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(Received for publication, June 26,
1995; and in revised form, September 5, 1995) From the
Receptor activation and agonist-induced desensitization of the
human neurokinin-2 (NK2) receptor expressed in Xenopus oocytes
have been investigated. When neurokinin A (NKA) was applied repeatedly
at 5-min intervals, the second and subsequent applications gave no
responses. This desensitization was not observed with the specific
agonists (Lys
The NK2 ( When expressed in Xenopus oocytes, the NK2 receptor causes release of Ca
Figure 1:
Putative structure
of human NK2 receptor and mutants. Schematic diagram showing the F248S
point mutation in the third intracellular loop (black dot) and
NK2 ligands used in this study are listed in Table 1.
The decapeptide NKA and its elongated form NPK are natural ligands
acting on NK2 receptor. The modified peptides GR64349 (4) and
(Nle
Figure 2:
Ca
Figure 3:
Agonist-induced
Ca
Functional activity of NK2 and mutant
F248S was further evaluated by Ca
Figure 4:
Effects of NK2 agonists on
To characterize NK2 and mutant receptors expressed
in Xenopus oocytes, equilibrium binding studies with the
specific NK2 antagonist [
Figure 5:
Attenuation of NK2 receptor
desensitization by heparin. Conditions are as in Fig. 3except
that oocytes were injected with 1 pmol of heparin (+) or
water(-), 30 min prior to application of 1 µM NKA.
Assuming an oocyte volume of 1 µl, the putative concentration of
heparin was about 1 µM.
Figure 6:
Effects of staurosporine on
desensitization of wild-type NK2 and mutant F248S. Chloride current
elicited by NKA and GR64349 in oocytes was preincubated and stimulated
in the presence (+) or absence(-) of 1 µM
staurosporine. Conditions are as in the legend of Fig. 3.
Figure 7:
Effects of Ro-31-8220 on
desensitization of wild-type NK2 and mutant F248S. Chloride current
elicited by NKA and GR64349 in oocytes preincubated and stimulated in
the presence (+) or absence(-) of 5 µM Ro-31-8220 is shown. Conditions are as in the legend of Fig. 3.
Figure 8:
Agonist-induced
Ca
In this study, we have compared the activity of different NK2
agonists (Table 1) on the NK2 receptor expressed in Xenopus oocytes. The functional activity of G protein-coupled and
phosphoinositide-coupled NK2 receptors was detected by the widely
employed technique of inward current recording. Though all agonists
tested elicited chloride currents, these compounds showed marked
differences in their ability to induce desensitization of NK2 ( Fig. 2and Fig. 3). Both NKA and GR64349 are potent
agonists able to stimulate either wild-type NK2 or F248S mutant with
identical maximum efficacy in Ca The main structural difference
between NKA and GR64349 or N(4-10) is the size of the peptide (Table 1). The latter are short agonists containing the
COOH-terminal portion minimum for tachykinin receptor activation. In
comparison, NKA can be viewed as a long agonist with amino-terminal
extension. The amino-terminal moiety in NKA may make additional
contacts with the NK2 receptor, favoring one conformation that is
recognized by receptor kinases. In support of this hypothesis, the
extended NKA analog NPK behaves as NKA. In contrast, short agonist
lacking this extension is unable to confer this conformation.
Alternatively, the amino-terminal portion of the long peptides may
lock, or modulate, the binding conformation of the COOH-terminal moiety
by intramolecular interactions. Our results do not allow us to
distinguish between these two possibilities. Thus, the amino-terminal
portion of NKA may have a dual role in, on one hand, stabilizing a
conformation leading to receptor kinase activation and desensitization
and, on the other hand, conferring selectivity for NK2 over NK1 and NK3
receptor subtypes. The lack of NK2 desensitization in response to
either GR64349 or N(4-10) and the reversal of this effect in
presence of staurosporine suggests the involvement of some
phosphorylation event hindering receptor phosphorylation by G protein
receptor kinases. The absence of effect of Ro-31-8220 in
enhancing GR64349-mediated desensitization suggests that this
phosphorylation is probably mediated by protein kinase A rather than
PKC. The protein kinase recognizing the GR64349-activated NK2 conformer
remains yet unidentified. Recently, it has been shown that agonists of
different structures bind to The substance P receptor (NK1) was shown to
be a substrate of First, truncations in the COOH-terminal part of the
receptor ( Second, injection of heparin, a known inhibitor
of receptor-specific kinases, caused a partial resistance to
desensitization. This effect was small (10-20%) and occurred only
at high heparin concentration ( A
phenylalanine to serine mutation in the COOH-terminal part of the third
cytoplasmic loop of the NK2 receptor reveals the existence of a
functional domain important for desensitization. This region had
already been shown to be an important determinant for G protein
interaction. Our data indicate that specific change of functional
groups, or conformational changes, in the cytoplasmic region proximal
to the sixth transmembrane segment may alter G protein receptor kinase
recognition or activation. The F248S mutation creates a consensus site
for phosphorylation by protein kinase C ( . . . S In
summary, our results indicate that agonist-induced conformational
changes of NK2 in oocytes leading to G protein activation and
desensitization can be dissociated at the molecular level. We have
identified one site in the third cytoplasmic loop that is important for
NK2 receptor desensitization. Also, we have shown that the
COOH-terminal tail is a functional domain for desensitization.
