Central effects of calcitonin receptor-stimulating
peptide-1 on energy homeostasis in rats
The CT-R [calcitonin (CT)
receptor] is expressed in the central nervous system and is
involved in the regulation of food intake, thermogenesis, and
behaviors. CT-R-stimulating peptide-1 (CRSP-1), a potent ligand
for the CT-R, was recently isolated from the porcine brain.
In this study, we first confirmed that porcine CRSP-1 (pCRSP-1)
enhanced the cAMP production in COS-7 cells expressing recombinant
rat CT-R, and then we examined the central effects of pCRSP-1
on feeding and energy homeostasis in rats. Intracerebroventricular
(icv) administration of pCRSP-1 to free-feeding rats suppressed
food intake in a dose-dependent manner. Chronic icv infusion
of pCRSP-1 suppressed body weight gain over the infusion period.
Furthermore, icv administration of pCRSP-1 increased body temperature
and decreased locomotor activity. The central effects of pCRSP-1
were more potent than those of porcine CT in rats. In contrast,
ip administration of pCRSP-1 did not elicit any anorectic or
catabolic effects. Administration icv of pCRSP-1 also induced
mild dyskinesia of the lower extremities and decreased gastric
acid output. Fos expression induced by icv administration of
pCRSP-1 was detected in the neurons of the paraventricular nucleus,
dorsomedial hypothalamic nucleus, arcuate nucleus, locus coeruleus,
and nucleus of solitary tract, areas that are known to regulate
feeding and energy homeostasis. Administration icv of pCRSP-1
increased plasma concentrations of ACTH and corticosterone,
implying that the hypothalamic-pituitary-adrenocortical axis
might be involved in catabolic effects of pCRSP-1. These results
suggest that CRSP-1 can function as a ligand for the CT-R and
may act as a catabolic signaling molecule in the central nervous
system.
Sawada H, et al. Endocrinology. 2006 Apr;147(4):2043-50.
Epub 2006 Jan 12.
Structure and biological properties of
three calcitonin receptor-stimulating peptides, novel members
of the calcitonin gene-related peptide family
In this review, we describe the structure
and biological properties of calcitonin receptor-stimulating peptide-1
(CRSP-1), CRSP-2 and CRSP-3, the novel members of the CGRP family.
CRSP-1, which has been identified in the pig, cow, dog, and horse,
is a specific ligand for the calcitonin (CT) receptor, and porcine
CRSP-1 elicits a 100-fold greater effect on a recombinant porcine
CT receptor than porcine CT, although this peptide has high structural
similarity with CGRP. CRSP-1 is expressed and synthesized mainly
in the central nervous system (CNS), pituitary and thyroid gland.
In an in vivo experiment, bolus administration of CRSP-1 into
rats reduced the plasma calcium concentration, but did not alter
blood pressure, indicating its action as a CT receptor agonist
in the peripheral circulation. In the CNS, CRSP-1 is also deduced
to be an endogenous agonist for the CT receptor. CRSP-2 has been
identified in the pig and dog, and CRSP-3 has been identified
only in the pig. They are expressed and synthesized mainly in
the CNS and thyroid gland. However, their endogenous molecular
forms, receptors, and biological activity remain unidentified.
Katafuchi T, Minamino N. Peptides. 2004 Nov;25(11):2039-45
Identification of second and third calcitonin receptor-stimulating
peptides in porcine brain
We identified two cDNAs encoding new
calcitonin receptor-stimulating peptides (CRSPs) in porcine hypothalamus
cDNA library by cross-hybridization with the CRSP cDNA, and designated
the second and third peptides as CRSP-2 and CRSP-3. The putative
amino acid sequences of prepro-CRSP-2 and prepro-CRSP-3 showed
higher identity with that of prepro-CRSP-1 than that of prepro-calcitonin
gene-related peptide (CGRP), respectively, and these three CRSPs
are considered to form a new family in the CGRP superfamily. RT-PCR
analysis demonstrated that both CRSP-2 and CRSP-3 gene transcripts
were expressed mainly in the central nervous system and thyroid
gland. Synthetic CRSP-2 and CRSP-3 stimulated cAMP production
very weakly in LLC-PK(1) cells compared with CRSP and calcitonin
(CT). Furthermore, CRSP-2 and CRSP-3 did not elicit a cAMP elevation
at all in the COS-7 cells expressing CT receptor or CT-like receptor
with or without one of receptor activity-modifying proteins. These
results suggest the presence of still unidentified action mechanisms
and functions of the peptides in the CGRP superfamily.
Katafuchi T,
Hamano K, Kikumoto K, Minamino N. Biochem Biophys Res Commun.
