From the
The G protein-linked receptor for neurokinin A (NKA) couples to
stimulation of phospholipase C and, in some cells, adenylyl cyclase. We
have examined the function of the C-terminal cytoplasmic domain in
receptor signaling and desensitization. We constructed C-terminal
deletion mutants of the human NK-2 receptor (epitope tagged) to remove
potential Ser/Thr phosphorylation sites, and expressed them in both
mammalian and insect cells. When activated, truncated receptors mediate
stronger and more prolonged phosphoinositide hydrolysis than wild-type
receptor; however, the amplitude and kinetics of the NKA-induced rise
in cytosolic Ca
Signal transduction via G protein-coupled receptors is
characterized by rapid desensitization, i.e. attenuation of
cellular responses upon prolonged or repeated agonist exposure
(1, 2, 3) . The mechanisms of desensitization
have been extensively studied in the
We are interested
to examine how signal transduction via PLC-coupled receptors and their
cellular actions are altered when the putative ``desensitization
domain'' of the receptor is deleted or mutated. One would expect
that such desensitization-defective receptors, when activated, mediate
prolonged rather than short-lived generation of second messengers.
Prolonged signal generation is likely to affect long-term cellular
responses such as cell growth and differentiation. Our approach makes
use of the receptor for the decapeptide neurokinin A (NKA, also known
as substance K; tachykinin receptor subtype, NK-2)
(7, 8) . The neurokinins (tachykinins) are a family of
neuropeptides involved in diverse physiological and pathological
processes, ranging from inflammation to neurotransmission. Where
tested, the neurokinin receptors couple to stimulation of PLC with
subsequent mobilization of Ca
To address the possible role of the cytoplasmic
tail in NK-2 receptor signaling and desensitization, we have
constructed and selected mutant receptors that lack various parts of
the C-terminal tail and yet remain coupled to PLC activation in an
agonist-dependent manner. We expressed these mutant receptors in
various cell systems, including Rat-1 fibroblasts, COS cells, and Sf9
insect cells, and examined how their signaling properties are altered.
Our results indicate that truncated receptors mediate enhanced and
prolonged activation of signaling pathways, apparently due to loss of
regulatory phosphorylation sites in the C-terminal domain.
The kinetics of receptor-mediated PLC activation by
NKA are shown in Fig. 4 B. The response to wt receptor
leveled off after about 5 min, whereas activation of
Taken together, these results
indicate that deletion mutant receptor mediates enhanced and sustained
activation of PLC as compared with wt receptor.
We also sought to monitor receptor-mediated
Ca
As shown in Fig. 6 B, inhibition of PKC by
Ro31-8220 or long-term (24 h) treatment with TPA strongly inhibited
NKA-induced cAMP formation but did not affect the response to
isoproterenol, which acts on the G
We also examined receptor phosphorylation in
baculovirus-infected Sf9 cells expressing wt or
Desensitization of G protein-coupled receptors is a complex
process involving various molecular mechanisms. Deletion or mutation of
putative phosphorylation sites in G protein-coupled receptors has
indicated the importance of receptor phosphorylation in mediating
desensitization
(28, 29, 30, 31) . In
particular, agonist-induced Ser/Thr phosphorylation of the cytoplasmic
tail has been implicated in receptor desensitization. We have used the
NK-2 receptor as a model system for investigating the role of the
C-terminal domain in receptor signaling, desensitization, and
phosphorylation. Our major findings can be summarized as follows: (i)
C-terminal deletion of 60 or 70 residues results in a receptor that is
still functional but fails to undergo desensitization in response to
agonist; (ii) truncated receptors are resistant to inhibition by
PKC-activating phorbol ester; and (iii) wild-type receptor, but not
truncated receptor, is phosphorylated upon addition of either NKA or
phorbol ester. From these results, we conclude that C-terminal tail
phosphorylation is responsible for both homologous and heterologous
desensitization of the human NK-2 receptor.
