(Received for publication, June 22, 1995; and in revised form, September 11, 1995)
From the
A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from
the PKSV-PCT renal proximal tubule cell line which expressed peptide YY
(PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A.,
and Laburthe, M.(1993) J. Biol. Chem. 268, 20547-20554).
In order to identify G protein(s) coupled to PYY receptors,
antisense G
protein RNAs were expressed in Cl.10 cells
by transfecting the pcDNA3 vector into which were inserted 39 bases of
the 5`-noncoding region of G
or G
used as specific antisense templates. A
Cl.10/
clone was selected which
displayed a drastic decrease (>90%) of the expression of
G
without changes of G
,
G
, and G
subunits (G
is not
present in Cl.10 cells) as evidenced by Western blots. When compared to
untransfected cells, this clone exhibited: (i) an increase in the
dissociation constant of PYY receptors (5.3 versus 0.6
nM) identical to that observed in pertussis toxin-treated
untransfected cells; (ii) an absence of inhibition of
I-PYY binding by guanosine
5`-O-(thiotriphosphate) (GTP
S); and (iii) the failure of
PYY to inhibit cAMP levels and to stimulate
[methyl-
H]thymidine incorporation into
DNA. A clone was also selected which exhibited a specific decrease
(>80%) of G
as compared to untransfected cells.
The sensitivity to GTP
S and the dissociation constant of PYY
receptors as well as PYY-mediated inhibition of cAMP were identical to
those observed in untransfected cells. These findings support an
exclusive coupling of PYY receptors to G
.
Following its discovery in rat intestinal epithelial
cells(1) , the peptide YY (PYY) ()receptor has been
characterized in dog adipocytes (2) and the proximal tubule
PKSV-PCT cell line derived from kidneys of transgenic mice(3) .
This receptor is PYY-preferring since it binds the intestinal hormone
PYY (4) with high affinity and the neuropeptide NPY (4) with a 10-fold lower
affinity(1, 2, 3, 4) . PYY and NPY
triggers several biological effects through interaction with PYY
receptors, including inhibition of adenylyl cyclase
activity(2, 3, 4, 5) , inhibition of
Cl
secretion in the small
intestine(4, 6, 7) , inhibition of lipolysis
in fat cells(2) , and stimulation of epithelial cell
growth(3, 8, 9) . The PYY receptor resembles
the Y2 subtype of NPY receptor (9, 10, 11) which does not discriminate
between PYY and NPY but, like the PYY receptor, binds long
COOH-terminal fragments of PYY or NPY (5, 7) . Its
pharmacology is clearly different from that of other receptors for the
PP-fold family of peptides, including the Y1 and Y3 subtypes of NPY
receptors and PP receptors(9, 10, 11) . Like
most receptors for this family of peptides(9) , with the
exception of the Y1 subtype of NPY
receptor(12, 13, 14) , the PYY receptor is
not yet cloned. However, it has been characterized as a M
44,000 glycoprotein by cross-linking experiments
and hydrodynamic studies (15) .
Recent studies characterized
PYY receptors in the PKSV-PCT cell line (3) derived from
microdissected proximal convoluted tubules of kidneys from transgenic
mice harboring the simian virus 40 (SV40) large T antigen placed under
the control of the rat L-type pyruvate kinase 5`-regulatory
sequence(16, 17) . PYY receptor-mediated events are
triggered through interaction of PYY receptors with pertussis
toxin-sensitive G proteins in PKSV-PCT cells. Indeed,
preincubation of cells with pertussis toxin completely reverses the
PYY-induced inhibition of cAMP production and stimulation of cell
growth and converts PYY receptors to a low affinity state(3) .
In view of the fact that (i) multiple G
proteins including
G
, G
, and G
can contribute to
heptahelical receptor-mediated inhibition of adenylyl cyclase (18) and (ii) pertussis toxin-sensitive G proteins have been
shown to be crucial for the mitogenic action of several
agents(19) , the characterization of the pertussis
toxin-sensitive G
protein(s) coupled to PYY receptors is an
important step leading to further understanding of the mechanism of
action of this recently discovered receptor.