2003 Aug 29;308(3):445-51.
Calcitonin Receptor-Stimulating Peptide (CRSP), a new member of
the calcitonin gene-related peptide family: Its isolation from
porcine brain, structure, tissue distribution and biological activity
We isolated a novel biologically active peptide, designated Calcitonin
Receptor-Stimulating Peptide (CRSP), from the acid extract of
the porcine brain by monitoring cAMP production in the porcine
kidney cell line LLC-PK1. Determination of the amino acid sequence
and cDNA analysis encoding a CRSP precursor showed that this peptide
has approximately 60% identity in the amino acid sequence with
human calcitonin gene-related peptide type-a (aCGRP ), type-b
(bCGRP ), and porcine CGRP. Northern blot analysis and radioimmunoassay
demonstrated that CRSP is expressed mainly in the thyroid gland
and the central nervous system, in which the calcitonin receptor
was abundantly expressed. Synthetic CRSP elicited a potent stimulatory
effect on the cAMP production in LLC-PK1 cells. Although it shows
significant sequence similarity with CGRPs, this peptide did not
elicit cAMP elevation in cells that were endogenously expressing
a CGRP receptor or an adrenomedullin receptor, or were transfected
with either of these recombinant receptors. Administration of
CRSP into anesthetized rats did not alter the blood pressure,
but induced a transient decrease in the plasma calcium concentration.
In fact, this peptide potently increased the intracellular cAMP
concentration in COS-7 cells that expressed the recombinant calcitonin
receptor. These unique properties indicate that CRSP is not a
porcine counterpart of bCGRP and probably elicits its biological
effects via the calcitonin receptor. Katafuchi
T, et al. J. Biol. Chem., 278,12046-12054 ( 2003)
Novel calcitonin-(8-32)-sensitive adrenomedullin receptors
derived from co-expression of calcitonin receptor with receptor
activity-modifying proteins
We tested whether heterodimers comprised of calcitonin (CT) receptor
lacking the 16-amino acid insert in intracellular domain 1 (CTR(I1-))
and receptor activity-modifying protein (RAMP) can function not
only as calcitonin gene-related peptide (CGRP) receptors but also
as adrenomedullin (AM) receptors. Whether transfected alone or
together with RAMP, human (h)CTR(I1-) appeared mainly at the surface
of HEK-293 cells. Expression of CTR(I1-) alone led to significant
increases in cAMP in response to hCGRP or hAM, though both peptides
remained about 100-fold less potent than hCT. However, the apparent
potency of AM, like that of CGRP, approached that of CT when CTR(I1-)
was co-expressed with RAMP. CGRP- or AM-evoked cAMP production
was strongly inhibited by salmon CT-(8-32), a selective amylin
receptor antagonist, but not by hCGRP-(8-37) or hAM-(22-52), antagonists
of CGRP and AM receptors, respectively. Moreover, the inhibitory
effects of CT-(8-32) were much stronger in cells co-expressing
CTR(I1-) and RAMP than in cells expressing CTR(I1-) alone. Co-expression
of CTR(I1-) with RAMP thus appears to produce functional CT-(8-32)-sensitive
AM receptors. Kuwasako K, et al. Biochem
Biophys Res Commun., 301(2):460-4 (2003)
Identification, structural determination,
and biological activity of bovine and canine calcitonin receptor-stimulating
peptides
We have recently identified in porcine brain a
series of new peptides, designated calcitonin receptor-stimulating
peptide-1 (CRSP-1), CRSP-2, and CRSP-3, but failed to find their
counterparts in humans and rodents by either database searching
or experimental cross-hybridization. In this study, we isolated
cDNAs encoding precursors of bovine CRSP-1, canine CRSP-1, and
canine CRSP-2 from their thyroid cDNA libraries. Although the
deduced mature amino acid sequences of bovine and canine CRSP-1s
and canine CRSP-2 showed identity with their respective porcine
CRSP counterparts, none of them had a C-terminal amide structure.
In LLC-PK(1) cells endogenously expressing the calcitonin (CT)
receptor, bovine and canine CRSP-1s enhanced the cAMP production,
while canine CRSP-2 did not stimulate it at all. Equine CGRP-I
had a high identity in its amino acid sequence with porcine CRSP-1
and stimulated LLC-PK(1) cells at a potency comparable to that
of porcine CT. None of these CRSPs or equine CGRP-I stimulated
the CT-like receptor, even in the presence of receptor activity-modifying
proteins. These results demonstrate that CRSP-1, a new class of
biologically active peptide, is present in animals evolutionarily
close to pigs and induces its activity through the calcitonin
receptor, suggesting a wide existence and common properties of
this peptide in mammals.
