Apela regulates fluid homeostasis by binding to the APJ receptor to activate Gi signaling

18 Pages·2015·0.9 MB·English

JBC Papers in Press. Published on May 20, 2015 as Manuscript M115.648238
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.648238
Apela regulates fluid homeostasis by binding to the APJ receptor to
activate Gi signaling#
Cheng Deng1,2*, Haidi Chen1, Na Yang1, Yi Feng2, Aaron J. W. Hsueh2*
1Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal
University, Nanjing 210023, China
2Program of Reproductive and Stem Cell Biology, Department of Ob/Gyn, Stanford University School of
Medicine, Stanford, CA 94305-5317, USA
* Corresponding author: Program of Reproductive and Stem Cell Biology, Department of Ob/Gyn, Stanford
University School of Medicine, Stanford, CA 94305-5317, USA. Tel.: [phone]; Fax: [phone]
7102; Email: [email protected]
Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal
University, Nanjing 210023, China. 1 Wenyuan Rd., Nanjing 210023, China. Tel.: [phone]; Fax: 86-
25-8589-1040; E-mail: [email protected]
#Running title: Apela regulates fluid homeostasis
Key words: apela, apelin, APJ receptor, Gi pathway, diuresis, water intake, fluid homeostasis
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Background: Apela, a newly identified apela to APJ with high affinity w
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peptide hormone, is important during (Kd=0.51nM) and the ability of apelin, loa
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zebrafish embryogenesis. the known peptide ligand for APJ, to ed
compete for apela binding. Apela, fro
Results: Apela binds directly to APJ and m
acts through the Gi pathway. Apela is similar to apelin, acts through the http
expressed exclusively in adult kidney and inhibitory G protein pathway by ://w
regulates fluid homeostasis. inhibiting forskolin-stimulated cAMP ww
production and by inducing ERK1/2 .jb
Conclusion: Apela regulates fluid c.o
homeostasis through Gi signaling pathway. pexhporsepshsoerdy leaxticoluns. iIvne alyd uinlt trhaets k, iadpneelya, is byrg/
Significance: Apela is a kidney ligand unlike the wide tissue distribution of gu
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more potent than apelin in regulating fluid apelin. In vivo studies demonstrated st o
homeostasis. the ability of apela to regulate fluid n A
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ABSTRACT homeostasis by increasing diuresis ril 9
and water intake. Dose-response , 20
Apela (APJ early endogenous ligand, 1
studies further indicated that apela 9
also known as elabela or toddler) is a
induces 2- and 5-fold higher maximal
recently discovered peptide hormone.
responses than apelin ERK1/2
Based on genetic studies in zebrafish,
phosphorylation and diuresis/water
apela was found to be important for
intake, respectively. After designing an
endoderm differentiation and heart
apela antagonist, we further
development during embryogenesis.
demonstrated the role of endogenous
Although common phenotypes of apela
ligand(s) in regulating APJ-mediated
and APJ null zebrafish during
fluid homeostasis. Our results
embryonic development suggested
identified apela as a potent peptide
that apela interacts with the APJ
hormone capable of regulating fluid
receptor, kinetics of apela binding to
homeostasis in adult kidney through
APJ and intracellular signaling
coupling to the APJ-mediated Gi
pathways for apela remain unknown.
signaling pathway.
The role of apela in adults is also
uncertain. Using a chimeric apela
ligand, we showed direct binding of
1

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
INTRODUCTION adult rats, and the ability of apela to
activate the Gi signaling pathway. In
Apelin, originally isolated from the bovine
addition, apela regulated diuresis and
stomach (1,2), encodes a mature peptide
water intake, and a newly designed apela
hormone of 13 amino acids conserved in
antagonist modulated fluid homeostasis in
vertebrates. Apelin was found to be
vivo.
