Báo cáo Y học: Relaxin-like bioactivity of ovine Insulin 3 (INSL3) analogues doc

Relaxin-like bioactivity of ovine Insulin 3 (INSL3) analogues
Antonia A. Claasz
1
, Courtney P. Bond
2
, Ross A. Bathgate
1
, Laszlo Otvos Jr
3
, Nicola F. Dawson
1
,
Roger J. Summers
2
, Geoffrey W. Tregear
1
and John D. Wade
1
1
Howard Florey Institute, University of Melbourne, Victoria, Australia;
2
Department of Pharmacology, Monash University,
Victoria, Australia;
3
The Wistar Institute, Philadelphia, USA
Relaxin is an insulin-like peptide consisting of two separate
chains (A and B) joined by two inter- and one intrachain
disulfide bonds. Binding to its receptor requires an Arg–X–
X–X–Arg–X–X–Ile motif in the B-chain. A related member
of the insulin superfamily, INSL3, has a tertiary structure
that is predicted to be similar to relaxin. It also possesses an

Arg–X–X–X–Arg motif within its B-chain, although this is
displaced by four amino acids towards the C-terminus from
the corresponding position within relaxin. We have previ-
ously shown that synthetic INSL3 itself does not display
relaxin-like activity although analogue (Analogue A) with an
introduced arginine residue in the B-chain giving it an Arg
cassette in the exact relaxin position does possess weak
activity. In order to identify further the structural features
that impart relaxin function, solid phase peptide synthesis
was used to prepare three additional analogues for bioassay.
Each of these contained point substitutions within the
arginine cassette. Analogue D contained the full human
relaxin binding cassette, Analogue G consisted of the native
INSL3 sequence containing an Arg to Ala substitution, and
Analogue E was a further modification of Analogue A, with
the same substitution. Each analogue was fully chemically
characterized by a number of criteria. Detailed circular
dichrosim spectroscopy analyses showed that the changes
caused little alteration of secondary structure and, hence,
overall conformation. However, each analogue displayed
only weak relaxin-like activity. These results indicate that
while the arginine cassette is vital for relaxin-like activity,
there are additional, as yet unidentified structural require-
ments for relaxin binding.
Keywords: cAMP; ligand binding; receptor; solid phase
peptide synthesis; THP-1 cells.
Relaxin is a 6-kDa peptide with two chains linked by one
intra- and two interchain disulfide bonds which is charac-
teristic of the insulin superfamily of peptide hormones.
Relaxin displays a wide variety of biological effects,

including connective tissue remodelling in the female
reproductive tract [1], stimulation of chronotropic and
inotropic responses in heart atria [2], and regulation of fluid
balance [3]. It has also been shown to be a potent
vasodilator [4,5] as well as an antifibrotic agent [6]
suggesting that it may have potential therapeutic applica-
tions. Extensive structure–function studies undertaken over
the years have led to the identification of the key structural
elementsthatareinvolvedintheinteractionofrelaxinwith
its receptor [7,8]. In particular, a motif in the B-chain,
consisting of two arginine residues in positions B13 and B17
[9,10], forming an arginine cassette (Arg–X–X–X–Arg), has
been demonstrated to be essential for receptor binding.
More recently it has been found that the B20 isoleucine
(IleB20) residue is also crucial for receptor binding [11].
When it is replaced with another residue, such as Ala or Thr,
the ability of relaxin to bind to its receptor is greatly reduced
[11]. One exception to this effect is when Ile is replaced with
Val. This substitution results in very similar bioactivity to its
IleB20 counterpart; indeed, some native relaxins contain a
Val in this position [12]. This B20 Ile/Val residue, along with
the previously mentioned Arg cassette, forms an extended
Arg–X–X–X–Arg–X–X–Ile/Val cassette, which is now
regarded as the principal relaxin receptor binding motif.
The primary structure of INSL3 (also known as relaxin-
like factor and Leydig cell insulin-like peptide), a recently
identified member of the insulin-like superfamily, possesses
striking similarity to relaxin with a predicted A-chain of 26
residues and a B-chain of 32 residues. Intriguingly, the
INSL3 B-chain contains an Arg–X–X–X–Arg binding

