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Departments of Medicine and Physiology, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106-4951
Address all correspondence and requests for reprints to: Dr. Marc Thibonnier, Room BRB431, Division of Clinical and Molecular Endocrinology, Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4951. E-mail: mxt10{at}po.cwru.edu
| Abstract |
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The affinity of the V3R for 21 peptide and nonpeptide AVP analogs was clearly distinct from that exhibited by the human V1R and V2R. AVP triggered stimulation of phospholipase C in CHO-V3 cells (partially sensitive to treatment with pertussis toxin) with a potency directly proportional to receptor density. V3R-mediated arachidonic acid release also was also sensitive to pertussis toxin and more efficacious in cells exhibiting medium than in those with high receptor density. AVP also stimulated the pertussis toxin-insensitive uptake of [3H]thymidine in CHO-V3 cells. The concentration-response curves for this effect were monophasic in cells expressing low and medium levels of V3Rs; on the contrary, a biphasic curve was observed in cells with high V3R density. Coupling of V3R to increased production of cAMP was only observed in CHOV3 high cells, suggesting a negative relationship between increased cAMP production and DNA synthesis. Activation of mitogen-activated protein kinases by V3R was pertussis toxin insensitive, but was dependent on activation of phospholipase C and protein kinase C; both the level and duration of activation were a function of the receptor density.
Thus, the human V3R has a pharmacological profile clearly distinct from that of the human V1R and V2R and activates several signaling pathways via different G proteins, depending on the level of receptor expression. The increased synthesis of DNA and cAMP levels observed in cells expressing medium and high levels of V3Rs, respectively, may represent important events in the tumorigenesis of corticotroph cells.
| Introduction |
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AVP is an important physiological regulator of the hypothalamo-pituitary-adrenal axis. AVP stimulates ACTH secretion by binding to a unique anterior pituitary receptor, distinct from the V1R and the V2R, which has been named the V3 or V1b receptor (3, 4, 5, 6, 7, 8, 9). Studies of the binding characteristics and signal transduction pathways activated after binding of AVP to its V3Rs have been hampered by the limited availability of this protein and were performed using either animal (rat, pig, and sheep) freshly isolated cells (3, 4, 10, 11, 12) or samples of human corticotroph adenomas (13). In these studies, occupancy of V3Rs by agonists triggered the sequential activation of phospholipase C (PLC) and protein kinase C (PKC), the phosphorylation of the myristoylated alanine-rich C kinase substrate, and secretion of ACTH (13, 14, 15). Conflicting data regarding coupling of the V3R to adenylyl cyclase have been also reported (11, 12, 16). No information is available regarding the nature of the G protein(s) and the kinases-phosphatases coupled to the V3R as well as the eventual mitogenic role of this receptor. Recently, different groups cloned several members of the family of human and animal AVP/oxytocin (OT) receptors (7, 17, 18, 19, 20, 21). Stable expression of these cloned receptors in mammalian cells now allows detailed characterization of the signal transduction pathways coupled to activation of a given receptor subtype by AVP.
Functional characterization of GPCRs (e.g. ß1- and ß2-adrenergic receptors, LH receptor, and the AVP V2-renal receptor) in mammalian cell lines indicates that a single receptor type can activate multiple second messenger pathways through interaction with one or more G proteins (22, 23, 24). In this context, the endogenous human TSH receptor activates not only Gs and Gq/11, but also members of the Gi and G12 families, indicating a complex modulation of downstream pathways (25). This pluripotential appears to be modulated by receptor density (22, 23, 24), as shown by the direct correlation between increased expression of Gs-coupled receptors and their ability to stimulate PLC in stably transfected L fibroblasts (23). Thus, GPCRs have the potential to couple to multiple signaling pathways, with the activation of a specific pathway being dependent on both the receptor density and the agonist concentration. These multiple pathways may play an important role in regulating the effectiveness of the signals in different tissues.
ACTH-secreting tumors are associated with abnormal pharmacological responses to AVP (26) and exhibit increased expression of the V3R gene, a marker of the corticotroph phenotype (8, 9). These findings highlight the relevance of understanding the role of V3Rs in normal and abnormal growth and development of the corticotroph cell.