In the present work, we
have developed the stable expression of antisense G RNA in a clone PKSV-PCT Cl.10 (referred to as Cl.10 below)
isolated from the parent PKSV-PCT cells in order to identify G
proteins coupled to PYY receptors. By studying receptor affinity
and regulation of ligand binding by GTP
S, inhibition of cAMP
production and stimulation of cell growth in Cl.10 cell clones in which
endogenous G
proteins were permanently down-regulated
after transfection with antisense G
expression
vectors, we provide evidence for the exclusive coupling of the PYY
receptor to the G
protein.
Figure 1:
Peptide
specificity of PYY receptors and PYY-induced inhibition of cAMP in
Cl.10 cells. A, peptide specificity of PYY receptors was
investigated with membranes from Cl.10 cells. Membranes were incubated
with 0.05 nMI-PYY and increasing concentrations
of unlabeled PYY (
), NPY (
), or PP (
) as described
under ``Experimental Procedures.'' Nonspecific binding was
determined in the presence of 1 µM unlabeled PYY. Results
are the means ± S.E. from three experiments. B,
inhibition of forskolin-stimulated cAMP production in Cl.10 cells.
Cellular cAMP content was determined on cells pretreated (
) or not
(
) with 0.4 µg/ml pertussis toxin for 18 h. Thereafter, cells
were incubated in the presence of 10
M
forskolin and increasing amounts of PYY for 40 min at 37 °C. The
cAMP content was determined as described under ``Experimental
Procedures.'' Each value is the mean ± S.E. of three
determinations.
Figure 2:
Western blot analysis of G and G
subunits of G
and G
proteins in Cl.10 cells and
Cl.10/
cells. Cell membrane
proteins (50 µg/lane) were subjected to 10% acrylamide slab gel
electrophoresis. After transfer onto nitrocellulose sheets, bands were
revealed using antisera against the
/
,
/
,
, or
subunits of G proteins. Gels were calibrated with several molecular
weight marker proteins as described under ``Experimental
Procedures.'' For the sake of clarity, only two protein markers
are shown, i.e. ovalbumin (43 kDa) and carbonic anhydrase (29
kDa). The G
protein which would migrate above the
G
protein in these electrophoresis conditions (23) was not detected in Cl.10 cells. The same holds true for
the G
protein which is not expressed in epithelial
cells. For details, see ``Experimental
Procedures.''
Figure 3:
Localization of G and
G
subunits in Cl.10 cells by indirect
immunofluorescence using confocal laser scanning microscopy. Cells
grown on 12-mm glass coverslips were permeabilized with saponin and
then incubated with anti-
(left) or
anti-
(right) antibodies. After subsequent
incubation with fluorescein isothiocyanate goat anti-rabbit antibody,
cells were analyzed by confocal microscopy as described under
``Experimental Procedures.''
Figure 4:
Light microscopic appearance of Cl.10
cells and Cl.10/ cells. Top, light micrograph of Cl.10 cells (A) and
Cl.10/
cells (B) grown on
porous filters. Filters were fixed in Bouin's fluid and embedded
in paraffin, and cross sections were stained with hematein eosin and
examined. Bars, 10 µm. Bottom, phase-contrast
micrograph of confluent Cl.10 cells (C) and
Cl.10/
cells (D) grown on
plastic Petri dishes. Note the presence of numerous domes, indicating
the fluid transport capacities of both cell clones. Bars, 50
µm.
Figure 5:
PYY
binding to Cl.10 cells and after expression of antisense G RNA in the Cl.10/
clone:
Scatchard analysis and effect of GTP
S. A, saturation
analysis was conducted as described under ``Experimental
Procedures'' in the presence of a fixed concentration of
I-PYY (0.05 nM) and increasing concentrations of
unlabeled PYY. Nonspecific binding was determined in the presence of 1
µM unlabeled PYY. Binding experiments were performed on
membranes prepared from Cl.10 cells (
), Cl.10 cells pretreated
overnight with 0.4 µg/ml of pertussis toxin (
) or
Cl.10/
cells (
). Scatchard
plots were analyzed using the LIGAND computor program(22) .
