| Identification
of relaxin-3/INSL7 as a ligand for Orphan G-protein Coupled Receptors
GPCR135 and GPCR142
ChangLu Liu, et al. 4th International Conference
on Relaxin and Related peptides. Sep. 5-10, 2004, Wyoming, USA
Identification of relaxin-3/INSL7 as a ligand for GPCR142
We have recently identified the insulin-like peptide
relaxin-3 (aka INSL7) as the endogenous ligand for an orphan G-protein-coupled
receptor, GPCR135 (aka somatostatin- and angiotensin-like peptide
receptor). Analysis of possible receptors related to GPCR135 revealed
a single orphan receptor, GPCR142. Thus, we tested whether GPCR142
could also respond to relaxin-3 or related insulin-like molecules.
Surprisingly, GPCR142 was activated by nanomolar concentrations
of relaxin-3 but was completely unresponsive to all other known
insulin-like peptides. We evaluated by reverse transcriptase-PCR
the expression of GPCR142 mRNA in a variety of human tissues and
found expression in brain, kidney, testis, thymus, placenta, prostate,
salivary gland, thyroid, and colon. In an analysis of other species,
we were able to find a full-length mouse homolog of GPCR142, but
were unable to detect any complete GPCR142 transcripts in rat. With
respect to intracellular signaling, GPCR142 is similar to GPCR135
in that it potently inhibits adenylate cyclase and stimulates 35S-GTPgammaS
incorporation in response to relaxin-3. However, whereas GPCR135
signaling could be converted to calcium mobilization using a Gqi5
or Galpha16 G-proteins, GPCR142 was only capable of functioning
in the presence of Galpha16. In the accompanying article (Liu, C.,
Eriste, E., Sutton, S., Chen, J., Roland, B., Kuei, C., Farmer,
N., Jornvall, H., Sillard, R., and Lovenberg, T. W. (2003) J. Biol.
Chem. 278, 50754-50764), we present the case that relaxin-3, which
has previously been shown to bind to the relaxin receptor LGR7,
is most likely the endogenous ligand for GPCR135. In this report,
we show an additional receptor, GPCR142, which is also selectively
activated by relaxin-3. However, the anatomical localization of
GPCR142 suggests that GPCR142 may have different physiological functions.
Liu C, et al. J Biol Chem. 2003 Dec 12;278(50):50765-70.
Epub 2003 Sep 30.
Physiological or pathological - a role for relaxin
in the cardiovascular system?
The omnipresent 6kDa polypeptide relaxin (RLX) is emerging as a
multi-functional endocrine and paracrine factor, with a broad range
of target tissues that includes the cardiovascular system. Humans
and other higher primates have three RLX genes, designated H1, H2
and H3, of which H2 RLX is the major stored and circulating form.
Rodents have only two RLX genes: relaxin-1 (equivalent to H2 RLX)
and relaxin-3 (equivalent to H3 RLX). The recent cloning of the
human RLX receptor (LGR7), a member of the leucine-rich repeat family
of G-protein-coupled orphan receptors, and detection of LGR7 gene
transcripts in the heart confirm this organ as a target for RLX
(H2). However, evidence for production of the ligand within the
cardiovascular system is limited, and few studies have clearly identified
the physiological effects of RLX on cardiac function. To add to
the controversy, serum concentrations and expression of RLX in the
heart are elevated in chronic heart failure patients and animal
models of cardiomyopathy, implying that RLX may only be a marker
for pathological cardiovascular conditions, rather than normal physiology.
Samuel CS, Parry LJ, Summers RJ. Curr Opin
Pharmacol 2003 Apr;3(2):152-8
| Anti-Relaxin-3
Receptor-2 /GPCR 142 (310-330) (Human) Serum works in rat
brain tissues |
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| Relaxin-3
Receptor-2 Immunohistochestry Protocol |
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| Relaxin-3 stimulates
35S-GTPS binding in GPCR142 expressing cells. Different peptides
with various concentrations were added to the human GPCR142
expressing cell membranes to stimulate GTPS incorporation.
The specific 35S-GTPS incorporation was obtained by subtracting
counts without ligand from the counts with ligand.
Liu C, et al. J. Biol. Chem., Vol.
278, 50765-50770, December 12, 2003 |
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A, inhibition of forskolin-stimulated
cAMP production by relaxin-3. Cells stably expressing GPCR142
were established in SK-N-MC cells. Different peptides with various
concentrations were added to the cell cultures. Forskolin was
then added to all samples at a final concentration of 5 µM.
Forskolin-stimulated cAMP accumulation was measured using cAMP
Flash Plates (PerkinElmer Life Sciences).
Liu C, et al. J. Biol. Chem., Vol. 278, 50765-50770, December
12, 2003 |
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Relaxin-3 stimulates Ca2+ mobilization
in HEK293 cells co-expressing GPCR142 and G16. HEK293 cells,
either transfected with G16 alone (G16), or co-transfected by
human GPCR142 and G16 (GPCR142/G16), were used for Ca2+ mobilization
assays using different concentrations of relaxin-3 as the ligand.
Ligand-stimulated intracellular Ca2+ mobilization was monitored
by FLIPR. HEK293 cells co-transfected by G16 and GPCR135 (GPCR135/G16)
were used in the same experiment for comparison.
Liu C, et al. J. Biol. Chem., Vol. 278,
50765-50770, December 12, 2003 |
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Reverse transcriptase-PCR detection of GPCR142
mRNA expression profiles in different human tissues. The PCR
products were run in 2% agarose gels, stained with ethidium
bromide, and visualized under UV irradiation.
Liu C, et al. J. Biol. Chem., Vol. 278,
50765-50770, December 12, 2003 |
Restricted, but abundant, expression of the
novel rat gene-3 (R3) relaxin in the dorsal tegmental region of
brain
Relaxin is a peptide hormone with known actions associated with
female reproductive physiology, but it has also been identified
in the brain. Only one relaxin gene had been characterized in rodents
until recently when a novel human relaxin gene, human gene-3 (H3)
and its mouse equivalent (M3) were identified. The current study
reports the identification of a rat homologue, rat gene-3 (R3) relaxin
that is highly expressed in a discrete region of the adult brain.
The full R3 relaxin cDNA was generated using RT-PCR and 3' and 5'
RACE protocols. The derived amino acid sequence of R3 relaxin retains
all the characteristic features of a relaxin peptide and has a high
degree of homology with H3 and M3 relaxin. The distribution of R3
relaxin mRNA in adult rat brain was determined and highly abundant
expression was only detected in neurons of the ventromedial dorsal
tegmental nucleus (vmDTg) in the pons, whereas all other brain areas
were unlabelled or contained much lower mRNA levels. Relaxin binding
sites and relaxin immunoreactivity were also detected in the vmDTg.
These together with earlier findings provide strong evidence for
a role(s) for multiple relaxin peptides as neurotransmitters and/or
modulators in the rat CNS.
Burazin TC, Bathgate RA, Macris M, Layfield
S, Gundlach AL, Tregear GW. J Neurochem 2002 Sep;82(6):1553-7
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