important in the regulation of water and
food intake (3-6) by binding and activating EXPERIMENTAL PROCEDURES
APJ (apelin receptor), a 7-transmembrane
Peptides, plasmids and animals-
G-protein-coupled receptor. APJ and its
Apela-32 (all apela peptides in this paper
ligand apelin play diverse roles in the
used is apela-32 except as otherwise
central and peripheral regulation of the
indicated), apela-21, angiotensin (AngII),
cardiovascular system, the release of
apelin, apelin-FA and other related
hormones/neuropeptides, the modulation
peptides were purchased from Phoenix
of immune functions, as well as the
Pharmaceuticals, Inc. and Chinese
maintenance of cardiac contractility during
Peptide Company (China). Apela analogs
pressure overload and aging (7-11). D
(apela-FA, PA, AA) were synthesized by o
w
n
Apela (APJ early endogenous the PAN facility at Stanford University and lo
a
d
ligand, also known as elabela or toddler) Chinese Peptide Company (China). APJ, e
d
is a recently identified gene, encoding GPR15 and GPR25 full-length cDNAs fro
m
conserved C-terminal mature peptide were sub-cloned into the pcDNA3.1 (+) h
ttp
hormones of 32 or 21 amino acids (12). plasmid and verified by DNA sequencing. ://w
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Two groups independently showed that Apela C-terminal coding sequences were w
apela is an embryonic hormone capable of subcoloned into the AP-tag vector .jbc
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regulating heart development and (GenHunter's AP-TAG technology) and rg
b/
promoting the movement of verified by DNA sequencing. Sprague- y
g
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mesendodermal cells during gastrulation, Dawley rats were used for in vivo studies. es
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respectively (13,14). Because the APJ null All procedures involving animals were n
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apela was identified as the cognate laboratory animals. 1
9
peptide ligand for the APJ receptor (13,14).
Real-time q-RT-PCR-Sprague-
In addition, apela binds to APJ and
Dawley rats were sacrificed for tissue
induces the internalization of APJ in
preparations. Total RNAs were extracted
receptor-transfected cell lines (13,14).
from stomach, lung, heart, muscle,
Although genetic studies intestine, testis, bladder, kidney, brain,
demonstrated the important role of apela spleen, liver, ovary and uterus. 500 ng of
during embryogenesis in zebrafish, the total RNA were reverse-transcribed to
expression pattern and function of apela in cDNAs using the following master mix: 6
adult mammals still remain unknown. In µl RNAse-free water, 2 µl 5x buffer
addition, whether apela functions through (Takara, China), 0.5 µl random 6 mers
coupling to APJ to modulate the G protein (100 µM) (Takara, China), 0.5µl oligo dT
signaling pathways has not been primer (50 µM) (Takara, China), 0.5µl
investigated. Here, we demonstrated the primer Script RT Enzyme Mix I and total
exclusive expression of apela in kidney of RNA (500ng). A master mix of the
2
following reaction components was gently agitated for 10 min. before
prepared: 6.8 µl water, 0.4 µl forward centrifugation at 13,000g for 15 min.
primer (10µM), 0.4 µl reverse primer Same amount of proteins was fractionated
(10µM), 10 µl SYBR Premix Ex Taq (Tli on a 10% SDS-PAGE gel before
RnaseH Plus) (Takara, China), and 0.4 µl immunoblotting. Anti-p44/42 ERK1/2 and
ROX Reference Dye I before adding 2 µl 44/42 ERK1/2 antibodies were from
PCR templates. The following real-time Beyotime Company (China). Experiments
PCR protocol was used: denaturation for were repeated independently at least 3
30 sec. at 95°C, 40 cycles of a three times. The density of the bands
segmented amplification and corresponding to 44 kDa and 42 kDa was
quantification program (denaturation for quantified with an imaging densitometer.
30 sec at 95°C, annealing for 5 sec. at the Immunoblotting data are expressed as
primer specific temperature, elongation for percentages of the maximal value and
30 sec at 60°C), and a melting step by represent the mean±SEM of three
slow heating from 60 to 99°C. independent experiments.
