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
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