In this study, we compared the ligand-binding characteristics of the human V3R with those of the human V1R and V2R and studied the cellular signals coupled to activation of different densities of human pituitary V3Rs as a prerequisite to understanding the role played by this receptor in the regulation of corticotropic cell function.
| Materials and Methods |
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-6
-diphenylglycoluril) was purchased from
Pierce Chemical Co. (Rockford, IL). Restriction and modification
enzymes were obtained from Boehringer Mannheim (Indianapolis, IN) or
New England Biolabs (Beverly, MA). [125I]Na (SA, 131
mCi/ml), [3H]AVP (SA, 48 Ci/mmol),
myo-[2-3H]inositol (SA, 20 Ci/mmol),
[3H]arachidonic acid (SA, 1 mCi/ml), and
[3H]thymidine (SA, 1 mCi/ml) were obtained from New
England Nuclear-DuPont (Wilmington, DE). The pBluescript II phagemid KS
and XL2-Blue Escherichia coli strain were obtained from
Stratagene (La Jolla, CA). The expression vector pZeoSV and the
antibiotic zeocin were purchased from Invitrogen (San Diego, CA). The
DNA sequencing kit was obtained from U.S. Biochemical Corp. (Cleveland,
OH). AVP, OT, arginine vasotocin, lysine vasopressin, and most of the
peptide V1, V2, V1/V3,
and OT agonists and antagonists were obtained from Bachem (Torrance,
CA) unless indicated otherwise. The nonpeptide V1
antagonist SR 49059 (batch MY10075) was provided by Dr. C.
Serradeil-Le Gal, Sanofi Recherche (Toulouse, France). The nonpeptide
rat V1 antagonist OPC 21268 (batch 93F92M) and the
nonpeptide V2 antagonist OPC 31260 (batch 93D96M) were
provided by Dr. J. F. Liard, Otsuka America Pharmaceutical
(Rockville, MD). The linear V1 antagonists
4-hydroxyphenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2
(OHPhaaGln) and
tert-butylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-Arg-NH2,
the V2 antagonist
d(CH2)5[D-Ile2-Ile4-Ala-NH2]AVP,
and the linear V1/V3 antagonist
phenylacetyl-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2
were provided by Dr. Maurice Manning, Medical College of Ohio (Toledo,
OH). The linear V1 AVP antagonist
phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-Tyr-NH2
(TyrPhaa), custom synthesized by Bachem, was radioiodinated
([125I]TyrPhaa) with the Iodogen technique and purified
by HPLC as previously described (27).
Complementary DNA (cDNA) constructs
We have previously described the isolation of a V3R
cDNA clone from a human pituitary tumor (7). To rule out the
possibility of mutations within the receptor sequence because of its
tumoral origin, we generated by PCR the human V3R cDNA from
normal human pituitary gland cDNA (human pituitary gland QUICK-clone
cDNA from Clontech, catalog no. 71731, lot 46910). The sense primer
5'-TGCTTGAAGTCCTCTGAACG-3' (nucleotides -167 to -148 in 24 and
reverse primer 5'-AAGACAGCACCATCCTAGGC-3' (nucleotides 15781597 in
Ref. 24, downstream from the stop codon and a native SpeI
site) were prepared in a synthesizer (Applied Biosystems, Foster City,
CA) and purified over oligonucleotide purification cartridges following
the manufacturers recommendations. The PCR reaction (final volume,
100 µl) contained 20 pmol of each primer, 2.5 U Taq
polymerase, 5 ng normal human pituitary gland cDNA, 25 µM
of each deoxy-NTP in buffer (10 mM Tris-HCl, 50
mM KCl, 1.5 mM MgCl2, and 0.1%
gelatin, pH 7.4). After initial denaturation at 95 C for 5 min, 30
cycles were run (95 C for 1 min, 55 C for 1 min, and 72 C for 2 min)
with a final extension at 72 C for 15 min. The PCR product was purified
with the GeneClean II system (Bio 101, Vista, CA). An SpeI
restriction site was introduced 140 bp upstream of the initiation codon
by PCR, and the SpeI-SpeI cDNA fragment was
digested and purified. The V3R
SpeI-SpeI construct was ligated into pBluescript
II phagemid vectors before transformation of XL2-Blue E.
coli strain. Double stranded DNA sequencing was performed with the
Taq Dye Deoxy Terminator cycle sequencing kit and a model
373A sequencer from Applied Biosystems. Nucleotide sequences were
analyzed and compared with the computer package GeneWorks on a
Macintosh computer (IntelliGenetics, Mountain View, CA).
We also generated a human V2R clone by PCR from human kidney cDNA (Clontech human kidney QUICK-Clone cDNA, no. 71121, lot 48570) using the sense primer 5'-CATCATGGGCCCACCATGCTCATGGCG-3' (which introduces an ApaI restriction site upstream of the start codon and includes the first 12 nucleotides in the open reading frame) (23) and the antisense primer 5'-ACACCCAGCTCAGTGAGCTG-3' (downstream from the stop codon and a native ApaI site, including nucleotides 13471366 in 23 . The correct sequence of this 1381-nucleotide long clone was verified by sequencing both strands before cell transfection. The conditions of the PCR amplification were as indicated above for the V3R clones. The ApaI-ApaI fragment was subcloned into pBluescript II as described above.
