cells (Fig. 3C).
Figure 3:
Pharmacological characterization of the
recombinant CGRP receptor stably expressed in 293 cells. A,
representative saturation curve for I-CGRP binding to the
membranes prepared from 293 cells expressing the recombinant CGRP
receptor.
, total binding;
, nonspecific binding; and
, specific binding. Inset, corresponding Scatchard plot
of the data. B, competition binding profile of CGRP (
)
and its analogs CGRP-(8-37) (
), Cys(ACM2,7)CGRP (
),
ADM (
), salmon CT (
), salmon CT-(8-32) (
),
human CT (
), angiotensin II (
), and vasoactive intestinal
peptide (
) for specific
I-CGRP binding to the
membranes. C, competition binding profile of CGRP and its
analogs for specific
I-CGRP binding to SK-N-MC cell
membranes.
Previous studies have shown
that CGRP mediates its responses by activation of adenylyl cyclase and
the generation of cyclic AMP(4, 5) . Treatment of
vector-transfected 293 cells with CGRP induced less than a 2-fold
increase in the accumulation of cAMP (Fig. 4A).
Addition of CGRP to 293 cells expressing the recombinant receptor
resulted in an increased accumulation of cAMP that was
concentration-dependent (Fig. 4B). The threshold,
half-maximal, and maximal concentrations of CGRP required to stimulate
cAMP accumulation in these cells were 0.1, 0.9, and 10 nM,
respectively. The maximal stimulation in response to agonist was
60-fold over basal levels of cAMP. Again, these results were comparable
to those achieved using SK-N-MC cells containing endogenous CGRP
receptors(35) . Treatment of cDNA-transfected 293 cells with
increasing concentrations of CGRP-(8-37), a selective CGRP
receptor antagonist, shifted the CGRP concentration response curve for
cAMP accumulation to the right in a parallel manner, indicating
competitive inhibition with a calculated pA value of 7.57 (Fig. 4C). Except at high concentrations (1
µM), the CGRP
receptor-selective agonist
Cys(ACM2,7)CGRP failed to stimulate cAMP in both receptor-transfected
cells (Fig. 4B) and SK-N-MC cells (data not shown). The
effect of Cys(ACM2,7)CGRP on the recombinant CGRP receptor in
inhibiting only
I CGRP binding but not a functional cAMP
response to CGRP is consistent with the properties of the native rat
CGRP
receptor present in spleen membranes(36) .
Thus, the receptor described in the present study is a CGRP
receptor.
Figure 4:
Functional characterization of the
recombinant CGRP receptor. A and B,
concentration-dependent effects of CGRP (), ADM (
), human
CT (
), and Cys(ACM2,7)CGRP (
) on cAMP accumulation in CDN
vector-transfected 293 cells (A) and CGRP receptor-transfected
293 cells (B). C, effect of CGRP-(8-37) on
CGRP-mediated cAMP response in the absence or presence of 100 or 300
nM CGRP-(8-37). The antagonist effect was studied by
performing CGRP concentration response curves in the absence (
)
and presence of 100 (
) and 300 (
) nM CGRP-(8-37).
At high concentrations, ADM evoked a specific
response (Fig. 4B), which was not surprising since this
agonist has been reported to interact weakly with the CGRP
receptor(37) . In contrast, human CT does not interact with the
recombinant receptor, since this ligand stimulated a nearly identical
activity in both CGRP receptor- and vector control-transfected cells (Fig. 4, A and B). Collectively, the
pharmacological and functional results confirm that the receptor cDNA
encodes a human CGRP receptor that is functionally coupled
to the activation of adenylyl cyclase.
Although originally
identified as an orphan rat CT-like receptor (18) and, very
recently, classified as an orphan human CT-like receptor (despite
testing for interaction with CGRP in addition to other ligands (19) ), the results presented within this report clearly
demonstrate that we have cloned a CGRP receptor. As to the
reason why previous investigators were unable to identify the reported
CT-like receptor as the CGRP
receptor is only a matter of
speculation. It is possible that the recombinant CGRP receptor can be
functionally expressed only in certain cell types. In this study we
used HEK 293 cells, whereas in other reports it appears that only COS
and OK cells were used for receptor evaluation. We have recently cloned
the porcine CGRP
receptor ortholog of the human receptor
and found it to exhibit essentially identical pharmacological
properties, which provides further supporting evidence that we have
identified the CGRP
receptor. (
)The availability
of the CGRP
receptor should facilitate the study of the
physiology and pathophysiology of CGRP as a neurotransmitter,
neuromodulator, local hormone, and inflammatory mediator.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L76380[GenBank].