That truncated receptors
(
Generation of cAMP by deletion mutants follows a pattern
similar to IP
In a recent study on desensitization of endothelin and NK-2
receptors, Cyr et al. (14) reported that a truncated
human NK-2 receptor (
Our
results strongly suggest that receptor phosphorylation occurs at the
C-terminal tail and is causally related to the observed receptor
desensitization. Basal and agonist-induced phosphorylation of wt and
mutant receptors is readily detected in COS cells and
baculovirus-infected Sf9 cells. In both systems, the unstimulated wt
receptor exists in a phosphorylated state. It remains to be seen
whether this represents a normal physiological state, since our efforts
to measure receptor phosphorylation at ``normal'' expression
levels (as in Rat-1 transfectants) were unsuccessful. In COS cells and
Sf9 cells, both NKA and phorbol ester induce a significant increase in
basal phosphorylation of wt but not mutant receptors. We also find that
the deletion mutants become resistant to inhibition by PKC and that
inhibition or down-regulation of PKC results in enhanced NKA-induced
PLC activity in stable transfectants. From these results, we conclude
that by phosphorylating one or more C-terminal Ser/Thr residues, PKC
mediates (feedback) inhibition of receptor signaling and thus
participates in both homologous and heterologous desensitization.
The apparent involvement of PKC does not, of course, exclude the
involvement of specific receptor kinases in mediating receptor
phosphorylation and desensitization. The deleted part of the NK-2
receptor C terminus contains several consensus phosphorylation sites
for both PKC (three sites) and
In conclusion, our results indicate a close link
between C-terminal tail phosphorylation and signal attenuation of the
human NK-2 receptor. Phosphorylation somehow leads to decreased
coupling efficiency to the relevant G protein(s). The exact
phosphorylation sites and the protein kinase(s) responsible remain to
be identified in further experiments. Another outstanding question is
whether sustained PLC stimulation mediated by truncated NK-2 receptors
leads to altered long-term cell behavior, such as cell cycle
progression and induction of mitogenesis. We hope to address these
questions in further studies.
We thank R. Kris for providing the full-length cDNA of
the human NKA receptor and M. Gebbink, L. van der Voorn, and G.
Hateboer for expression vectors.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
remain unaltered. Protein kinase C
(PKC)-activating phorbol ester abolishes wild-type receptor signaling
but not mutant receptor signaling. Mutant receptors also mediate
enhanced and prolonged cAMP generation, at least in part via PKC
activation. When expressed in COS cells or Sf9 insect cells, the
wild-type receptor is phosphorylated; receptor phosphorylation
increases after addition of either NKA or phorbol ester. In contrast,
mutant receptors are not phosphorylated by either treatment. Our
results suggest that C-terminal Ser/Thr phosphorylation sites in the
NK-2 receptor have a critical role in both homologous and heterologous
desensitization. Removal of these phosphorylation sites results in a
receptor that mediates sustained activation of signaling pathways and
is insensitive to inhibition by PKC.
-adrenergic receptors and the
visual receptor rhodopsin. In those systems, signal attenuation occurs
through receptor phosphorylation by various Ser/Thr kinases, in
particular protein kinase A, protein kinase C (PKC),
(
)
and specific receptor kinases
(4) . Much less is
known about the desensitization mechanisms of PLC-coupled receptors,
although the available evidence suggests that, again, phosphorylation
may play a critical role
(5, 6) .
from intracellular
stores
(9, 10, 11, 12) . In some cell
systems, there is additional coupling to stimulation of adenylyl
cyclase, either directly via G
(9) or secondary to
Ca
mobilization
(10) , or arachidonate
metabolism
(13) . Like other G protein-coupled receptors, the
neurokinin receptors undergo agonist-induced desensitization, but the
molecular basis is not known, although C-terminal phosphorylation would
be a plausible mechanism. However, a recent study
(14) reports
that the C-terminal domain of the NK-2 receptor is not involved in
desensitization.
Materials
The PKC inhibitor Ro31-8220
was obtained from Roche Research Center (Welwyn Garden City, United
Kingdom). Endoglycosidase H was purchased from Boehringer Mannheim
GmbH, indo-1 acetoxymethylester was from Molecular Probes Inc.,
[2-[I]iodohistidyl]neurokinin A (2000
Ci/mmol), myo-[
H]inositol (80-120
Ci/mmol),
L-[
S]methionine/
L-[
S]cysteine
labeling mix (>1000 Ci/mmol), and
P
(10
mCi/ml) were from Amersham, as well as the ECL kit. Prot A-Sepharose
(CL-4B) beads were from Pharmacia Biotech Inc., and
peroxidase-conjugated antibodies were from Dako. All other chemicals
were from Sigma.