Katafuchi T, Hamano K, Minamino
N. Biochem Biophys Res Commun. 2004 Jan 2;313(1):74-9
Antibody
for Calcitonin Receptor-Stimulating Peptide-3 (CRSP-3)
Immunohistochemistry in rat brain
Effects of icv administration
of pCRSP-1 on food intake. A, Cumulative food intake
in
free-feeding rats in the dark phase (n = 12 per group)
was measured following icv
administration of saline, pCRSP-1 (0.3, 0.5, 1.0 nmol)
or pCT (1.0 nmol). B, Cumulative food intake in 14
h-fasted rats in the light phase (n = 8 per group)
was measured following icv administration of saline,
pCRSP-1 (0.3, 0.5, 1.0 nmol) or pCT (1.0 nmol) at
the light phase. Control rats were given 0.9% saline.
*, P < 0.05; **, P < 0.001 (vs. saline controls).
a, P < 0.05; b, P < 0.001 (vs. pCT). C, Effect
of ip administration of pCRSP-1 (10 nmol) in the dark
phase to rats (n = 6 per group). Control rats were
given 0.9% saline by ip injection. D, Conditioned
taste aversion test. Conditioned rats (n = 10 per
group) received icv administration of pCRSP-1 (0.5,
1.0 nmol) or saline and ip administration of LiCl
or saline. *, P < 0.05 (vs. ip saline controls).
E, Cumulative food intake in free-feeding rats in
the dark phase (n = 9 per group) was measured following
icv administration of saline or pCRSP-2 (1.0 nmol).
F, Cumulative food intake in free-feeding rats in
the dark phase (n = 8 per group) was measured following
icv administration of saline or pCRSP-3 (1.0 nmol).
Sawada H, et al. Endocrinology. 2006 Apr;147(4):2043-50.
Epub 2006 Jan 12.
Effects of chronic icv administration of pCRSP-1 on
rats. One-day food intake (A) and
cumulative body weight gain (B) were measured during
icv injection (2.0 nmol/day) for 7 days (n = 7 per group).
Each pair-fed rat was given the same amount of food
as the paired pCRSP-1-administered rat consumed on the
previous day. Control rats were given 0.9% saline. *,
P < 0.05; **, P < 0.001 (vs. saline controls).
a, P < 0.05 (vs. pair-fed). Sawada H, et al. Endocrinology.
2006 Apr;147(4):2043-50. Epub 2006 Jan 12.
Effects of icv administration
of pCRSP-1 on body temperature and locomotor activity.
A, Body temperature was measured from -30 min to 240
min after administration of pCRSP-1 (0.1, 1.0 nmol)
or pCT (0.1 nmol) (n = 6 per group). Control rats
were given 0.9% saline. *, P < 0.05; **, P <
0.001 (vs. saline controls). a, P < 0.05 (vs. pCT).
B, Body temperature was measured up to 240 min after
administration of pCRSP-1 (1.0 nmol), pCRSP-2 (5.0
nmol), or pCRSP-3 (5.0 nmol). Control rats were given
0.9% saline. **, P < 0.001 (vs. saline controls).
C, Locomotor activity was measured for 12 h after
administration of pCRSP-1 (0.3, 0.5, 1.0 nmol) or
pCT (1.0 nmol) (n = 5 per group). Control rats were
given 0.9% saline. *, P < 0.05; **, P < 0.001
(vs. saline controls). a, P < 0.05 (vs. pCT). Sawada
H, et al. Endocrinology. 2006 Apr;147(4):2043-50.
Epub 2006 Jan 12.
Plasma levels of ACTH 15 min
after the administration of pCRSP-1 (1.0 nmol) or
saline (n = 5 per group). B, Plasma levels of corticosterone
30 min after the administration of pCRSP-1 (1.0 nmol)
or saline (n = 5 per group). *, P < 0.05 (vs. saline
controls). Sawada H, et al. Endocrinology. 2006 Apr;147(4):2043-50.
Epub 2006 Jan 12.
Fig. 1. Nucleotide and deduced amino acid
sequences of bovine (A) and canine (B) CRSP-1, and alignment
of deduced mature peptides of bovine and canine CRSP-1
with equine CGRP-I, porcine CRSP-1, and porcine CGRP
(C). (A,B) Nucleotide and amino acid numbers are shown
on the right. Putative signal peptides are shown in
italics. The mature amino acid sequence of each CRSP-1
is boxed. (C) The deduced mature amino acid sequences
of bovine (bCRSP-1) and canine CRSP-1 (cCRSP-1) are
aligned with those of equine CGRP-I (eCGRP-I), porcine
CRSP-1 (pCRSP-1), and porcine CGRP (pCGRP). The amino
acids identical to porcine CRSP-1 are shaded. T.