Selection and stable expression of AVP receptors in CHO
cells
Stable transfection of CHO cells with the V3R cDNA
clone ligated into the eukaryotic expression vector pZeoSV was
performed using the calcium phosphate precipitation method as described
previously (17). Clones were grown in medium F-12 supplemented with
10% FCS, selected with the neomycin analog zeocin, and purified by the
limiting dilution technique. Clones (CHO-V3) expressing
various densities of V3Rs were studied by radioligand
saturation and competition binding experiments as well as measurement
of inositol phosphate production, arachidonic acid release, cAMP
production, thymidine uptake, and mitogen-activated protein (MAP)
kinase activation as described below. Similarly, CHO cells were also
transfected with the pZeoSV expression vector containing the sequence
of the human V1R (CHO-V1) (17) or
V2R (CHO-V2) cDNAs.
Radioligand binding assays
Control and transfected CHO cells were grown to confluence in
24-well dishes and washed twice with PBS, 10 mM
MgCl2, and 0.2% BSA, pH 7.4. Competition-binding
experiments were performed as described previously (17), using one
fixed concentration of [3H]AVP for CHO-V2
cells and CHO-V3 cells or [125I]TyrPhaa for
CHO-V1 cells, in the presence or absence of increasing
concentrations of unlabeled AVP or peptide and nonpeptide analogs
(n = 35 for each analog) for 30 min at 30 C. IC50
values were derived from nonlinear least square analysis, and
Ki values were calculated by the equation of Cheng and
Prusoff: Ki = IC50/(1 +
Lf/Kd), where Lf and Kd
represent the concentration and dissociation constant of the
radioligand, respectively.
Also, saturation binding experiments of AVP receptors of transfected CHO cells were performed in 24-well dishes in duplicate with increasing concentrations of [3H]AVP with or without 1 µM unlabeled AVP (17). The affinity (Kd) and binding capacity (Bmax) of the AVP receptors were calculated by a nonlinear least square analysis program (28, 29). The protein concentration was measured with Pierces BCA reagent using albumin as an internal standard.
Inositol phosphate production
Subconfluent monolayer cultures of CHO-V3 cells were
grown for 48 h in 12-well dishes, washed with serum- and
inositol-free medium (Life Technologies, no. 885090 EC) and labeled
overnight with myo-[2-3H]inositol (1 µCi/well) in same
medium. The effect of pertussis toxin (PTX) on inositol phosphate
production was assessed by treatment with 0.5 µg/ml toxin during the
overnight labeling period. Thereafter, the cells were preincubated for
1 h in serum-free, inositol-containing medium, followed by 15-min
preincubation in HBSS containing 10 mM glucose, 1.2
mM CaCl2, and 10 mM LiCl.
Incubations were carried out for 30 min at 37 C in the presence of
increasing concentrations of AVP. The reaction was stopped by the
addition of 1 ml ice-cold methanol-HCl (100:1), followed by 400 µl
H2O and chloroform, to obtain a ratio of
chloroform/methanol/HCl of 200:100:1. Agonist-stimulated release of
inositol monophosphate was determined as described previously (17, 30).
The amount of inositol phosphate released was expressed as a percentage
of total labeled phosphoinositides. Typical incorporation into
phosphoinositides amounted to approximately 3.55 x 104
3H dpm/well.
[3H]Arachidonic acid release
Subconfluent monolayer cultures of transfected CHO cells grown
in 12-well plates were labeled overnight with 0.5 µCi/well
[3H]arachidonic acid in serum-free medium containing
0.1% BSA. In some experiments, PTX (0.5 µg/ml) was also added during
the labeling period. After labeling, the cells were washed five times
with serum-free medium containing 0.1% BSA. Incubation at 37 C for 10
min was started by the addition of 1 ml medium/well containing 0.1%
BSA, 100 µM unlabeled arachidonic acid, and increasing
concentrations of AVP. At the end of this period, 700-µl samples of
incubation medium were centrifuged at 14,000 x g for 2
min at room temperature. The release of [3H]arachidonic
acid was measured by counting 500-µl samples of the supernatants.
cAMP production
Subconfluent monolayer cultures of control and transfected CHO
cells were grown for 48 h in 12-well dishes and serum depleted in
F-12-HEPES medium for 4 h, followed by labeling in medium
containing 1 µCi/ml [3H]adenine for 2 h. Cells
were washed and incubated in medium containing 0.5 mM
isobutylmethylxanthine in the absence or presence of 10
µM forskolin and/or different amounts of AVP for 10 min.