Cell Culture
Rat-1 fibroblasts, human
embryo kidney (HEK293) cells, and COS-7 cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
7.5% fetal bovine serum and antibiotics. Spodoptera frugiperda (Sf9) insect cells were obtained from the ATCC and were grown in
Grace's insect medium supplemented with 10% fetal calf serum and
antibiotics at 27 °C.
Construction of Mutant NK-2 Receptors and Recombinant
Baculovirus
The cDNA of the human NK-2 receptor (kindly
provided by Dr. R. Kris
(12) ) was subcloned into a modified
pMT2 vector (with altered multiple cloning site constructed by M.
Gebbink) containing an epitope-tag sequence derived from the
VSV-glycoprotein
(15) directly preceded by a NotI site
(construct designed and made by L. van der Voorn and G. Hateboer).
Polymerase chain reaction fragments were generated up and until the
desired last amino acid of the NK-2 receptor (amino acids 398, 338,
328, and 309, respectively), containing a NotI overhang for in
frame cloning with the tag sequence. This resulted in C-terminal
extension of full-length and mutant receptors with the sequence
SGRPYTDIEMNRLGK. The constructs coding for wt and 328 were
subcloned into the pVL1393 vector and cotransfected with lethal virus
DNA (BaculoGold kit by PharMingen) into Sf9 cells. Recombinant virus
was plaque-purified and amplified. Protein expression was determined by
Western blot using monoclonal antibody P5D4 against the epitope tag.
DNA Transfections
Cells were transfected
with 10 µg of purified recombinant pMT2 plasmid by the calcium
phosphate precipitation method. For stable transfection of Rat-1 cells,
0.1 µg of pSVneo was included in the precipitate. The next day,
cells were split 1:10 and selected in DMEM supplemented with 1 mg/ml
G418. Resistant clones were isolated and screened for expression by
radioligand binding assay and subsequently subcloned by single cell
dilution. Comparison of tagged versus non-tagged NK-2 receptor
expressed in Rat-1 cells revealed no differences in behavior of the
receptors as measured by Camobilization (not shown).
For stable transfection of HEK293 cells, 0.5 µg of pSV2
3.6,
containing the cDNA for murine
Na
,K
-ATPase that confers ouabain
resistance
(16) , was included in the precipitate. The HEK293
cells were selected with 1 µ
M ouabain, and resistant
clones were picked and screened for NK-2 receptor expression and
subcloned in conditioned medium from untransfected HEK293 cells. COS
cells were grown in 10-cm dishes and transfected with 10 µg of DNA
by the DEAE-dextran method, trypsinized the next day, and divided over
a 6-well plate. They were serum-starved overnight, labeled with
P
or
S-labeled Met/Cys for 3 h,
and stimulated with the agonists required.
Radioligand Binding
Cells were grown to
confluency in 24-well plates, washed with Hepes-buffered DMEM, and
incubated on ice with the same buffer containing 0.1% bovine serum
albumin, 1 m
M Mn, and 0.1 n
M
I-NKA for at least 1 h. Cells were then washed four
times with ice-cold phosphate-buffered saline containing 1 m
M Ca
and 1 m
M Mg
,
dissolved in 0.1
N NaOH, and monitored for
radiation.
Nonspecific binding was measured in the presence of 0.5 µ
M unlabeled NKA.
Inositol Phosphates
Cells were grown in
6-well plates to near confluency and labeled overnight in serum-free
medium containing 2 µCi/ml
myo-[H]inositol. Cells were washed with
DMEM-Hepes and equilibrated for 2 h. After stimulation in the presence
of 10 m
M LiCl, inositol phosphates were extracted and isolated
using AG 1-X8 anion exchange columns (formate form; Bio-Rad) as
described
(17) . Total inositol phosphates
(IP
) were eluted with 1
M ammonium
formate, 0.1
M formic acid.
Ca
Rat-1 cells were plated on rectangular
glass coverslips and grown to 80% confluency. The cells were loaded
with 5 µ
M indo-1 acetoxymethylester in serum-free
Hepes-buffered DMEM for 20-30 min, washed with HBS (10 m
M Hepes, pH 7.4, 140 m
M NaCl, 5 m
M KCl, 1 m
M CaClMeasurements
, 1 m
M MgCl
, 10 m
M glucose), and incubated at 37 °C in HBS in quartz cuvettes.