Depending on the structure of the ligand, agonist-occupied receptor may
adopt distinct conformations that are functionally active with respect
to G protein activation but show different susceptibility to kinases
involved in desensitization. Because desensitization is a regulatory
mechanism controlling cellular activity, the effect of analog may last
longer compared to the natural ligand and alter long term cell
behavior. Therefore, the results reported here may have important
implications in pharmacology.
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27601-27605
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DIVERGENT CONFORMATIONAL REQUIREMENTS FOR NK2 RECEPTOR SIGNALING
AND AGONIST-INDUCED DESENSITIZATION IN XENOPUS OOCYTES (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,
Gly
-R--lactam-Leu
)NKA(3-10)
(GR64349) or (Nle)-NKA(4-10). However, in the
presence of the protein kinase inhibitor staurosporine, stimulation
with GR64349 or (Nle
)-NKA(4-10) induced receptor
desensitization. In contrast, the protein kinase C inhibitor
Ro-31-8220 was not able to enhance GR64349-mediated
desensitization. We created a mutation (F248S) in the third cytoplasmic
loop of NK2 that impairs NKA-induced desensitization. In the presence
of either staurosporine or Ro-31-8220, the mutant receptor was
desensitized in response to NKA application but not to GR64349. Also,
truncation mutants
62 and 87, lacking serine and threonine
residues in the cytoplasmic COOH-terminal tail, were functionally
active and were partially resistant to desensitization. These
observations indicate that 1) there are different conformational
requirements for NK2 receptor signaling and agonist-induced
desensitization, 2) the third intracellular loop and the cytoplasmic
tail of NK2 are functional domains important for agonist-induced
desensitization, and 3) some agonists at the NK2 receptor cause much
more desensitization than others and suggest that this might result
from phosphorylation by receptor-specific kinases and other
non-identified protein kinases.
)receptor is a cell surface receptor
mediating the actions of the tachykinin peptide neurokinin A (NKA,
substance K) in the central and peripheral nervous system(1) .
NK2 belongs to the superfamily of receptors coupled to G proteins.
Stimulation of G protein-coupled receptors by specific ligands triggers
intracellular signaling pathways via activation of G proteins, which
regulate several effector enzymes and second messenger production.
Prolonged or repeated agonist stimulation induces desensitization of
receptor activity. For several G protein-coupled receptors, the
mechanism of desensitization has been shown to involve agonist-induced
phosphorylation of the receptor by a receptor-specific kinase and
uncoupling of the receptor from the effector enzyme(2) .
Binding of agonist by receptors stabilizes an active conformation that
couples to G protein. Likewise, an agonist-induced conformational
mechanism is believed to promote interaction and activation of
receptor-specific kinase(3) . from intracellular stores as a result of stimulation of
phospholipase C and production of inositol triphosphate. Elevation of
intracellular Ca
concentration activates
Ca
-dependent chloride channels, which can be
electrophysiologically recorded. In this report, we have investigated
the signaling and acute desensitization of NK2 receptor expressed in Xenopus oocytes in response to agonists of different chemical
structures. We have examined the roles of a point mutation in the third
cytoplasmic loop (F248S) and deletions in the cytoplasmic tail (
62
and 87) in G protein activation and desensitization.
Materials
NKA, neuropeptide K (NPK) and
(Nle)-NKA(4-10) (N(4-10)) were from Bachem
(Bubendorf, Switzerland). GR64349 was synthesized as
described(4) . SP6 and T7 polymerases were from Promega. XhoI and BsmAI endonucleases were from New England
Biolabs. Staurosporine, bacitracin, chymostatin, leupeptin,
benzamidine, MS222, and collagenase were from Sigma. Ro-31-8220
was from Calbiochem. [
H]SR48968 (28 Ci/mmol) was
obtained from Amersham (Buckinghamshire, United Kingdom).Expression Vectors for NK2 and
Mutants
The gene encoding the human ileum NK2 receptor (5) was subcloned, downstream of the SP6 polymerase initiation
site, in a transcription vector (pGEMNK2) (6) . The pGEMNK2
vector was optimized for efficient transcription, containing a short
5`-non-translated region with low secondary structure forming potential (7) and a Kozak eucaryotic consensus sequence for efficient
initiation of translation at AUG(8) . NK2 deletions mutants
62 and 87 were obtained by cutting pGEMNK2 vector at unique
sites using restriction endonucleases XhoI or BsmAI,
respectively. NK2 site-specific mutants F248S and F248A were
constructed by polymerase chain reaction and subcloned into
pBluescript. F248E mutant was constructed by the method of Kunkel (25) using the following primer (sense strand):
5`-GCCAAGAAGAAGGAAGTGAAGACCATG (mutation underlined). All
constructs were confirmed by restriction mapping and DNA sequencing.