The reaction was stopped by the removal of medium and the addition of
0.5 ml ice-cold 5% trichloroacetic acid (TCA) containing 1.5
mM cAMP. Separation of [3H]cAMP was carried
out as described by Evans et al. (31). The amount of
[3H]cAMP synthesized during the incubation with agonists
was expressed as a fraction of the total labeled nucleotides present in
the cell extracts (10-4 x cAMP dpm/total dpm in acid
extract).
[3H]Thymidime uptake
Subconfluent monolayer cultures of transfected CHO cells were
grown in 24-well plates to measure thymidine uptake in the presence of
AVP as described previously (32). In some experiments, the effect of
treatment with either PTX (0.5 µg/ml) or forskolin (10
µM) was assessed by adding these reagents during the last
24 h of culture in serum-free medium. Cells were washed with 500
µl F-12 medium and grown for 72 h in 500 µl F-12 medium
supplemented with 25 mM HEPES and 0.1% BSA. Cells were
treated with increasing concentrations of AVP (ranging from
10-1210-6 M) for 18 h,
followed by incubation with 0.5 µCi [3H]thymidine for
45 min. The cells were subsequently transferred on ice, washed twice
with 0.5 ml ice-cold PBS, fixed with 1 ml ice-cold 10% TCA for 30 min
at 4 C, washed twice with 1 ml ice-cold 5% TCA solution, and
solubilized with 250 µl 0.1 N NaOH0.1% SDS.
Radioactivity present in samples of the extracts was measured using a
scintillation counter. Similar experiments were conducted with a
[3H]thymidine pulse for 6 h.
Cell proliferation assay
Subconfluent monolayer cultures of transfected CHO cells were
grown in 96-well plates to measure cell proliferation in the presence
of AVP using the CellTiter 96 cell proliferation assay from Promega
(Madison, WI). Cells were washed with F-12 medium and grown for 72
h in 100 µl F-12 medium supplemented with 25 mM HEPES and
0.1% BSA. Cells were treated with 10% FBS or AVP for 18 h,
followed by incubation with 15 µl dye solution for 4 h according
to the manufacturers instructions. Subsequently, 100 µl
solubilization solution were added, and after mixing for 1 h at
room temperature, absorbance was recorded at 570-nm wavelength using an
enzyme-linked immunosorbent assay plate reader (650-nm reference
wavelength). Values recorded for positive control (cells not stimulated
by AVP or FCS) were subtracted.
Phosphorylation of MAP kinase (MAPK)
Phosphorylation of p42 and p44 MAPKs by AVP was measured in
subconfluent monolayer cultures of transfected CHO cells grown in
12-well plates. Cells were serum starved for 48 h before agonist
stimulation. In some experiments, pretreatment with PTX (0.5 µg/ml)
took place during the last 18 h of culture in serum-free medium.
After stimulation with the various compounds described in
Results, cells were washed twice with ice-cold PBS and lysed
in buffer containing 50 mM HEPES-Tris (pH 7.5), 150
mM NaCl, 1.5 mM MgCl2, 1
mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM
phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, 10 µg/ml
pepstatin, 50 µg/ml bestatin, 200 µM
Na3PO4, and 1 mM NaF. The lysed
cells were scraped and centrifuged at 14,000 x g for
20 min. The supernatants were subjected to SDS-PAGE. After
electrophoresis, proteins were transferred to Immobilon-P membrane
(Millipore, Bedford, MA) and immunoblotted overnight at 4 C with the
phospho-specific p44/42 MAPK (Tyr204) antibody (rabbit
polyclonal IgG affinity-purified antibody from New England Biolabs,
Beverly, MA). Immunodetection was carried out with the ECL kit
(Amersham, Arlington Heights, IL) following the manufacturers
recommendations. Quantification of MAPK phosphorylation was performed
by scanning densitometry of the autoradiograms with a U.S. Biochemical
Corp. SciScan 5000 automated scanning system (Cleveland, OH).
Data analysis
Nucleotide and amino acid sequences were analyzed with the
computer package GeneWorks on a Macintosh computer (IntelliGenetics).
Binding parameters (Kd and Bmax) of AVP
receptors and the pharmacodynamic profile of stimulation of
intracellular signals by AVP (EC50 and Emax)
were calculated using a nonlinear least square analysis program (28).