Katafuchi et al. Biochemical and Biophysical Research
Communications 313 (2004) 74–79 75
Fig. 2. Nucleotide and deduced amino acid
sequences of canine CRSP-2 (A), and alignment of deduced
mature peptide of canine CRSP-2 with canine CRSP-1 and
CGRP, and porcine CRSP-2, CRSP-3, and CRSP-1 (B). (A)
Nucleotide and amino acid numbers are shown on the right.
Putative signal peptide is shown in italics. The mature
amino acid sequence of canine CRSP-2 is boxed. (B) The
deduced amino acid sequences of mature canine CRSP-2
(cCRSP-2) are aligned with those of canine CRSP-1 (cCRSP-1),
canine CGRP (cCGRP), porcine CRSP-2 (pCRSP-2), porcine
CRSP-3 (pCRSP-3), and porcine CRSP-1 (pCRSP-1). The
amino acids identical to canine CRSP-2 are shaded.
Fig. 3. Dose–response elevation of cAMP production
in the culture medium of LLC-PK1 cells. LLC-PK1 cells
were stimulated with the indicated concentrations of
bovine CRSP-1 (closed circle), canine CRSP-1 (closed
triangle), equine CGRP-I (open circle), porcine CRSP-
1 (open square), porcine CT (closed square), and canine
CRSP-2 (open triangle). Each point represents the meanSEM
of three separate determinations. T.
Katafuchi et al. / Biochemical and Biophysical Research
Communications 313 (2004) 74–79
Nucleotide sequences and deduced amino
acid sequences of CRSP-2 (A) and CRSP-3 (B) cDNA,
and alignments of amino-acid sequences of porcine
prepro-CRSPs and prepro-CGRP (C). (A, B) Nucleotide
and amino acid numbers are shown on the right. Putative
mature amino acid sequences of CRSP-2 and CRSP-3 are
boxed. The donor glycine of the C-terminal amide is
shaded and the termination codon is marked with an
asterisk. The signal sequences are shown in italics.
(C) The amino acid sequences of prepro-CRSP-2 and
CRSP-3 are compared with those of prepro-CRSP-1 and
prepro-CGRP. The deduced amino acid sequences for
mature peptides are shown by upper cases and prepro
peptides are indicated by lower cases. The putative
prohormone convertase cleavage sites are boxed. The
residues that are in common with and distinct from
CRSP-2 are shown by bold and gray characters, respectively.
Katafuchi T, Hamano K,
Kikumoto K, Minamino N. Biochem Biophys Res Commun.
2003 Aug 29;308(3):445-51
Measurement of mRNA levels of CRSP-1,
CRSP-2, CRSP-3, CGRP, CT, and GAPDH by RT-PCR. RT-PCR
analyses were performed as described in "Materials
and methods" with total RNA from each of the 19 organs
listed at the top. The PCR products of CRSP-1 (306 bp),
CRSP-2 (305 bp), CRSP-3 (329 bp), CGRP (640 bp),
CT (570 bp), and GAPDH (378 bp) were resolved
by electrophoresis in agarose gels, stained with ethidium
bromide, and visualized using an FLA-2000 fluorescent
image analyzer. The bottom panel shows the RT-PCR
amplification of GAPDH mRNA as proof of the integrity
of each RNA preparation. Katafuchi
T, Hamano K, Kikumoto K, Minamino N. Biochem Biophys
Res Commun. 2003 Aug 29;308(3):445-51
Effects of CRSPs and its related peptides
on the cAMP production in the LLC-PK1 cells.
LLC-PK1 cells were stimulated with the
indicated concentrations (0, 10-11–r10-6 M)
of porcine CRSP-1 (closed square), CRSP-2 (open circle),
CRSP-3 (closed circle), CT (open square), CGRP (open
triangle), and human AM (closed triangle). LLC-PK1
cells were harvested on a 48-well plate, cultured
for 24 h, and then used for the measurement of
cAMP production. Each point represents the means ±r SEM
of three separate determinations. Katafuchi
T, Hamano K, Kikumoto K, Minamino N. Biochem Biophys
Res Commun. 2003 Aug 29;308(3):445-51
Effects of CRSPs and its related peptides
on CT receptor or CL receptor in the absence or presence
of RAMPs. CT receptor (CTR) or CL receptor (CLR) cDNA
inserted into pcDNA was transfected into COS-7 cells
with one of RAMP1, RAMP2, and RAMP3 cDNA inserted
into pcDNA or insert-free pcDNA, which is indicated
at the top-right of each figure, using Lipofectamine
Plus reagent. The cells were then cultured for 24 h
and used for the experiments. The cells were stimulated
with 10-7 M of the indicated peptides.
Each point represents the means ±r SEM
of three separate determinations. Katafuchi
T, Hamano K, Kikumoto K, Minamino N. Biochem Biophys
Res Commun. 2003 Aug 29;308(3):445-51