Infected Sf9 cells were harvested 24 h postinfection, gently washed in
HBS (pH 6.5), loaded in HBS containing 2 µ
M indo-1
acetoxymethylester for 45 min, and transferred to quartz cuvettes
(10
cells/ml) kept at 27 °C. Fluorescence was measured
with 355 nm excitation and 405 nm emission wavelength as described
(18) .
cAMP Measurements
Confluent serum-starved
cells were stimulated in the presence of 1 m
M isobutylmethylxanthine and assayed for cAMP content using a
[H]cAMP assay system (Amersham Corp.) according
to the manufacturer's instructions.
Infection and Labeling of Sf9 Insect
Cells
Insect Sf9 cells were infected with recombinant
baculovirus for 30 min in Grace's insect medium without fetal
calf serum in a suspension of 25 10
cells/ml and
then plated in Grace's medium with fetal calf serum in 10-cm
dishes. After 40 h of infection, cells were radiolabeled by incubating
them in synthetic Sf9 medium (50 m
M PIPES, pH 6.5, 55 m
M KCl, 10 m
M MgCl
, 5 m
M CaCl
, 5 m
M NaHCO
, and 100 m
M sucrose) containing 750 µCi/ml
P
for
3 h. Immunoprecipitation of wt and mutated NK-2 receptors was carried
out as described for mammalian cells (see below). The
immunoprecipitates were dissolved in sample buffer containing 6
M urea, separated on an 11% gel, transferred to nitrocellulose, and
probed with the P5D4 antibody followed by the ECL reaction.
Immunoprecipitations
Cells labeled with
either S-labeled Met/Cys or
P
were lysed in Nonidet P-40-lysis buffer (0.5% Nonidet P-40, 50
m
M Tris, pH 7.4, 150 m
M NaCl, 5 m
M MgCl
, 1 m
M EGTA, 0.1 m
M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10
µg/ml aprotinin; phosphatase inhibitors, in the case of
P labeling, were 0.1 m
M Na
VO
, 10 m
M NaF, and 10 m
M Na
P
O
). Lysates were precleared
twice with normal mouse serum precoupled to fixed Staphylococcus
aureus or to Sepharose-protein A beads. Specific precipitation was
performed with the P4D5 monoclonal antibody recognizing the tag. P5D4
monoclonal antibody was used as culture supernatant from the hybridoma
(15) . Precipitates were washed six times in the same buffer.
For endoglycosidase H digestion, immunocomplexes were resuspended in 25
µl of citrate buffer (50 m
M sodium citrate, pH 5.5, 0.2%
SDS) with or without 0.5 milliunits endoglycosidase H and incubated for
1 h at room temperature. SDS-sample buffer containing 6
M urea
was added; samples were not boiled to avoid protein aggregation and
were immediately loaded on a polyacrylamide gel. The amount of
immunoprecipitated material in each lane was assessed by Western
blotting. The blots were probed with P5D4 and peroxidase-conjugated
swine anti-mouse immunoglobulin, subjected to the ECL procedure,
briefly washed, dried, and exposed to film.
Construction of Truncated Receptors
To
examine the importance of the cytoplasmic tail in NK-2 receptor
signaling and desensitization, we set out to generate mutant receptor
cDNAs. The predicted secondary structure of the 398-amino acid human
NKA receptor (NK-2 receptor) is shown in Fig. 1 A. Several
consensus sites for post-translational modifications such as
glycosylation, palmitoylation, and phosphorylation are indicated. There
are 25 intracellular serine/threonine residues, 13 of which occur in
motifs similar to known consensus sequences for PKC, cAMP-dependent
protein kinase, or members of the ARK family
(19, 20) . We chose to delete 60, 70, or 89 amino acids
from the C-terminal tail (Fig. 1 A; truncation sites indicated
by arrows), yielding three distinct mutant receptors termed
338,
328, and
309 lacking 18, 17, and 15 Ser/Thr
residues, respectively. All receptor constructs were extended at their
C terminus with a 15-amino acid epitope tag derived from the
VSV-glycoprotein (Ref. 15, see also ``Experimental
Procedures''), as illustrated in Fig. 1 B. The cDNA
constructs were cloned into the pMT2 expression vector and transfected
into COS cells and Rat-1 fibroblasts.