Cell culture, cAMP assay and Binding assay-The assay for the D
o
immunoblotting-Chinese hamster ovary production of alkaline phosphatase (AP)- w
n
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(CHO) cells were grown in Dulbecco's tagged proteins and binding affinity a
d
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Modified Eagle's medium (DMEM) measurements have been described (15). d fro
supplemented with 10% fetal bovine Briefly, the AP-apela plasmid was m
h
serum (FBS). APJ was transfected into transiently transfected into HEK293T cells ttp
CHO cells using lipofectamine 2000 and, after 24h, cells were cultured in ://w
w
w
(Invitrogen) before selection for their serum-free medium for 2 days. The .jb
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resistance to the antibiotic G418. G418- supernatant containing AP-apela was .o
rg
resistant clones were screened for the quantified using the AP activity assay b/
y
expression of APJ. For cAMP assay, CHO (GenHunter Corp.). For the cell-based g
u
e
cells expressing APJ were pretreated for binding assay, CHO cells expressing APJ st o
n
30 min. with apela or apelin at the were grown to near confluence and A
p
indicated concentration before treatment incubated with AP-apela for 90 min. at ril 9
with 1uM forskolin (FSK) in the presence room temperature. At the end of , 2
0
1
of 0.2 mM IBMX. At 30 min. later, total incubation, cells were washed with HBSS 9
cAMP levels were determined. To containing 0.5 mg/ml BSA and 20 mM
determine the effects of PTX pretreatment, HEPES (pH 7.0), and bound AP activities
cells were pretreated overnight with PTX were determined by using AP assay
(200 ng/ml) before cAMP assay. For reagents (GenHunter Corp.). Data were
determining the effects of apela-PA, cells analyzed using Graphpad Prism 5.0 and
were pretreated with apela-PA at the Kd values were calculated. For peptides
indicated concentration for 30 min. before competition assays, the same APJ-
cAMP assay. For immunoblotting, CHO expressing CHO cells were incubated with
cells expressing APJ at subconfluence AP-apela (5nM) and different peptides
were serum-deprived or pretreated with analogs with indicted concentration for 90
pertussis toxin at 200ng/ml for 12h before min. at room temperature. Finally the
peptide exposure for different times. Cells same AP activity assay was determined.
were washed once in PBS and lysed for Data were analyzed using SigmaPlot.
15 min. on ice in a RIPA Lysis and
Extraction Buffer, and the mixture was
3
Measurement of urine flow rate, groups. Data are expressed as mean ±
water intake and food intake-All SEM. Calculations were done with a
experiments were performed using adult standard statistical package (SPSS for
male rats (Sprague-Dawley). For animals Windows, version 21). Statistical
in urine flow rate tests, a stabilizing period significance was defined as a P value
of 30 min. was allowed after anesthesia <0.05 (*) or P value <0.01(**).
(using 2, 2, 2-tribromoethanol by
RESULTS
intraperitoneal injection), and an
intravenous (tail vein) injection of saline Apela binds to APJ with high
(500ul) was performed to determine affinity-Apela induces the internalization of
baseline values of urine flow rates (two APJ in transfected cell lines over-
hours basal period). Some rats received expressing APJ (13,14). However, binding
500ul of different doses of apelin or apela kinetics between apela and APJ has not
or apela-PA by intravenous (tail vein) been demonstrated. Peptide ligands with
injection for two hours (the experimental alkaline phosphatase (AP) appended at its
period). Urines were collected to tube by N-terminal end allows easy detection of
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pipet from plastic membrane at the bottom chimeric probes for demonstrating direct o
w
of cage when urines were generated. Two ligand-receptor binding (16). To directly nlo
a
hours post-injection was taken as the end analyze the ligand-receptor relationship of de
d
point and then urines are measured and apela and APJ, we introduced the alkaline fro
m
calculated. Urine flow rate is expressed as phosphatase tag (AP-tag) to apela-32 and h
ttp
ul/min per 100g body weight. For animals generated a chimeric apela ligand, AP- ://w
in water intake and food intake tests, apela. In addition, we isolated CHO cells ww
either saline or different doses of apelin, stably expressing APJ for binding tests. .jbc
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or apela, or apela-PA was administered As shown in Fig. 1A, incubation of rg
into rats using intraperitoneal injections. increasing levels of AP-apela led to a by/
g
24h post-injection was taken as the end dose-dependent binding of the ligand to ues
point, and water/food intake were CHO cells expressing APJ. However, AP- t on
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Water intake was expressed as ul/h per and GPR25 (17) (Fig. 1A). Scatchard plot 01
9
100g body weight and food intake was analyses (Fig. 1B) indicated an
expressed as g/day per 100g body weight. equilibrium-binding constant (Kd) of
Every group included at least five animals 0.51nM for AP-apela, a high affinity
for tested independently. comparable to the interaction between
apelin-13 and APJ (Kd=0.4nM) (18).