Data were expressed as the mean ± SEM.
| Results |
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The binding profile of the human V3R clone expressed in CHO
cells (CHO-V3) is clearly distinct from those of the human
V1 vascular and V2 renal AVP receptor subtypes
expressed in the same CHO cell line (Table 1
). None of the 20 AVP analogs tested in
our experiments had a higher affinity for the human V3R
than the endogenous ligand AVP. OT, the other endogenous ligand in
humans, has a very low affinity for the V3R (1782
nM). Interestingly, the two V3 antagonists we
tested exhibited a significantly higher affinity for the
V1R than for the V3R, thus confirming the
current lack of potent and selective antagonists for the V3
receptor. The V1 antagonist
4-hydroxyphenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2,
which displays an excellent affinity for the V1R
(Kd = 0.45 nM) also binds to the
V3R with a good affinity (Kd = 2.22
nM). Although the two V2 agonists tested in
these experiments (dDAVP and dVDAVP) indeed show a good affinity
for the human V2R (2.7 and 0.8 nM,
respectively), they are not very selective, as they also bind to the
human V1R and V3R with dissociation constants
in the 1020 nM range. Along the same line, the two
peptide V2 antagonists tested in these experiments
(d(CH2)5[D-Ile2-Ile4-Ala-NH2]AVP
and
d(CH2)5[D-Ile2-Ile4-Arg8-Ala9-NH2]AVP)
show a rather low affinity for the human V2R (88 and 76
nM), which is even weaker than that for the human
V1R (53 and 30 nM). This lack of selectivity
for the human receptors may explain some of the discrepancies with the
subtype of AVP receptor involved in the vasodilatory action of dDAVP or
dVDAVP and the inhibitory properties of the antagonists tested (34).
The two peptide OT antagonists tested in our experiments
(d(CH2)5[O-Me-Tyr2-Thr4-Orn8]vasotocin
and
d(CH2)5[O-Me-Tyr2-Thr4-Orn8-Tyr9-NH2]vasotocin)
also display high affinity for the human V1R, but bind
poorly to the human V3R. Finally, the three nonpeptide
V1R or V2R antagonists tested in our cell lines
(SR49059, OPC-21268, and OPC-31260) have a low affinity for the human
V3R. These results underscore the need for developing
specific and potent analogs, interacting with the various human AVP
receptor subtypes.
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| Discussion |
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Before the cloning and functional expression of the human pituitary V3R, the pharmacological characterization of this receptor has been very limited, relying upon experiments conducted in primary cultures of pituitary or corticotroph cells of human or animal origin. Anterior pituitary cells present in dispersed primary cultures constitute a heterogeneous mixture of well differentiated secretory cell types, including somatotropes, lactotropes, thyrotropes, gonadotropes, and corticotropes. Because of this cellular heterogeneity and the demonstrated presence of several subtypes of AVP/OT receptors in the anterior pituitary gland, development of a homogeneous cell line expressing a defined receptor subtype was central to exploring its signaling properties. No information was available in the literature regarding coupling of V3Rs to G protein coupling, MAPK activation, and cell proliferation in normal and tumorous human corticotroph cells. Although there has been no direct assessment of the level of V3R density in normal and tumorous human corticotroph cells, Dahia et al. recently reported a 2- to 5-fold increase in V3R messenger RNA levels in human ACTH-secreting tumors (8). Although it is possible to infer from the level of expression of human endogenous vascular V1Rs and renal V2Rs that CHO-V3Low clones express endogenous levels of V3Rs presumably present in normal human corticotroph cells, the exact relationship of the expression level in CHO-V3Medium and CHO-V3High to human pathophysiology remains to be established.
Stable expression of the human pituitary V3R in CHO cells
allowed us to carry out a complete characterization of the
ligand-binding properties of this AVP receptor subtype in comparison to
the other two subtypes, i.e. the vascular V1R
and the renal V2R. Our studies show that the linear
V1/V3 antagonist
phenylacetyl-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2,
which displayed the highest affinity for the porcine V3R
(Ki = 37.2 nM) among AVP analogs recently
available, also exhibits a reasonable affinity for the human
V3R in our studies (Ki = 15.9
nM); however, it behaves as a more potent V1R
analog in both species. In this context, it appears that the
V1R antagonist 4-OH-Phaa, the most potent V1R
compound tested in our series of experiments, also displays a high
affinity for the human V3R (Ki = 2.2
nM). As this compound can be radioiodinated, it may replace
tritiated AVP as a radioligand with high specific activity to carry out
further ligand binding characterization of the human V3R.