Figure 1:
Schematic representation of the human
NK-2 receptor. A, predicted secondary structure. The standard
one-letter amino acid code is used. Bold circles indicate cytoplasmic Ser/Thr residues; arrows indicate
sites of truncation in the C-terminal tail; shaded Asn residues in the N terminus are consensus sequences for
N-linked glycosylation; the potential myristoylation site
(Cys-323) is marked with a zigzag; B, constructs of
wt and deletion mutant NK-2 receptors. Transmembrane ( TM)
domains are shaded; the epitope tag sequence is indicated with
a flag. The proline ( P) in the tag sequence does not
belong to the original VSV-glycoprotein epitope as described
(15).
Expression of Wild-type and Truncated NK-2 Receptors
in COS Cells
Expression of the NK-2 receptor constructs was
examined by COS cell transfections. At 48 h after transfection, the
cells were metabolically labeled with S-labeled Met/Cys
for 3 h followed by immunoprecipitation and SDS-PAGE analysis of
precipitated proteins (Fig. 2). Proteins of various molecular masses
are detected, ranging from about 44 kDa for wt receptor to 35 kDa for
309. It is seen that each transfected cDNA translates into a
doublet of protein bands, differing about 6 kDa in size, as well as a
band of roughly twice the predicted mass. After treatment of the
immunoprecipitates with endoglycosidase H to remove high mannose
N-linked sugars, the higher molecular weight forms shift back
to the lower molecular weight form ( lanes marked with +).
Lower endoglycosidase H doses yielded a partial digestion resulting in
a third protein product of intermediate size (not shown). Thus, the
upper band represents a double glycosylated form of
the receptor, the lower band represents the
non-glycosylated form, and the middle band (poorly visible in
Fig. 2
) contains one N-linked glycan. The
endoglycosidase H-sensitive proteins in the upper region of the gel presumably represent receptor aggregates (dimers).
Figure 2:
Expression of wt and mutant NK-2 receptors
in COS cells. COS cells transiently transfected with the constructs
depicted in Fig. 1 B were metabolically labeled at 48 h after
transfection with S-labeled Met/Cys for 3 h and then
lysed; NK-2 receptors were immunoprecipitated with anti-tag monoclonal
antibody P5D4. Immunoprecipitates were treated with endoglycosidase H
(+) (Endo H) as indicated and analyzed by SDS-PAGE and
autoradiography as described under ``Experimental
Procedures.''
Coupling of the receptors to phospholipase C was measured by the
accumulation of IP. Agonist stimulation of
transfected COS cells showed a 2.5-3-fold increase in
IP
formation with all constructs except for the
309 mutant (results not shown). This indicates that mutant
309 is not functional; therefore, this mutant was not used in
further experiments.
Expression of Wild-type and Truncated NK-2 Receptors
in Rat-1 Cells
To investigate the signaling properties of
the wt and mutant NK-2 receptors, stable transfectants of Rat-1 cells
were generated. Clones were selected on the basis of
I-NKA binding. Agonist displacement analysis (Fig. 3)
reveals that truncated and wild-type receptors have similar affinities
for NKA. The IC
value for displacement by unlabeled NKA is
estimated at 3-6 n
M, in agreement with reported affinity
values for endogenous and transfected NK-2 receptors
(10, 21, 22, 23) . Analysis of these
data
(24) yields receptor densities of about 8,000/cell for wt
receptor-expressing cells, 11,000/cell for
328, and 26,000/cell
for
338. In our further signaling studies, we focused mainly on wt
and
328 receptors, given their comparable expression levels in
Rat-1 cells. Yet, where tested, mutant
338 behaved qualitatively
similar to
328.
Mutant Receptors Mediate Sustained Inositol Phosphate
Accumulation
To characterize PLC activation by wt and
truncated receptors, we measured NKA-induced inositol phosphate
formation in the various Rat-1 transfectants. Stimulation of wt
receptor with 1 µ
M NKA for 5 min resulted in an
approximate 3-fold increase in IP(Fig.
4 A). Brief pretreatment of the cells with the PKC-activating
phorbol ester TPA (100 ng/ml) completely inhibited NKA-induced
IP
formation in wt receptor-expressing cells
without affecting control IP
levels. Conversely,
when PKC activity was inhibited by the selective PKC inhibitor
Ro31-8220 (3 µ
M), NKA-induced IP
formation was consistently elevated when compared with control
cells (Fig. 4 A); similar results were obtained after
down-regulation of PKC by prolonged TPA treatment (100 ng/ml for 24 h).