Statistics-Experiments for In vitro
tests include cAMP assay, binding assay To analyze the specificity of apela
and immunoblotting were repeated and apelin binding to APJ, we also
independently at least 3 times. Every checked for binding competition by related
animal group for in vivo experiments peptides. As shown in Fig. 1C, apela-32
included at least five animals for tested and apela-21 competed for AP-apela
independently. Differences between two binding to APJ with equal potency,
groups were compared using two-tailed whereas apelin only partially competed for
Student’s t-test. One way ANOVA AP-apela binding. In contrast, AngII
followed by a Fisher’s LSD post-hoc test (angiotensisn II), a peptide ligand capable
to evaluate the differences among multiple
4
of binding to the angiotensin receptor, with In addition, we monitored apela
sequence similarity to APJ (19), was induction of the phosphorylation of p42/44
ineffective (Fig. 1C). Although both apela ERKs, known to be downstream of Gi/Go
and apelin have mature peptides in their signaling (23). As shown in Fig. 2C and
C-terminal region, they do not have 2D, both apela and apelin treatment of
obviously homologous sequences APJ-expressing cells promoted a time-
(Supplementary Fig. 1A). After alignment dependent phosphorylation of p42/44
of their gene synteny in vertebrates, we ERKs with the highest stimulation at 7 min,
also could not find their common which is consistent with previous study
origination in vertebrates (Supplementary (24). Dose-dependency tests further
Fig. 1B). Thus, our data suggested that indicated that treatment of APJ-expressing
APJ binds to two phylogenetically- CHO cells with either apela or apelin for 7
unrelated peptide ligands, apela and min. promoted a dose-dependent
apelin. phosphorylation of p42/44 ERKs (Fig. 2E),
with apela showing a 2-fold higher
Based on an early report showing
maximal increase than apelin (Fig. 2E).
the importance of the C-terminal region of D
Furthermore, PTX pretreatment abrogated o
apelin for receptor signaling (20), we w
this activation (Fig. 2F). Also, the newly nlo
designed apela analogs by modifying key a
designed apela analog (apela-PA) dose- de
residues in its C-terminal region (Fig. 1D). dependently reduced effects of both apela d fro
As shown in Fig. 1C, both apela and m
and apelin on ERKs phosphorylation (Fig. h
apelin analogs competed for AP-apela ttp
binding to the APJ receptor (apelin=apela- 2G and 2H), further indicating apela-PA is ://w
an antagonist of apela/apelin actions and w
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PA>apela-FA=apelin-FA>apela-AA). also APJ receptor functions. In addition, .jb
c
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Apela activates Gi signaling by other G protein pathways (Gs, Gq and rg
interacting with the APJ receptor-Apelin G12) were tested but apela show no by/
g
binds to APJ and activates downstream Gi stimulation (Data not shown).Taken ues
signaling (1,21,22). We also checked together, our results suggest that apela t on
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inhibited forskolin-stimulated cAMP 1
Apela is exclusively expressed in 9
production in APJ-expressing CHO cells in
kidney of adult rats-Both apelin and APJ
a dose-dependent manner with IC50 value
are expressed in the central nervous
of 0.27nM, showing a similar potency as
system and diverse other tissues including
apelin (IC50=0.29nM) (1). Furthermore,
stomach, gastrointestinal tract, heart,
pretreatment of APJ-expressing cells with
kidney, adipose and lung (10,25-27). As
pertussis toxin (PTX), which selectively
showed in Fig. 3A and 3B, we confirmed
ADP-ribosylates Gi/Go proteins and
the wide tissue expression pattern of
uncouples them from their associated
apelin and APJ in adult rats. As another
receptors, abrogated the inhibitory effects
natural peptide ligand for APJ, apela was
of apela on cAMP production (Fig. 2B).