cassette, however, it is displaced towards the C-terminus
by four residues. Although INSL3 displays its own distinct
physiology with important roles in testis descent and
ovarian function [13], previous work indicated that INSL3
may interact with relaxin receptors [14]. Studies from our
laboratory using synthetic ovine INSL3 showed that this
peptide has no relaxin-like activity in the rat atrial bioassay
[15], and did not cause a significant reduction in the
response to relaxin when the two peptides were assayed
together. Modelling of bombyxin, an insulin-like protein
from the silkworm, together with the known tertiary
structures of insulin and relaxin show a distinctive insulin-
like structure, consisting of two a-helices in the A-chain and
three disulfide bonds [16]. INSL3 is predicted to possess a
similar insulin-like structure, so its lack of relaxin-like
activity is probably a consequence of the absence of specific
amino acids required for the correct relaxin-like binding
Correspondence to J. D. Wade, Howard Florey Institute of Experi-
mental Physiology and Medicine, The University of Melbourne,
Victoria 3010, Australia.
Fax: 61 3 9348 1707, Tel.: 61 3 8344 7285,
E-mail: j.wade@hfi.unimelb.edu.au
Abbreviations: INSL3, insulin-like peptide 3; LGR7/8, leucine-rich
repeat G-protein coupled receptor 7/8.
(Received 19 June 2002, revised 1 September 2002,
accepted 5 November 2002)
Eur. J. Biochem. 269, 6287–6293 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03348.x
motif in the correct position. An analogue with an Arg
residue replacing the His in position B12, thus creating an
Arg–X–X–X–Arg cassette in the same position as in relaxin

(as aligned by cysteine residues) was then chemically
synthesized (Analogue A). It produced weak relaxin-like
activity in the isolated rat atria [15], clearly reinforcing the
importance of the Arg binding cassette in the relaxin
conformation. Thus, INSL3 and the effect of this single
amino acid substitution, provides us with a useful molecular
template with which to study any further structural
requirements for full relaxin binding. However, as the
relaxin-like activity produced by this analogue was com-
paratively weak, it is clear that there are further require-
ments besides the Arg cassette for full interaction of relaxin
with its receptor. This prompted us to examine further
modifications of the INSL3 B-chain in comparison with
relaxin. Firstly an analogue was produced with a full human
relaxin Arg cassette (RELVR) placed into the B-chain
(Analogue D, Fig. 1) in the relaxin position. Secondly, as
mentioned above, IleB20 is also a vital residue for relaxin-
like bioactivity and this corresponds to a Val residue (B19)
in INSL3. However, an additional Arg residue is located in
the B20 position of INSL3, adjacent to this Val and may
have the effect of hindering the interaction of the Val residue
with the relaxin receptor. To further investigate this, we
undertook the chemical synthesis of two new INSL3
analogues where the ArgB20 residue was substituted for a
less bulky Ala residue (Analogues E and G; Fig. 1).
Removal of the ArgB20 might be expected to have the
effect of unmasking the ValB19 residue, allowing it to
interact with the receptor. Each of these newly designed
analogues (D, E and G), together with the previously
prepared analogue (A), were assayed in the established

cAMP bioassay [17], and relaxin receptor binding assay.
Both assays are based on the THP-1 human monocytic cell
line [18], which expresses the human relaxin receptor.
MATERIALS AND METHODS
Peptide synthesis
For each INSL3 analogue, both the A- and B-chains
were separately synthesized by the continuous flow Fmoc
solid-phase method on a 0.15-mmol scale. Each A-chain
was assembled manually on a CRB Pepsynthesiser, using
Fmoc-His(Trt)-Novasyn PA 500 (Novabiochem, Switzer-
land) as support. Amino acid acylation was performed with
HOBt-catalyzed Fmoc-amino acid pentaflourophenyl esters
(Auspep, Melbourne, Australia), with the exception of
arginine whichwas activated withO-(7-aza-benztriazol-1-yl)-
N,N,N,N¢-tetramethylammonium hexaflourophosphate
(HATU) and DIEA in dimethylformamide. B-chain syn-
theses were carried out using an automated MilliGen 9050
synthesiser on PAC-PEG-PS support. Amino acid acylation
was performed with HOBt in dimethylformamide with the
addition of 1,3-diisopropylcarbodiimide. For both chains,
N
a
-Fmoc deprotection was with 20% piperidine in dimethyl-
formamide. All amino acid couplings were of 30 min
duration.
Cleavage and purification
Protected A- and B-chain resins were each treated with
82.5% trifluoroacetic acid/5% phenol/5% H
2
O/5% thioan-