These results strongly suggest that NKA-induced PLC activation via wt
NK-2 receptors is subject to feedback inhibition by PKC.
Figure 4:
Agonist-induced phosphoinositide
hydrolysis in transfected Rat-1 cells. A, confluent cells were
labeled with myo-[H]inositol for 20 h
and stimulated for 5 min in the presence of Li
with
either buffer (control) or NKA (1 µ
M). Where indicated,
cells were pretreated with 100 ng/ml TPA for 5 min or with the PKC
inhibitor Ro31-8220 (3 µ
M) for 10 min. B, time
course of NKA-induced inositol phosphate formation.
[
H]Inositol-labeled cells were stimulated with
NKA for the indicated periods of time and assayed for inositol
phosphate formation. Each data point represents mean ± S.E.
C, duration of NKA-induced PLC activity.
myo-[
H]Inositol-labeled cells were
stimulated with NKA at time point zero. LiCl (10 m
M) was added
at 0, 30, or 60 min after NKA addition, and inositol phosphates were
allowed to accumulate for 30 min. Incubations were terminated, and
inositol phosphate levels were determined as described under
``Experimental Procedures.'' Data represent the amount of IP
accumulated during a 30-min incubation with Li
.
C, control.
In cells
expressing mutant receptor 328, the relative increase in
IP
accumulated during 5 min of agonist stimulation
was consistently higher than observed with wt receptor (Fig.
4 A). Strikingly, in
328-expressing cells, NKA-induced
phosphoinositide breakdown was completely insensitive to inhibition by
TPA (Fig. 4 A). Furthermore, neither the PKC inhibitor
Ro31-8220 (Fig. 4 A) nor prolonged TPA treatment (not
shown) had any potentiating effect on NKA-induced IP
formation via truncated NK-2 receptor. Collectively, these
results suggest that (feedback) inhibition of NK-2 receptor signaling
by PKC is mediated by phosphorylation of the receptor's
C-terminal tail.
328 gave a
steadily increasing formation of inositol phosphates. To examine the
duration of PLC activation in further detail, we measured
IP
accumulation during 30-min Li
pulses at various times after agonist addition
(17) .
After 1 h of stimulation, the mutant receptor still mediated a 5-fold
increase in IP
following a Li
pulse, whereas the PLC activation by wt receptor was almost back
to control levels (Fig. 4 C). Thus, signal termination as
observed with wt receptor is not attributable to depletion of
agonist-sensitive phosphoinositide pools but rather reflects receptor
signaling shut-off (homologous desensitization), a phenomenon not
observed with truncated receptor.
Ca
Does sustained
PLC activation lead to altered CaMobilization by NK-2
Receptors Expressed in Rat-1 and Sf9 Cells
signaling kinetics?
Fig. 5 A shows typical time courses of NKA-induced
Ca
mobilization in stably transfected Rat-1 and human
293 cells, expressing either wt or
328. No significant differences
are observed in the amplitude and duration of the NKA-induced
Ca
transient mediated by either wt or mutant receptor
in both cell types.
mobilization in insect Sf9 cells expressing either
wt or
328 receptor. Previous work has indicated that certain
mammalian or avian receptors can effectively couple to endogenous G
proteins in Sf9 cells
(21, 25) . At 24 h after infection
with recombinant baculovirus, cells were loaded with indo-1
acetoxymethylester, and agonist-induced changes in intracellular
Ca
concentration were measured. As shown in
Fig. 5B, NKA evokes a rapid Ca
transient in Sf9 cells expressing either wt receptor or
328
in the presence of EGTA, indicative of receptor-mediated Ca
release from intracellular stores. As in Rat-1 cells, the NK-2
receptor expressed in Sf9 cells appears to be sensitive to inhibition
by PKC, since addition of TPA for 15 min blocks NKA-induced
Ca
mobilization. In contrast, the Ca
transient mediated by mutant
328 remains unaffected after
TPA addition.
Figure 5:
NKA-induced Ca
mobilization in Rat-1 and Sf9 cells expressing NK-2 receptors.