Recently, Barberis et al. (35) reported that the same
compound displays a high affinity for the rat V3R
(Ki = 4.5 nM) and significantly blocked
AVP-induced ACTH release in cultured rat pituitary cells. The results
in Table 1
indicate that no compound displays higher affinity for the
human V3R than the native hormone AVP itself, and none is
selective for this receptor subtype. This underscores the need to
develop new selective agonists for this receptor subtype. The
availability of the CHO-V3 cells should facilitate the
screening and development of such selective ligands. As summarized
above, little is known about the characteristics of endogenous human
V3Rs expressed in corticotroph cells or the level of
expression of V3Rs in human normal and tumorous
corticotroph cells.
The pathway(s) linking activation of AVP receptors to regulation of
specific effectors through G protein(s) is the object of our
investigations. For instance, two pathways of activation of
phosphoinositide hydrolysis mediated through G proteins can be
distinguished by their sensitivity or insensitivity to PTX. Gß
dimers have been proposed to mediate PTX-sensitive phosphoinositide
hydrolysis, whereas members of the Gq/11 family participate
in the PTX-insensitive activation of PLCß (36). Experiments by Exton
and colleagues showed that the rat liver V1 AVP receptor
couples to two distinct G proteins (37), and our data with human
platelets are consistent with the human V1-vascular AVP
receptor coupling to Gq/11 (38). In human myometrium, OT
activates PLCß by interacting with at least two G proteins of the
Gq and Gi families (39). The G protein(s)
linked to the human V3R is currently unknown. Our results
suggest that the human V3R is coupled to several G proteins
of the Gq, Gi, and Gs families,
depending on the level of expression of this receptor. Moreover, it
appears that the coupling of V3R to different effectors
(PLC, PLA2, MAPKs, and DNA synthesis) proceeds through
different combinations of G proteins. PLC activation after AVP binding
to the V3R is only partially sensitive to PTX treatment,
thus suggesting the involvement of proteins of the Gq/11
and Gi families. Moreover, the degree of stimulation of PLC
seems to be positively related to the receptor expression level,
without intervention of counterregulatory mechanisms. On the other
hand, activation of arachidonic acid release after AVP binding to the
V3R was dependent on the level of receptor expression. Both
low and medium densities of V3Rs stimulated the release of
arachidonic acid with a monophasic concentration-response pattern,
whereas activation of CHO-V3High resulted in a diminished
response at relatively high concentrations of agonists compared with
that in cells expressing medium receptor density. This result suggested
the recruitment of additional regulatory mechanisms after activation of
a high number of V3Rs. The difference in PTX sensitivity of
arachidonic acid release in CHO cells expressing different levels of
V3R could be interpreted as indicative of the recruitment
of different forms of PLA2 or different G proteins.
Examination of these issues as well as identification of different
forms of PLA2 coupled to V3R require further
studies.
The uptake of [3H]thymidine triggered by the
V3R was clearly dependent on the level of receptor
expression. The decreased stimulation of thymidine uptake by AVP
observed in the CHO-V3High cells appears to represent
differential coupling of this receptor to adenylyl cyclase, as
suggested by both the inhibition of this response driven by occupancy
of the Gs-coupled V2R and the significant
increase in cAMP production that was only observed in agonist-treated
CHO-V3High. It is possible to hypothesize that after
expression of high levels of V3Rs, coupling of activated
receptors to G proteins becomes less specific or relaxed, leading to
the recruitment of additional transducers that are not engaged at lower
receptor densities. This multiple signaling potential seems to
represent a shared feature among G protein-coupled receptors, as shown
by stimulation of both adenylate cyclase and PLC by four classical
Gs-coupled receptors (LH, V2,
ß1-, and ß2-receptors) expressed at high
levels in L cells (23). Nonetheless, the signaling pluripotential of G
protein-coupled receptors is not necessarily caused by receptor
overexpression, as exemplified by the endogenously expressed human TSH
receptor that couples to all four G protein families, Gs,
Gi, Gq/11, and G12 (25),
and by the dual coupling of endogenous ß-adrenergic receptors in
COS-7 cells to adenylyl cyclase and MAPK activation (due to release
of
s- and ß
-subunits, respectively) (40).