isole/2.5% ethanedithiol (v/v/v/v) plus three drops of
triethylsilane for 3 h. The trifluoroacetic acid solution was
then removed under a stream of nitrogen, followed by
diethyl ether extraction twice. The resulting peptide pellet
was then resuspended in 0.1% (v/v) aqueous trifluoroacetic
acid and lyophilized. Crude peptides were purified using a
Waters HPLC system using a Vydac C
18
reverse-phase (RP)
column (10 · 250 mm 218TP), with a solvent system of
0.1% (v/v) aqueous trifluoroacetic acid (buffer A) and 0.1%
(v/v) trifluoroacetic acid in acetonitrile (buffer B) in linear
gradient mode. Fractions were collected and lyophilized.
Chain combination
Disulfide bond formation between purified chains was
performed in 0.15
M
3-(cyclohexamino)propanesulfonic
acid (CAPS), pH 10.5, 1
M
guanidine hydrochloride, 10%
(v/v) methanol, 2.5 m
M
dithiothreitol buffer, vigorously
stirring at 4 °C. The reaction was monitored by RP-HPLC
at hourly intervals, and terminated by addition of trifluoro-
acetic acid when no starting B-chain was remaining (after
approximately 24 h). Preparative RP-HPLC, as described
above, was then used to isolate and purify the product.
Characterization

Purity of both peptides was analyzed using MALDITOF
MS, performed in the linear mode on a Bruker BIFLEX
instrument, analytical RP- and cation-exchange HPLC.
Cation-exchange HPLC was performed using a Poly CAT
column and a 0–70% gradient (Buffer A: 25 m
M
KH
2
PO
4
,
pH 7, and Buffer B: 0.5
M
KCl + 25 m
M
KH
2
PO
4
,pH7).
Peptide quantitation was performed by amino acid analysis
on a GBC automatic amino acid analyzer (Melbourne,
Australia).
Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy was carried out using
a Jasco J-720 spectropolarimeter using a 0.2-mm path
length cell in the 180–260 nm range. The peptides were
dissolved in double-distilled water at room temperature.
Four scans were averaged, and the resulting crude spectra
were smoothed by the algorithm provided by Jasco. Mean

residue ellipticity is expressed in deg cm
2
Ædmole
)1
with using
a mean residue mass of 110. Alpha helicity was calculated
from the ellipticity values at 208 nm, according to the
algorithm of Greenfield and Fasman [19]. The peptide
Fig. 1. Sequence of INSL3 B chain, analogue B-chain sequences,
and relaxin B-chain. Underlined residues in the relaxin B-chain
indicate the relaxin binding motif. Underlined residues in INSL3
and analogue B-chains indicate the displaced Arg cassette. High-
lighted residues indicate amino acid substitutions in comparison to
INSL3.
6288 A. A. Claasz et al.(Eur. J. Biochem. 269) Ó FEBS 2002
concentrations were 0.17–1.38 mgÆmL
)1
, as determined by
amino acid analysis (above).
cAMP bioassay
Peptides were assayed for relaxin-like activity in THP-1
cells, using a cAMP ELISA (Biotrak), as previously
described [17]. In short, THP-1 cells were cultured in
suspension, and plated out in a 96-well plate, at a density of
40 000 cells per well. Cells were then incubated with 1 l
M
forskolin and 50 l
M
IBMX (in 200 lL RPMI 1640,
containing 10% foetal calf serum, penicillin/streptomycin

and 2 m
ML
-glutamine), plus the peptide of interest for
30 min at 37 °C. Analogues were assayed alongside 5 n
M
H2(B29) relaxin and medium with IBMX and forskolin
only (used as control). The 96-well plate was then centri-
fuged and the supernatant immediately aspirated. Cells were
lysed in 200 lL lysis buffer (from kit) per well, and 100 lL
of the lysate per well used in the ELISA to measure cAMP
levels. Each analogue was tested in at least three experiments
in quadruplicate and the data analyzed by one-way
ANOVA followed by Newman–Keuls multiple comparison
test.
Binding assay
THP-1 cells were grown in modified RPMI 1640 medium,
spun down and resuspended in binding buffer [20 m
M
Hepes, 50 m
M
NaCl, 1.5 m
M
CaCl
2
,1%(w/v)BSA,
0.1 mgÆmL
)1
lysine, 0.01% NaN
4
,pH7.5][14]togive