Results shown are from a typical experiment. Two other experiments gave
similar results. B, effect of GTP
S on PYY binding to
membranes from Cl.10 cells (
) and
Cl.10/
cells (
). Experiments
were carried out in the presence of a fixed concentration of
I-PYY (0.05 nM) and increasing concentrations of
GTP
S. Nonspecific binding was determined in the presence of 1
µM unlabeled PYY and substracted from total
binding.
As PYY
inhibited cAMP production and stimulated cell growth in the mouse
proximal tubule cell line PKSV-PCT(3) , we further investigated
the influence of expression of G antisense RNA on
both processes in Cl.10/
and
control Cl.10 cells. As shown in Fig. 6, PYY inhibited both
basal and forskolin-stimulated cAMP production in Cl.10 cells. In
contrast, PYY failed to alter basal and forskolin-stimulated cAMP
levels in Cl.10/
cells (Fig. 6). Therefore, it appeared that the down-regulation of
G
expression in Cl.10/
cells completely reversed the PYY receptor-mediated inhibition of
cAMP production. As previously observed in F9 teratocarcinoma
cells(20) , the suppression of G
did not
change basal or forskolin-stimulated cAMP levels which were identical
in Cl.10/
and control Cl.10 cells.
This suggested that G
itself does not play a major
role in the control of cAMP production unless it is activated by
receptors such as the PYY receptor. As shown on Fig. 7, PYY
stimulated the incorporation of
[methyl-
H]thymidine into DNA of Cl.10
cells, whereas it had no effect in Cl.10/
cells. This is in line with the fact that cell growth in Cl.10
cells is a forskolin sensitive cAMP-dependent process (data not shown).
Altogether these data support the notion that G
is
responsible for the coupling of PYY receptors to adenylyl cyclase and
the subsequent stimulatory effect on cAMP-dependent incorporation of
[methyl-
H]thymidine into DNA. However,
the decrease in [methyl-
H]thymidine
incorporation into DNA in Cl.10/
cells (Fig. 7), where there is no change in basal cAMP
level (Fig. 6) suggested that G
might also
participate in cAMP-independent pathway(s) for the control of DNA
synthesis in Cl.10 cells. The microtubule-associated protein kinase
cascade which can be activated by G
(28, 29, 30) is a good candidate.
Figure 6:
Effect of PYY on basal and
forskolin-stimulated cAMP levels in Cl.10 cells or after expression of
antisense G RNA in
Cl.10/
cells or antisense
G
RNA in Cl.10/
cells. PYY effect on cAMP production was investigated in Cl.10,
Cl.10/
cells or
Cl.10/
cells. Cells were incubated
with (right) or without (left) 10
M forskolin in the absence (hatched bars) or in
the presence (solid bars) of 1 µM PYY for 30 min
at 37 °C. The cellular cAMP content was then determined as
described under ``Experimental Procedures.'' Each value is
the mean ± S.E. of three determinations. *p < 0.001 versus control without PYY; NS,
nonsignificant.
Figure 7:
Effect of PYY on
[methyl-H]thymidine incorporation into
DNA of Cl.10 cells or after expression of antisense G
RNA in Cl.10/
cells. Two days
after seeding, cells were cultured in fetal calf serum-deprived medium
in the presence (solid bars) or absence (hatched
bars) of 0.1 µM PYY for 18 h. Cells were then pulsed
for the last 6 h of incubation with 0.5 µCi/ml
[methyl-
H]thymidine as described under
``Experimental Procedures.'' Values are the means ±
S.E. from 12 experiments. *p < 0.005 versus control without PYY; NS,
nonsignificant.
Figure 8:
Western blot analysis of G and G
proteins and Scatchard plot of PYY
binding after expression of antisense G
RNA in the
Cl.10/
clone. A, Western
blot analysis of G
and G
subunits
of G
proteins in membranes prepared from
Cl.10/
and control Cl.10 cells.