found to be expressed during zebrafish
Also, the newly designed apela analog
embryogenesis (13,14). To investigate
(apela-PA) dose-dependently reduced
apela expression in adults, we performed
inhibitory effects of apela on cAMP
quantitative-PCR screening of diverse
production (Fig. 2B), indicating apela-PA
tissues in adult rats, and found exclusive
is an antagonist of Apela-APJ functions.
expression of apela in the kidney (Fig. 3C),
5
suggesting apela may play important roles importance role of the APJ receptor in
in fluid homeostasis, in addition to its role fluid regulation by the kidney.
in heart development during
Combined with the high expression
embryogenesis.
pattern of apela in adult kidney, our results
Effects of apela on diuresis and suggested that apela and APJ may play
water intake-Earlier studies demonstrated important roles in fluid regulation by the
that the apelin-APJ pair regulates kidney kidney.
functions (3-5). Because apela is
DISCUSSION
exclusively expressed in the kidney during
adult life (Fig. 3C), we further checked its Our results demonstrated binding kinetics
regulation of fluid homeostasis. As shown of apela to its cognate receptor-APJ and
in Fig. 4A, urine flow rate was increased apela inhibition of forskolin-stimulated
following intravenous injection of adult rats cAMP production and induction of ERK1/2
with apelin as compared with saline- phosphorylation though coupling of APJ to
injected animals, consistent with earlier the PTX-sensitive Gi pathway. In addition,
results (3). Of interest, apela injection we identified the exclusive expression of D
o
induced about 5-fold higher maximal apela in kidney of adult rodents and the wn
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increases in plateau-responses of urine role of apela in regulating diuresis and ad
e
flow rates than apelin (Fig. 4A). water intake. d fro
m
Previous study showed the role of Apela and apelin, two http
apelin in water and food intake (6). phylogenetically unrelated, natural peptide ://w
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However, contrary data also exists (28). ligands for APJ, are both conserved in w
To further elucidate apela function in vivo, vertebrates and represent a rare case of .jbc
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we investigated the effect of apela and GPCR ligand-receptor pairs showing one brg/
apelin on water and food intake. As shown receptor interacting with two distinct y g
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in Fig. 4B, intraperitoneal injection of adult ligands (29). Because apelin can compete es
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rats with either apela or apelin increased for the binding of AP-apela to the APJ n A
wfoaldte hr iginhtaekr em waxitihm aapl einlac rsehaoswe iinng p alabtoeuatu 5 - rtwecoe ppetoprt i(dFeigs .b 1inCd) ,t oit iosv leikrelalyp pthinagt tshiteesse i n pril 9, 2
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response than apelin (Fig. 4B). In contrast, APJ and it is interesting to investigate the 19
no effect of either apela or apelin on food exact binding sites of APJ for apela and
intake was found (Fig. 4C). apelin based on structural analysis.
To further confirm the roles of Angiotensin, bradykinin, and apelin
endogenous APJ ligands on urine flow bind to angiotensin II receptor types 1/2,
rate and water intake, we treated adult bradykinin receptor B1/2, and APJ,
rats with the antagonistic analog apela-PA. respectively (19). Receptors for these
Interestingly, urine flow rates measured three ligands constitute a subfamily of
after apela-PA treatment were significantly peptide GPCRs due to their close
lower than saline-treated animals (Fig. 5A). sequence homology (30). Early studies
Likewise, treatment with apela-PA dose- demonstrated the role of this GPCR
dependently decreased water intake of subfamily in fluid homeostasis. Treatment
rats following intraperitoneal injections with angiotensin II in mice inhibits drinking
(Fig. 5B). Our results further confirmed of water or saline, whereas administration
of angiotensin II receptor antagonists
6
stimulates diverse responses including Furthermore, apela stimulated a 5-fold
water drinking, vasopressin secretion and higher maximal response than apelin in
natriuresis (31,32). In addition, regulating diuresis and water intake,
experiments on dogs showed that suggesting apela of kidney origin is more
bradykinin regulates proximal tubular important than apelin in controlling APJ
sodium reabsorption (33). functions in the kidney (3-5). Also, apela
showed 2-fold higher plateau responses in
Although apelin is the cognate
stimulating phosphorylation of ERK1/2
ligand for APJ and abnormal fluid
than apelin (Fig. 2E), although both of
homeostasis was found in APJ null mice
them inhibited FSK-stimulated cAMP
(34,35), the exact role of apelin in the
production with the same potency (Fig.