2 · 10
6
cellsÆwell
)1
in a 96-well plate. The cells were
incubated in binding buffer with 100 p
M
33
P-labeled human
gene 2 (B33) relaxin [H2 (B33)], [20], at 25 °Cfor90minin
the absence or presence of increasing concentrations of
unlabelled human gene 2 (B29) relaxin [H2(B29)]; 100 p
M
to
30 n
M
), INSL3 (30 n
M
to 30 l
M
), Analogue D and
Analogue E (10 n
M
to 300 l
M
). Nonspecific binding was
definedwithH2(B29)(1l
M
). Cells were harvested using a
Packard 96-well plate cell harvester and Whatman GF/C

glass fibre filters treated with 0.5% poly(ethylenimine). The
filters were washed three times with modified binding buffer
(20 m
M
Hepes, 50 m
M
NaCl, 1.5 m
M
CaCl
2
),driedina
37 °C oven, 30 lL of scintillant (Microscint O, Packard)
added and the radioactivity counted by liquid scintillation
spectrometry (TopCount
TM
,Packard).
RESULTS
The chemical synthesis of the individual chains proceeded
without difficulty, and following cleavage and purification,
were obtained in good overall yield. Each of the A- and
B-chains were subjected to detailed chemical characteriza-
tion prior to their combination in solution. Analogues E
and G were obtained in modest yield due to the difficulty in
preventing B-chain dimerization during the process of chain
combination.
Analysis of both peptides by MALDITOF MS showed
principal products with the expected molecular weights, but
also the trace presence of A-chain (not shown). No other
contaminants were detected. Analytical cation-exchange
HPLC was used to further assess the purity of the

analogues, as free A-chain coeluted with the combined
A- and B-chain on reverse-phase HPLC. Cation exchange
HPLC produced a single peak, and no free A-chain was
detected (data not shown) indicating that only trace
amounts of free A-chain were present. Mass spectral
analysis of the main peak for each analogue contained the
target disulfide bonded A- and B-chain product (not
shown).
Secondary structure analysis by CD spectroscopy showed
the presence of a positive band for all three INSL3 peptides
at 187 nm and a negative band at 205 nm. An additional
negative shoulder was seen at around 222 nm (Fig. 2). The
helical content of these peptides were around 25% and 11%
for Analogues E and G, respectively, although these figures
need to be treated with caution due to the low peptide
concentration which may influence the accuracy of the
results. However, there were clearly no major spectral
differences found compared to unmodified INSL3, but all
three ovine sequences exhibited significantly reduced alpha-
helicity compared to the human relaxin peptide. While the
CD spectrum of relaxin showed bands characteristic for
alpha helices (192 nm positive, and 208 nm and 223 nm
negative bands), the helix for INSL3 analogs were signifi-
cantly less alpha helical, as indicated by the blueshift of the
pi-pistar bands to 187 nm and 205 nm. These CD spectra
were remarkably similar to those of the N-methyl-
D
-aspartate-antagonist peptide conantokin G [21], and
featured characteristics of 3–10 helices, which can be
considered as repeating b turns [22]. Indeed, high resolution

NMR spectra of conantokin G verified the 3–10 helix
structure [23]. Hence, the intensity differences between the
INSL3 peptide spectra may reflect differences in the turn-
forming potential. However, the very similar spectral
characteristics argue for simple inaccuracies in concentra-
tion determination. In this regard it is interesting to note
that the amino acid changes in the INSL3 to more closely
Fig. 2. CD spectra for human relaxin (solid line), sheep INSL3 (dashed
line), Analogue E (dots and dashes) and Analogue G (dotted line) in
water.
Ó FEBS 2002 Relaxin activity of B chain analogues of ovine INSL3 (Eur. J. Biochem. 269) 6289
mimic the relaxin structure were not accompanied by more
alpha helical spectral transitions in water (Fig. 2).
Analogues D, E and G, along with previously synthesized
Analogue A were subjected to a cAMP bioassay using
THP-1 cells. These cells have been shown to express the
relaxin receptor at a density of  275 receptors per cell [17].
A preliminary competitive whole-cell binding assay, using
33
P-labelled H2(B33) relaxin with increasing concentrations
of unlabelled H2 (B29) relaxin, was performed in order to
verify the receptor number in our batch of cells, before these
were used to assay any peptides. Scatchard analysis showed
that receptors were expressed at a density of  265
receptors/cell (data not shown), with a dissociation constant
(K
d
¼ 0.13 n
M
), which is similar to what has previously