Capped mRNA transcripts for NK2 and mutants were obtained by run-off
transcription of linearized pGEMNK2 or pBluescript plasmids with SP6
polymerase or T7 polymerase, respectively, in the presence of capping
nucleotide using the RNA capping kit from Stratagene. The yield was
approximately 10 µg of capped mRNA from 1 µg of plasmid DNA.
The mRNA was used directly for injection into oocytes.Preparation and Microinjection of
Oocytes
The female frogs (Xenopus laevis) were
anesthetized by immersion in 0.4% MS222 at room temperature for about
15-30 min and killed by decapitation. To remove follicle cells,
oocytes were collected and incubated with collagenase 0.2% in 50 ml of
OR2 medium without Ca and Mg
(OR2
= 82.5 mM NaCl, 2.5 mM KCl, 1 mM
Na
HPO
, 15 mM HEPES, 2 mM
CaCl, 1 mM MgCl, pH adjusted to 7.6)
in tubes under slow agitation for 1 h at room temperature. The oocytes
were washed with OR2 medium and incubated again for 1 h with a fresh
solution of collagenase (0.2%) in OR2. When inspection indicated that
most of the oocytes were free from their follicles, the oocytes were
rinsed carefully with OR2 medium and then with ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl, 1
mM MgCl, 5 mM sodium pyruvate, 5 mM HEPES, adjusted to pH 7.5 with NaOH). Oocytes were allowed
1-2 h for recovery after collagenase treatment before selecting
stages V-VI for injections. Selected oocytes were incubated in
filtered ND96 containing 100 µl/ml penicillin, 100 µg/ml
streptomycin, overnight at 18 °C before injection. Oocytes were
microinjected using an Inject+Matic air pump (Gabay). Injection
needles were made from calibrated Drummond capillaries of 6 µl
volume. mRNA (25 ng in 50 nl) was injected in the cytoplasm. Oocytes
were individually transferred to wells of 96-well flat bottom culture
plates and incubated in ND96 at 18 °C for 24 h before voltage-clamp
recording or for 22 h before Ca efflux assays.
Electrophysiology
Electrophysiological
recordings were carried out on oocytes superfused with OR2 medium at
18-20 °C under voltage-clamped conditions using two
microelectrodes (1-2 M, both filled with 3 M KCl).
The membrane potential was clamped routinely at -100 mV using a
Gene Clamp 500 (AXON) instrument. Each test substance was dissolved in
OR2 medium and applied by constant flow superfusion over 6 s.
Staurosporine (1 µM) and Ro-31-8220 (5
µM) were applied externally by addition to the medium 5
min before testing and to the superfusate.
Preparation of Oocyte Membrane
Fraction
The membranes were prepared according to Kobilka (9) with the following modifications. Oocytes were homogenized,
24 h after injection, in ice-cold buffer A (75 mM Tris-HCl, pH
7.4, 12.5 mM MgCl, 1 mM EDTA, 30%
sucrose, 0.15 mg/ml benzamidine, 0.1 mg/ml bacitracin) by passing the
mixture through a pipetteman equipped with a 200-µl tip ten times.
The homogenate was centrifuged at 3,000 
g for 10 min
to remove pigment granules. The supernatant fraction was centrifuged at
10,000 g for 10 min. The supernatant was then
centrifuged at 400,000 g for 30 min. The pellet,
containing the membranes, was resuspended in buffer A in a sonicator
bath for 5 min. Protein concentration was determined by the method of
Lowry et al.(10) using bovine serum albumin as a
standard. Typically, the yield was about 10 µg of total membrane
protein per oocyte. Membrane fractions were stored at -80 °C. Binding
Assays
[H]SR48968 binding assays on
membrane fraction from 12 oocytes were performed by incubation for 1 h
at ambient temperature in a 0.5-ml total volume of binding assay medium
comprising 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3
mM MnCl, 0.2 mg/ml bovine serum albumin, 40
µg/ml bacitracin, 0.8 mg/ml chymostatin, 1.6 mg/ml leupeptin.