A, NKA-induced Ca
mobilization in stable
transfectants of Rat-1 ( upper panel) or HEK293
( lower panel) expressing wt NK-2 receptor or
328. Cells were loaded with indo-1 acetoxymethylester, and
agonist-induced calcium signaling was monitored in the presence of 3
m
M EGTA; arrowheads indicate time point of NKA
addition. The concentration of NKA was 1 µ
M in all
experiments. B, Sf9 cells infected for 24 h with either wt
( upper panel) or
328 ( lower panel) receptor recombinant virus were loaded with indo-1
acetoxymethylester and stimulated with 1 µ
M NKA
( arrow) in the presence of EGTA (3 m
M). TPA (100
ng/ml) was added for 15 min.
These results indicate that there is no direct
relationship between the duration of PLC activation and that of the
resulting Casignal. Thus, termination of the
Ca
transient is not regulated at the level of
receptor desensitization; instead, the rate-limiting step is likely at
the level of IP
metabolism and/or IP
receptor
desensitization.
Truncated Receptors Mediate Increased cAMP Formation
in a PKC-dependent Manner
In Rat-1 cells expressing wt
receptor, NKA induces a rather small increase in cAMP content (Fig.
6 A). However, it is seen that stimulation of mutant receptors
results in a much more sustained cAMP production. Various mechanisms
can account for receptor-mediated cAMP production: (i) direct
activation of adenylyl cyclase (AC) via G, (ii) indirect
activation of AC via production of eicosanoids acting on their own G
protein-coupled receptors, (iii) activation of one or more AC isoforms
by Ca
calmodulin, i.e. secondary to
Ca
mobilization, and (iv) activation of an AC isoform
by PKC.
-coupled
-adrenergic receptor. Intracellular Ca
stores were depleted with either thapsigargin
(26) or
ionomycin in the absence or presence of EGTA. Neither of these
treatments affected NKA-induced cAMP formation (Fig. 6 B), nor
did the cyclo-oxygenase inhibitor indomethacin (20 µ
M)
(not shown), arguing against regulation by intracellular Ca
or prostaglandin production.
Figure 6:
Agonist-induced cAMP accumulation in Rat-1
transfectants. A, time course of NKA-stimulated cAMP formation
in two independently selected clones expressing wt, two clones
expressing 328, and one clone expressing
338 receptor.
B, effect of modulation of intracellular Ca
levels by thapsigargin or ionomycin and modulation of PKC
activity by TPA (100 ng/ml; 5 min or 24 h) on NKA-induced (1
µ
M, 5 min) cAMP formation in
328 cells; Ro31-8220 was
added 10 min prior to NKA. Depletion of intracellular Ca
stores was achieved by addition of thapsigargin 5 min prior to
NKA addition; ionomycin (1 µ
M) was added 30 s before NKA.
Data points represent mean ± S.E. C, nonstimulated
cells (control).
Together, our results are
consistent with enhanced cAMP production being mediated, either
directly or indirectly, by PKC. The recent cloning and expression of
adenylyl cyclases have shown that type II AC is stimulated by phorbol
ester, presumably via direct PKC phosphorylation of the enzyme
(27) . However, we were unable to raise cAMP levels in Rat-1
cells by addition of TPA alone. We therefore conclude that in addition
to PKC, other as yet unidentified mechanisms participate in NKA-induced
activation of AC.
Receptor Phosphorylation in COS Cells and Sf9 Insect
Cells
The possible link between signal attenuation and
receptor phosphorylation was examined in COS cells expressing wt or
truncated NK-2 receptor; expression levels in transfected Rat-1 cells
turned out to be too low for reliable receptor phosphorylation
experiments. Immunoprecipitated wt NK-2 receptor from
P
-labeled COS cells showed basal
phosphorylation of the wt receptor bands (both unglycosylated and
glycosylated forms) (Fig. 7 A). Stimulation with NKA for 20 min
resulted in increased receptor phosphorylation, as is also seen after
treatment with TPA. In contrast, mutant receptor showed little basal
phosphorylation, which did not increase after addition of either NKA or
TPA.
328 NKA receptor.
Radiolabeling experiments confirmed that surface expression of wt and
mutant receptors was about equal (not shown). Fig. 7 B shows that receptor phosphorylation was significantly increased
following addition of either NKA or TPA. In contrast, mutant receptor
328 showed only minor basal phosphorylation levels, which were not
increased after NKA or TPA treatment. Western blot analysis confirmed
that equal amounts of receptor protein were immunoprecipitated.