been found [17]. THP-1 cells responded to increased
concentrations of H2(B29) relaxin with increased cAMP
production, with an approximate EC50 of 0.1 n
M
. Maxi-
mum cAMP stimulation was achieved at 5 n
M
(Fig. 3), and
hence was taken as the 100% value used for comparison
with the analogues.
Each peptide was assayed alongside varying concentra-
tions of H2(B29) relaxin, as an internal control in each
assay, and also for the purpose of comparison. All
analogues were assayed at a concentration of 100 and
500 n
M
, and at additional concentrations where possible,
alone or in the presence of 5 n
M
H2(B29) relaxin to
ascertain whether these analogues could augment or
antagonize the response to relaxin. INSL3 itself was also
assayed at 500 n
M
, on its own as well as in the presence of
H2(B29) relaxin. Free A-chain was also assayed both in
isolation and in the presence of H2(B29) relaxin to ensure
that it did not have any effect on its own, in order to control
for the presence of free A-chain detected by mass spectro-
metry as a contaminant in trace amounts in both analogues

E and G. The THP-1 cAMP assay was responsive to
H2(B29) relaxin and analogues A, D, and E. However,
INSL3 produced no significant response, nor did it have any
effect on the response to relaxin (Fig. 4A). Analogue A
(Fig. 4B) produced approximately 60% of the maximum
relaxin response at 100 n
M
, and around 75% maximum
response at 500 n
M
, while a 55% maximum response at
100 n
M
and a 90% maximum response at 500 n
M
was
produced by Analogue D (Fig. 4C). Analogue E displayed a
response at 50% of maximum relaxin response at 100 n
M
,
and approximately 80% of maximum relaxin response at
500 n
M
(Fig. 4D). Furthermore, a  40% response was
observed at 50 n
M
, and no significant response was
observed at 10 n
M
. Therefore, while Analogue E did display

some degree of bioactivity the activity of the previous
analogues was not improved upon. Neither Analogue G
(Fig. 4E) nor free A chain (not shown) produced any
response.
Interestingly, Analogue A produced a significant reduc-
tion in the response to relaxin when the two were assayed in
combination (P < 0.05, by ANOVA followed by New-
man–Keuls multiple comparison test). Analogues D and E
also produced a slight reduction in the relaxin response,
however, these results did not reach statistical significance.
Due to the limited amounts of peptide available, higher
doses could not be assayed for antagonist activity.
The results obtained from the THP-1 binding assay
(Fig. 5) showed that INSL3 had low affinity for the relaxin
receptor (pK
i
6.0 ± 0.32, n ¼ 4) compared to H2(B29)
relaxin (pK
i
8.7 ± 0.11, n ¼ 11). Incorporation of the
relaxin Arg motif into INSL3 (Analogue D) increased the
affinity of the peptide fivefold for the relaxin receptor (pK
i
6.7 ± 0.18, n ¼ 4) but it was still 100-fold weaker than
H2(B29) relaxin. Removal of both Arg motifs from INSL3
(Analogue G) further reduced affinity for the receptor (pK
i
5.4 ± 0.20, n ¼ 4). Due to the limited amounts of available
peptide, analogues A and E could not be assessed in the
THP-1 binding assay.

DISCUSSION
While species variation in amino acid sequences of relaxin is
significant, and may be as great as 60%, relaxin peptides will
generally interact with relaxin receptors between species
[24,25]. The common elements to relaxin peptides between
species are the disulfide bond patterns, and the Arg–X–X–
X–Arg–X–X–Ile motif in the B-chain. Given that INSL3
has the former and a portion of the latter, we sought to
make it more relaxin-like by successively introducing all of
the Arg cassette components. Such a study would help
provide a clear indication of the role of this cassette for
relaxin function. As earlier studies showed that human,
ovine (unpublished data) and rat [26] INSL3, all show no
activity in the THP-1 cell bioassay and, in our hands, the
ovine peptide is slightly easier to chemically synthesize, we
chose to use the ovine sequence as the template for further
study.
The four INSL3 analogues that were selected for
preparation allowed examination of the amino acid residues
required for interaction of relaxin with its receptor. It was
previously shown that while native INSL3 is not a relaxin-
like hormone at physiological concentrations, replacement
of B-chain HisB12 with Arg did result in some relaxin-like
activity in isolated rat atria [15]. The interaction of
Analogue A with the receptor indicated that INSL3 must
have a similar secondary structure to relaxin. Although
active, Analogue A was still 100-fold less potent than
H2(B29) relaxin, clearly indicating that the arginine cassette
is probably only one of a number of requirements for full
relaxin-like bioactivity. This has been highlighted by the