Cell membrane proteins (50 µg/lane) were subjected to 10%
acrylamide slab gel electrophoresis. After transfer on nitrocellulose
sheets, bands were revealed using antisera against the
/
or
/
subunits of G proteins. Gels were calibrated with several
molecular weight marker proteins as described under ``Experimental
Procedures.'' For the sake of clarity, only two protein markers
are shown on the figure, i.e. ovalbumin (43,000) and
carbonic anhydrase (29,000). For details, see
``Experimental Procedures.'' B, PYY binding to
membranes from Cl.10/
cells (
)
and control Cl.10 cells (
). Membranes were incubated with
I-PYY (0.05 nM) and increasing concentrations of
unlabeled PYY. Nonspecific binding was determined in the presence of 1
µM unlabeled PYY. Results are from a typical experiment.
Another experiment gave similar data. See ``Experimental
Procedures'' for details.
The present investigation which takes advantage of the
powerful antisense RNA technology is the first to demonstrate the
coupling of PYY receptors to the G protein. This was
possible because selection of 39 bases of the 5`-noncoding region of
G
or G
(20) for use as
antisense templates provided the necessary nucleotide sequence
specificity, e.g. only 48% identity in this
region(31, 32) , whereas selection of templates in the
coding region with >85% identity (31, 32) would
have probably failed to ensure such specificity. In fact, the isolation
of clones after transfection of Cl.10 cells with antisense
G
or G
expression vectors resulted
in stable cell lines which showed a 90% decrease of G
(Cl.10/
cells) and 80% decrease of
G
(Cl.10/
cells),
respectively. The mechanism whereby the production of antisense RNA in
those cells blocks the expression of targeted proteins is not known but
hybridization of antisense RNA with the corresponding mRNA was shown to
prevent translation and/or to enhance mRNA degradation (reviewed in (33) ).
A great deal of evidence argues for an exclusive
role of G among other candidate G
proteins for
mediating PYY receptor signal transduction in the mouse kidney proximal
tubule cell clone Cl.10 isolated from PKSV-PCT cells. Thus, the
Cl.10/
clone in which the synthesis
of the G
protein was down-regulated by expression of
antisense G
RNA exhibits the following properties:
(i) an increase of the dissociation constant of PYY receptors which was
identical to that observed when the untransfected Cl.10 cells had been
pretreated with pertussis toxin. Since G
and
G
are substrates for pertussis toxin and
G
is not present in Cl.10 cells, these data alone
strongly suggest that G
does not participate
significantly in the direct coupling of PYY receptors with G proteins.
(ii) The inhibition of PYY binding by GTP
S could not be observed,
again ruling out a major role of G
in controlling the
dissociation of PYY from PYY receptors. (iii) Basal and
forskolin-stimulated cAMP levels as well as incorporation of
[methyl-
H]thymidine into DNA were
totally unaffected by PYY, suggesting that G
was also
crucial for PYY receptor-mediated events. Finally, the fact that the
Cl.10/
clone, in which the
synthesis of the G
protein was specifically
down-regulated by expression of antisense G
RNA, did
not exhibit any modification in the dissociation constant of PYY
receptor or in the sensitivity to GTP
S further confirmed that
G
was not coupled to PYY receptors in Cl.10 cells.
Furthermore, the subcellular distribution of G
and
G
in Cl.10 cells, as determined by confocal laser
microscopy, is consistent with the coupling of PYY receptors to
G
rather than to G
. Indeed,
G
is found associated mainly with plasma membranes
where PYY receptors (3) and adenylyl cyclase (18) are
located; in contrast G
is preferentially localized on
the perinuclear Golgi complex. The localization of G
is in line with recent observations indicating that G
is involved in intracellular processes in epithelial cells such
as autophagic sequestration (34) and Golgi
trafficking(35, 36, 37) . Finally, the
absence of G
not only in Cl.10 cells but also in
other PYY receptor-containing epithelia, such as the rat intestinal
epithelium(1, 23) , lends support to the fact that
G
is not coupled to PYY receptors, at least in
epithelial cells. By using antibodies to G
subunits,
the PYY-mediated inhibition of adenylyl cyclase, which occurs through
the Y2 subtype of NPY receptor in a neuronal cell line, was shown to
involve both G
and G
, with G
possibly playing the more important role(38) . This
contrasts with the exclusive coupling of PYY receptors with G
in renal proximal tubule cells. Whether the difference is related
to receptors, i.e. NPY Y2 receptors (38) versus PYY-preferring receptors (this study) and/or tissues, i.e. neuronal cells (38) versus epithelial cells
(this study), is not known. What is known is that both G
and G
participate in the inhibition of adenylyl
cyclase (39, 40, 41, 42) and that a
specific receptor may signal through distinct G
proteins to inhibit adenylyl cyclase(42) .