Figure 7:
Phosphorylation of NK-2 receptors
expressed in COS cells and insect Sf9 cells. A, COS cells were
transfected with wt or mutant 338 NK-2 receptor. Transfected
cultures were serum starved overnight, labeled for 3 h with 200
µCi/well
P
, stimulated with NKA (1
µ
M) or TPA (100 ng/ml) for 20 min, and then lysed and
immunoprecipitated with antibody P5D4. Immunoprecipitates were analyzed
by SDS-PAGE and autoradiography as described under ``Experimental
Procedures.'' B, receptor phosphorylation in
baculovirus-infected Sf9 cells. Cells were infected and labeled as
described under ``Experimental Procedures,'' stimulated, and
immunoprecipitated as in Fig. 7 A. The receptor
immunoprecipitates were analyzed by SDS-PAGE, transferred to
nitrocellulose, and probed with P5D4 antibody followed by the ECL
reaction to visualize the amount of receptor protein (lower part of the
autoradiograph). The same blot was then briefly washed, dried, and
exposed to film to detect
P-labeled proteins. C,
non-stimulated cells (control).
Together with the signal transduction data, these results
demonstrate that there is concordance between agonist- and phorbol
ester-induced receptor phosphorylation and signal attenuation.
328 and
338) are desensitization defective is indicated by
their sustained stimulation of both PLC and AC as compared with the
transient activation observed with wt receptor. It is noteworthy that
despite sustained PLC activation, the kinetics of NKA-induced
Ca
signal from intracellular stores (reflecting
IP
action) were the same for wt and mutant receptors. This
indicates that the rate-limiting step for termination of Ca
signaling occurs downstream of the receptor, most likely at the
level of IP
catabolism and/or IP
receptor
regulation.
formation. All our results point to
a pivotal role of PKC in stimulating AC activity, although
PKC-activating phorbol ester alone is incapable of raising cAMP levels
in Rat-1 cells; hence, additional mechanisms are likely to participate
in AC activation by (truncated) NK-2 receptors. In transfected Chinese
hamster ovary cells, the neurokinin receptors also couple to
stimulation of both PLC and AC, although it is not clear whether AC
activation is PKC dependent as it is in Rat-1 cells
(9) . In
another study on transfected Chinese hamster ovary cells, the NK-2
receptor reportedly mediates enhanced cAMP generation as a secondary
response to eicosanoid production
(13) . However, our results
argue against a role for eicosanoids in NKA-induced cAMP formation.
Whatever the exact mechanisms of enhanced cAMP generation, the data
show that at least two distinct effectors, PLC and AC, are
constitutively activated by truncated receptors, indicating the
importance of the C-terminal tail in controlling signal termination.
336), nearly identical to the
338
construct used in the present study, behaves like wild-type receptor;
the authors conclude that the C-terminal tail of the NK-2 receptor has
no apparent role in desensitization. The discrepancy with the present
study remains unexplained. However, direct comparison should be made
with caution because the expression systems used were very different
( Xenopus oocytes versus mammalian cells).
ARK (six sites). In fact, the
related NK-1 receptor can be phosphorylated by the
-adrenergic
receptor kinases
ARK1 and
ARK2
(32) , at least in
vitro. The visual receptor rhodopsin is a target for both PKC and
a specific rhodopsin kinase in a ligand-dependent manner
(33) .
Signal attenuation by the PLC-coupled thrombin receptor is also
mediated, at least in part, by
ARK
(34) . Furthermore, the
PLC-coupled receptor for platelet-activating factor may be a target for
ARK
(6) . Future studies should reveal whether the
ARKs, which recognize only the ligand-activated form of the
receptor, are also involved in NK-2 receptor phosphorylation and
desensitization.
ARK,
-adrenergic receptor kinase; VSV,
vesicular stomatitus virus; IP
, inositol
1,4,5-trisphosphate; IP, total inositol phosphates; PIPES,
1,4-piperazinediethanesulfonic acid; wt, wild type; PAGE,
polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified
Eagle's medium;
I-NKA,
[2-[
I]iodohistidyl]-neurokinin A.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.