The mechanisms underlying dual activation of G proteins are not completely understood. As far as AVP receptors are concerned, a recent publication by Liu and Wess provides useful information about the receptor domains involved in functional coupling (41). Construction of rat V1/human V2 AVP receptor hybrids showed that the second intracellular loop of the V1 receptor activates PLC, whereas receptors containing the third intracellular loop of the V2 receptor stimulate cAMP production. This bifunctionality of the hybrid AVP receptors was not due to a totally relaxed interaction with G proteins, as no coupling of Gi to this receptor was observed. Conversely, Wong and Ross (42) reported that chimeric muscarinic cholinergic:ß-adrenergic receptors (third intracellular loop of one receptor was replaced by that of the other one) were promiscuous in their coupling to G proteins. Obviously, additional studies are required to decipher the molecular domains responsible for multiple activation of G proteins after occupancy of different levels of cognate receptors by agonists.
Receptor tyrosine kinases are involved in the regulation of cell
proliferation and differentiation. Several kinases participate in this
process, including the phosphoinositide 3-kinase, S6 kinase, MAPK,
and Jak/STAT pathways. The role of each pathway in cell signaling
varies with the cell type and agonist studied. The MAPKs are a point of
convergence for mitogenic signals triggered by several classes of cell
surface receptors, including the GPCRs. Gi- and
Gq-coupled receptors stimulate MAPK activation via distinct
signaling pathways (43). In transfected COS-7 cells, MAPKs can be
stimulated by Gs, Gi, and Gq
through participation of both
- and ß
-subunits (44). AVP
stimulation of V1-vascular receptors transiently activates
MAPKs through a PTX-independent pathway (45). At least two major
pathways link V1-vascular receptors to MAPK (46): one
involves TPA-sensitive PKC, and the other involves wortmannin-sensitive
molecules such as phosphoinositide 3-kinase. Among other members of the
AVP/OT family of receptors, OT receptors of human uterine myometrium
stimulate MAPK phosphorylation via a PTX-sensitive G protein (47). In
the studies here described, we observed that activation of
V3Rs leads to stimulation of the MAPK pathway. This action
is specifically blocked by a V1/V3 antagonist,
whereas both V1- and V2-selective antagonists
are ineffective. This activation of MAPK after occupancy of
V3R is physiologically relevant, as the corresponding
EC50 values are in agreement with the Kd
measured in the binding experiments. The lack of effect of PTX on
AVP-induced MAPK phosphorylation suggests participation of a
PTX-insensitive G protein/s, presumably of the Gq/11
family. This toxin insensitivity is unlikely to reflect a partial
uncoupling of the V3R to Gi due to the fact
that this rather extensive treatment (0.5 µg/ml PTX, 1824 h)
results in almost complete inhibition of signaling driven by ATP
receptors expressed in the same CHO cell line (48) and in approximately
50% inhibition of AVP-dependent arachidonic acid release. MAPK
activation is PLC and PKC dependent, as suggested by the experiments
performed in the presence of neomycin or staurosporine. This profile is
different from that of angiotensin II, which activates MAPK in a
PKC-independent manner in vascular smooth muscle cells (49). The
down-regulation of PKC activity in CHO cells by chronic exposure to
phorbol ester completely blocks Gq/11-mediated mitogenic
signals, whereas Gi-mediated signals are unaffected (43).
These findings are in agreement with our own data.
Cellular responses are determined by the duration of MAPK activation. The ability of a receptor to produce longer or more sustained activation of MAPK has been shown to determine the ability of a cell to differentiate or proliferate, depending on the cell type. For instance in PC12 cells, sustained MAPK activation is associated with translocation of MAPKs to the nucleus, whereas transient activation does not lead to nuclear activation (50). Along the same line, differences in V3R receptor number markedly affected the duration of MAPK activation. Indeed, MAPK stimulation induced by AVP in CHO cells expressing high densities of V3Rs was more intense and long lasting than in the other cell types.
These findings raise the issue of the role of V3R in the development of ACTH-secreting tumors, which have been shown to express high levels of V3Rs (8, 9). Two of our observations appear linked to the characteristics of ACTH-secreting corticotroph tumors. First, initial up-regulation of V3Rs in these tumors and the augmented uptake of [3H]thymidine driven by medium range densities of this receptor, suggest that AVP may play at least a contributing role in uncontrolled corticotroph proliferation. Second, and considering the presence of cAMP-responsive elements in the POMC gene (51) and the recognized effect of cAMP-elevating agents in ACTH secretion, the increase in levels of this second messenger after activation of high densities of V3R may imply the participation of AVP in the augmented secretion of ACTH in these tumors.