recent discovery that IleB20 is crucial for binding of relaxin
to its receptor [11], which corresponds to ValB19 in INSL3
Fig. 3. Standard response to relaxin in the cAMP/THP-1 cell assay.
Data is expressed as percentage maximum response. pEC50 ¼ 10
(0.1 n
M
). Maximum response observed at  5n
M
.
6290 A. A. Claasz et al.(Eur. J. Biochem. 269) Ó FEBS 2002
(when sequence is aligned according to the cysteine
residues). It was shown that when IleB20 was replaced with
an Ala residue, it had much less affinity for the relaxin
receptor in mouse brain. However, when IleB20 was
replaced with a Val residue, binding was not affected. Val
is also present in this position in some native relaxins, such
as porcine relaxin [12]. This is of particular importance when
comparing the B-chains of relaxin and INSL3. In INSL3,
the corresponding residue to the relaxin IleB20 residue is a
Val. This means that yet another residue of importance for
relaxin-like activity is present in INSL3, however, the
adjacent ArgB20 residue may well sterically hinder the
interaction of this residue with the relaxin receptor. It was
speculated that activity of Analogue A [15] could be
improved upon by removing this additional Arg residue
found in INSL3 (ArgB20).
INSL3 demonstrated no significant relaxin-like activity in
the THP-1 cAMP assay. It also did not have any significant
effect on the cAMP response to H2(B29) relaxin when the
two were assayed in combination. In a different assay

system, the rat atrial bioassay, INSL3 also has no significant
effect on chronotropic or inotropic responses [15]. At the
time of writing this paper, the sequences of the relaxin and
INSL3 receptors became available, named LGR7 and
LGR8, respectively [27]. The two receptors are G-protein
coupled receptors, and share approximately 60% sequence
homology. Interestingly, both receptors bind relaxin.
Clearly aspects of the ligand-binding domain of both
receptors must share some similarity, while the ligands
themselves have subtle differences which affords them their
specificity. A better understanding of the binding require-
ments of these individual ligands may ultimately allow us to
design specific analogues to avoid cross-reactivity between
the two receptors. We have since confirmed, using RT-PCR,
that THP-1 cells only express LGR7 (data not shown).
Analogue A and D both displayed relaxin-like activity in
the THP-1 cAMP assay. Analogue A produced  75% of
the maximum response at 500 n
M
, while Analogue D at the
same concentration produced  80% of the maximum
response. In the rat atrial bioassay Analogue A at 1 l
M
produced 42% and 45% of the maximum response to
H2(B29) relaxin in the chronotropic and inotropic assay,
respectively [15]. Analogue E contains a human relaxin
binding motif, as well as having the Arg21 residue replaced
by Ala21. The replacement of the Arg residue failed to
increase relaxin activity. Analogue E produced 80% of the
maximum H2(B29) relaxin cAMP response at 500 n

M
and
was therefore equipotent to Analogues A and D. In
Analogue G ArgB20 was replaced by an Ala residue.
Analogue G would thus serve as a negative control, in case
this residue replacement was sufficient to induce relaxin-like
activity. Analogue G failed to have activity in the relaxin
Fig. 4. cAMP response to sheep INSL3, Analogues A, D, E and G. (A)
cAMP response in THP-1 cells to sheep INSL3, expressed as a per-
centage of maximum relaxin response. (B) cAMP response to Ana-
logue A, expressed as a percentage of maximum relaxin response.
* P < 0.05 vs. relaxin 5 n
M
. (C) cAMP response to Analogue D,
expressed as a percentage of maximum relaxin response. (D) cAMP
response to Analogue E, expressed as a percentage of maximum
relaxin response. (E) cAMP response to Analogue G, expressed as a
percentage of maximum relaxin response. Each analogue was tested in
at least three experiments in quadruplicate and the data analyzed by
one-way ANOVA followed by Newman–Keuls multiple comparison
test.
Ó FEBS 2002 Relaxin activity of B chain analogues of ovine INSL3 (Eur. J. Biochem. 269) 6291
bioassay, and furthermore showed a lower affinity for the
relaxin receptor in the binding assay, reinforcing the
importance of the Arg–X–X–X–Arg cassette B13–17 posi-
tion for relaxin-like bioactivity.
The circular dichroism spectroscopy studies performed
on these peptides showed that both INSL3 and analogues E
and G were less a-helical than human relaxin. As the
primary receptor binding site of relaxin is located in the