In view of
the fact that G protein subunits are generally considered to be
expressed in large excess over individual G protein-coupled
receptors(43) , it was intriguing to observe that a 90%
decrease of the expression of G in
Cl.10/
cells totally abolished the
regulation of PYY binding by GTP
S as well as PYY receptor-mediated
inhibition of cAMP production and stimulation of
[methyl-
H]thymidine incorporation into
DNA. We have no definitive answer to this issue. It can be hypothesized
that: (i) a threshold amount of G
protein is
necessary to interact significantly with PYY receptors. After
transfection of Cl.10 cells with the
pcDNA3/G
expression vector, we have
isolated two other cell clones which exhibited a 60% decrease in
G
content as compared to the parent Cl.10 cells. We
have examined PYY receptors and PYY-mediated inhibition of cAMP
production in one of these clones. We obtained essentially the same
data as in Cl.10/
cells with a 90%
decrease in G
content, i.e. an increase in
the dissociation constant of PYY receptors and the failure of PYY to
inhibit basal and forskolin-stimulated cAMP levels. (
)Although Western blotting cannot be considered as a
quantitative method for measuring protein levels, these data show that
partial inhibition of G
expression is sufficient for
uncoupling PYY receptors and suggest that a critical amount of
G
is necessary to maintain the functional response
and high affinity ligand binding. (ii) The remaining low amount of
G
protein in Cl.10/
cells is not localized to the plasma membrane where PYY receptors
are present and functionally coupled to adenylyl cyclase(3) .
Confocal laser microscopy of the remaining G
protein
in Cl.10/
cells (not shown) did not
favor this hypothesis. However, in view of the importance of membrane
organization in G protein mechanisms(44) , we cannot exclude
the possibility that the nonhomogeneous localization of G
to patches within the plasma membrane (45) was modified
in Cl.10/
cells.
Types V and VI
appear to be the dominant forms of adenylyl cyclase in peripheral
tissues(18) . The three isoforms of G have
been shown to be equally potent and efficacious in inhibiting
G
- and forskolin-stimulated type V and type VI
adenylyl cyclase(18, 46) . Therefore, it is not
surprising that down-regulation of the expression of G
in Cl.10/
cells abolished PYY
receptor-mediated inhibition of adenylyl cyclase and subsequent
cAMP-dependent effects. The reason why basal as well as
forskolin-stimulated levels of cAMP are identical in Cl.10 and
Cl.10/
cells is less clear. This
phenomenon has been previously observed in F9 teratocarcinoma cells
expressing G
antisense RNA (20) and could be
due to the fact that multiple G protein subunits, including
G
, G
, and also
(18) , participate in the inhibiting tonus of
adenylyl cyclase in Cl.10 cells and/or that G
-mediated
inhibition of adenylyl cyclase is strictly dependent on the activation
of inhibitory receptors, such as PYY receptors, by agonists.
In
conclusion, our antisense RNA technology studies indicate that PYY
receptors are coupled with a strict specificity to G in the proximal tubule Cl.10 cell clone and that G
is responsible for PYY receptor-mediated inhibition of adenylyl
cyclase and stimulation of cell growth. These findings further document
the mechanism of PYY receptor-mediated responses in epithelial cells.
Note Added in Proof-While this paper was under review, the cloning of a cDNA encoding a human Y2 subtype of NPY receptor was reported (J. Biol. Chem.270, 22661-22664, 1995).