Also, the question remains as to whether other kinases contribute to
the mitogenic response triggered by V3Rs and the role of
activation of these kinases by V3R in normal and
pathological corticotroph cell growth. A counterintuitive observation
was that although activation of both medium and high densities of
V3Rs triggered a significant activation of p44/p42 MAPKs,
only the former resulted in increased [3H]thymidine
uptake. Incubation of cells with a MAP kinase kinase inhibitor (10
µM PD98059) completely prevented AVP-induced activation
of p42/p44 on CHO-V3Medium cells, but resulted in only a
3050% inhibition of agonist-induced [3H]thymidine
uptake (data not shown). This indicates that the effect of AVP on cell
proliferation implies the recruitment of both p42/p44 MAPK-dependent
and -independent pathways. This opens the possibility that occupancy of
the V3R by agonist may trigger simultaneous activation of
other MAPK cascades, such as p38 or Jun-N-terminal
kinase/stress-activated protein kinase, which are more critical for the
proliferative response. In this context, the observation that
arachidonic acid mediates activation of Jun-N-terminal
kinase/stress-activated protein kinase (52) may explain the partial
proliferative response triggered by AVP in CHO-V3High
compared with CHO-V3Medium cells. It could be argued that
the reduced proliferative effects triggered by high concentrations of
AVP in CHO-V3High cells result from an increase in cAMP
levels, leading to activation of PKA and raf inhibition
(53). Indeed, there is a cross-talk between the MAPK pathway and the
cAMP signaling pathway, leading to a dual effect: inhibition by PKA and
stimulation by G-protein ß
dimers (54, 55). As expected, we have
observed a partial inhibition of AVP-dependent p42/p44 activation after
treatment of CHO-V3Medium with 10 µM
forskolin (results not shown). These observations indicate that
although increased levels of cAMP and PKA activity inhibit
agonist-dependent MAPK activation, this cannot explain the reduction in
cell proliferation by AVP in CHO-V3High cells due to the
combined evidence that these cells exhibit the highest stimulation of
p42/p44 and that complete inhibition of AVP-induced MAPK activation
(using PD98059 results only in partial inhibition of cell
proliferation. This suggests that 1) AVP recruits both MAPK-dependent
and -independent pathways leading to cell proliferation; 2) elevation
of cAMP levels blocks both pathways; and 3) the MAPK-independent
pathway appears quantitatively more important (based on both the
partial effect of the MAP kinase kinase inhibitor on thymidine uptake
in CHO-V3Medium cells and the robust activation of p42/p44
by AVP in CHO-V3High cells. Although these studies in
transfected CHO cells represent an important step in defining the
signaling pathways coupled to activation of different densities of
V3Rs, it is important to mention that the relevant
downstream effectors may vary in each host cell, reflecting conditions
such as relative expression of G proteins and/or effector subtypes.
Clearly, the host cell specificity is an important consideration in
extrapolating the conclusions from this study to the phenotype of
corticotroph tumors. This phenotypic modulation was recently
illustrated by Calleja et al., who showed that the effect of
cAMP on cell proliferation depended on both the type of tyrosine kinase
receptor present and the cell type involved (56). Comparative studies
examining AVP-dependent activation of multiple MAPK pathways after
stimulation of corticotroph cell lines (such as AtT20) expressing
different densities of V3Rs will define this aspect in more
detail.
From these experiments using CHO cells expressing various densities of
V3Rs, several conclusions can be drawn. More than one G
protein seems to participate in signal transduction pathways linked to
V3Rs. The pattern of activation of a given signal is
dependent on both the level of V3R expression and the
concentration of agonist. For some cellular responses, including
activation of PLC and MAPKs, the degree of stimulation by AVP is
directly proportional to both the concentration of agonist and the
level of expression of V3Rs. Moreover, only G protein(s) of
the Gq/11 class seem to be involved in these signals. For
other cellular responses, including activation of PLA2,
cAMP production, and thymidine uptake, high levels of V3R
expression seem to couple simultaneously to both stimulatory and
inhibitory components in the presence of high concentrations of AVP.
Also, several G proteins, including Gs, Gi and
Gq/11 classes, mediate recruitment of these pathways. The
increased synthesis of DNA and cAMP levels observed in cells expressing
medium and high levels of V3Rs, respectively, may represent
important events in the induction and phenotype maintenance of
ACTH-secreting tumors. The different intracellular pathways linked to
the human V3R are summarized in Fig. 10
.
|
| Acknowledgments |
|---|
| Footnotes |
|---|
Received February 10, 1997.
| References |
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G
s-cAMP-mediated inhibition. J Biol Chem 270:2525925265
subunits of heterotrimeric G proteins stimulate the
mitogen-activated protein kinase pathway in COS-7 cells. J Biol
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