regulation of fluid homeostasis has been
2A). Further study can investigate
controversial. Several reports showed the
additional apela effects on kidney
ability of apelin to regulate fluid
functions and the exact kidney cell types
homeostasis in rodents (3-6) whereas
expressing apela and APJ.
other studies showed that apelin does not
have reliable or robust effects on fluid Our results showed the newly D
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intake or blood pressure in rats (28). Our designed apela antagonist, apela-PA, w
n
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studies demonstrated that apela is antagonized ERKs activation of both apela a
d
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expressed exclusively in adult kidney and and apelin (Fig. 2G and 2H), and serving d fro
apela is more potent than apelin in as the antagonist for APJ functions. Also, m
h
regulating diuresis and water intake. In vivo, apela-PA inhibits the urine flow ttp
Because apelin is expressed in low levels rate and water intake after injection in ://w
w
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in the adult kidney, our findings suggest adult rats. Studies on the antagonistic .jb
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that apela is more important than apelin in functions of apela-PA and related peptides .o
rg
regulating fluid homeostasis in adults. could allow the formulation of new therapy b/
y
for kidney diseases. g
u
Both apelin and APJ are widely es
t o
expressed in the nervous systems in Author contributions: CD and AH designed the n
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astdriualttsu,m in, cmluiddbinragi nth, eh ifpropnotcaal mcoprutesx, ,m edulla sdteusdigyn aendd, pwerrofoter mtheed p aanpde ra.n CaDly,z HeCd athned NY pril 9, 2
0
pons, cerebellum, pituitary, olfactory experiments shown in Figure 1, Figure 2 and 19
tubercle, and septum. They are also Figure 3. CD and YF designed, performed and
expressed in peripheral tissues including analyzed the experiments shown in Figure 4
adrenal, vas deferens, testis, intestine, and Figure 5. All authors reviewed the results
and kidney (26). For the adult kidney, and approved the final version of the
apelin showed low expression levels, as manuscript. The authors declare that they
compared with the high and exclusive have no conflicts of interest with the contents
expression of apela (Fig. 3C). of this article.

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FOOTNOTES ://w
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#This work was supported by the National Natural Science Foundation of China (31401207), .jb
c
the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) .o
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and the One Hundred Person Project of Nanjing Normal University. b/
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1Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, e
s
Nanjing Normal University, Nanjing 210023, China t o
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p
2Program of Reproductive and Stem Cell Biology, Department of Ob/Gyn, Stanford ril 9
University School of Medicine, Stanford, CA 94305-5317, USA , 2
0
1
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* Corresponding author: Program of Reproductive and Stem Cell Biology, Department of
Ob/Gyn, Stanford University School of Medicine, Stanford, CA 94305-5317, USA. Tel.: +1
650 725 6802; Fax: [phone]; Email: [email protected]
Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing
Normal University, Nanjing 210023, China. 1 Wenyuan Rd., Nanjing 210023, China. Tel.: 86-
25-8589-1040; Fax: [phone]; E-mail: [email protected]

FIGURE LEGENDS
FIGURE 1: Direct binding of apela to the APJ receptor with high affinity. A, Saturation
curve for AP-apela binding to APJ and lack of binding to related receptors. CHO cells stably
expressing APJ were incubated with increasing concentration of AP-apela for 90 min. at
room temperature before determination of alkaline phosphatase activities. Specific binding
was calculated by subtracting the value from cells transfected with empty vectors. Similar
tests were performed using cells stably expressing GPR15 and GPR25. Experiments were
repeated independently at least 3 times. Data were analyzed using Graphpad Prism 5.0 and
10