a-helical region of the B-chain, any changes in the helicity in
this region could well change the orientation of the binding
cassette and hence have a considerable effect on the
receptor–ligand interaction. This could therefore be a major
contributing factor to the reduced bioactivity of these
analogues.
Of further interest is the reduction in relaxin response
seen when H2(B29) relaxin and Analogue A were assayed
together. This is in contrast to the rat atrial bioassay [15],
where no antagonist activity was observed for this analogue.
Analogues D and E show some potential for antagonist
activity, although this was not significant at the concentra-
tions assayed. A possible explanation for the antagonist
activity of Analogue A could be the presence of the
additional ArgB20 residue in the B-chain along with the lack
of a human relaxin Arg cassette (this analogue had the
RHFVR motif). In comparison to Analogue D, which has
the human relaxin Arg (RELVR) cassette, Analogue A
shows a trend towards causing a lesser relaxin-like response
in this assay. The complete RELVR sequence may cause a
slight change in the overall structure of this particular region
of the B-chain, allowing a better interaction of Analogue D
with the receptor compared to Analogue A, where the Arg
cassette has the sequence RHFVR. Furthermore, the
GluB13 residue in the complete Arg cassette, as in Analogue
D, has a negative charge, while the HisB13 residue in
Analogue A has a positive charge. This may also influence
the interaction of these analogues with the relaxin receptor.
This unfavourable effect on the structure of the B-chain in
Analogue A coupled with the additional ArgB20 residue

may be sufficient to give Analogue A weak antagonist
activity. Even if Analogue D still has the additional Arg, the
more favourable interaction of the human Arg cassette with
the receptor may serve to counteract any antagonist activity
of this analogue. Analogue E, while not having the complete
human relaxin Arg cassette (RHFVR, as in Analogue A),
does not possess the additional ArgB20 residue, reducing its
potential for antagonist activity.
Our data from this study has re-emphasized the import-
ance of an Arg cassette in the correct position for relaxin-
like activity. However, it has also clearly demonstrated that
there are other necessary factors for full relaxin-like bio-
activity. The overall structure of the INSL3 B-chain may
have subtle, yet vital differences compared to relaxin, which
necessarily prevents it from fully interacting with the relaxin
receptor. To investigate this aspect, other residues in the
INSL3 B-chain could be substituted, to give rise to further
INSL3 analogues. Furthermore, the INSL3 A-chain could
also display structural differences to the relaxin A-chain. It
has previously been shown that the relaxin A-chain is
essential for full bioactivity [28], most likely by acting as a
scaffold to hold the B-chain in its correct conformation.
Our study also suggests the distinct possibility of produ-
cing a relaxin antagonist by changing the structure of
INSL3. However, for this to be achieved, full elucidation of
the INSL3 tertiary structure will ultimately be required for a
better understanding of the essential differences between
relaxin and INSL3. Nevertheless, INSL3 is clearly not only
a useful tool for understanding the requirements for agonist
activity at the relaxin receptor, but it also has the potential

to assist us in designing and preparing a relaxin antagonist,
which will provide a useful molecular probe of relaxin
action. Given the recent confirmation of the potent vaso-
active effect of relaxin and the fact that vasodilator
pathways are key targets for new therapeutics, relaxin is
an attractive peptide for further development into both
agonists and antagonists. With the recent cloning of the
relaxin and INSL3 receptors [27] and the discovery that
both receptors bind relaxin, the subtle differences between
the two ligands is of even more importance, particularly in
the design of specific therapeutics.
ACKNOWLEDGEMENTS
The work described herein was supported by an Institute Block Grant
Reg Key Number 983001 from the National Health and Medical
Research Council of Australia. We thank Mare Cudic (Wistar Institute)
for performing the CD spectroscopy. We also thank Kathryn Smith of
the Howard Florey Institute for performing amino acid analysis.
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