Separation of protein-bound ligand from free ligand was achieved by
rapid filtration on Whatman GF/B filters, pretreated with ice-cold 0.5%
(w/v) polyethylenimine, using a Brandel cell harvester, followed by
three washes with ice-cold 50 mM Tris-HCl, pH 7.4. The filters
were then dried and counted in a Beckman LS-5000CE liquid scintillation
counter. Nonspecific binding was determined in the presence of 10
µM Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH (R396). K
and B
values were
determined by Scatchard analysis. Saturation binding curves and
Scatchard plots were calculated using the programs RadLig and Grafit.Ca
Mobilization
Assays
Ca
efflux assays were
performed as described (11) with the following modifications.
Pools of 10 oocytes were incubated in 0.2 ml of ND96 containing
Ca
(75 µCi/ml) for 2 h at 18 °C.
They were then carefully washed with 0.3 ml of ND96. This medium was
changed every 10 min for 50 min. NKA or GR64349 was added at 0.1
µM for 1 min. Oocytes were then washed twice and incubated
for 15 min in 0.2 ml of ND96. The medium was removed and added to 3 ml
of scintillation fluid and counted.
Receptors and Ligands
The predicted
structure of the human NK2 receptor and mutants is shown in Fig. 1. Mutants F248S, F248A, and F248E contain a single amino
acid change in the COOH-terminal portion of the third cytoplasmic loop,
a region that is known to be important for G protein interaction.
Mutants 62 and 87 are truncated forms of the receptor with 62
and 87 amino acids deleted from the COOH terminus, respectively.
62 lacks 15 serine/threonine residues, and 87 lacks all of
the 18 serine/threonine residues as well as the putative palmitoylation
site at cysteines 324-325 of the cytoplasmic COOH-terminal
domain.
62 and 87 truncation mutants in the cytoplasmic tail. Gray dots represent serine or threonine residues in
cytoplasmic loops that are potential sites for phosphorylation by G
protein receptor kinases.
)-NKA(4-10) (N(4-10)) (12) are
shorter forms of NKA that have high selectivity and potency at NK2.
Functional Expression of NK2 Receptors in Xenopus
Oocytes
Oocytes were injected with 25 ng of cRNA for NK2 or
mutants and stimulated by superfusion with NK2 agonist after 24 h,
under voltage-clamp at a holding potential of -100 mV.
Electrophysiological recordings shown in Fig. 2are
characteristic of inward Ca-dependent chloride
currents elicited by NK2 agonists at 1 µM. Similar results
were obtained at 0.1 µM. The waveforms generated by
wild-type NK2 and NK2 mutants (F248S, F248A, F248E,
62, or
87) were very similar. The onset of response for F248E was delayed
with a mean latency of 13 ± 3 s compared to 4 ± 1 s for
wild-type NK2 and the other mutants. The current amplitudes varied from
oocyte to oocyte, but the average values for NK2 and mutants F248S,
F248A, or F248E were not significantly different. Moreover, for a given
receptor, the amplitudes were independent of the structure of the
ligand used to elicit the signal at 1 µM (Fig. 3).
Oocytes not injected with cRNA did not elicit currents in response to
NKA. The amplitude of inward current peak was function of the amount of
cRNA injected. Typically, for NK2 after 24 h, the responses at 2.5 and
0.25 ng of cRNA were 20-50% and 2-10%, respectively, of the
response at 25 ng of cRNA.
-dependent chloride
current evoked by stimulation of NK2 receptor in X. laevis oocytes. Oocytes were injected with 25 ng of cRNA into the
cytoplasm and incubated at 18 °C for 24 h. Representative traces of
voltage-clamp recordings for wild-type NK2 and NK2 mutant F248S after
stimulation with the appropriate ligand (1 µM, 6 s) are
shown. The oocytes were washed for 5 min before the second stimulation.
In each panel, traces from left to right are recordings after the first and second stimulation,
respectively. Typical data obtained from three different oocyte batches
are shown.
-dependent Cl
currents elicited
by various ligands in oocytes injected with wild-type NK2 or F248S
mutant cRNA. Oocytes were stimulated with agonist (1 µM, 6
s) 24 h postinjection (empty bars). Oocytes were then washed
for 5 min and stimulated a second time in the same conditions (filled bars). Data are mean ± S.E. of 5 oocytes from
the same source. Comparable results were obtained in three to six
separate batches of oocytes.
efflux
assay(11) . Activation of either NK2 or mutant F248S by NKA or
GR64349, at 0.1-1 µM concentrations, produced
identical acceleration of
Ca
efflux (Fig. 4).
Ca
efflux from Xenopus oocytes
injected with wild-type NK2 or F248S mutant receptor cRNA. Oocytes were
loaded with
CaCl
22 h after injection and then
stimulated with 0.1 µM agonist for 1 min as described in (11) . Each bar represents the mean Ca
efflux ± S.E. from triplicates
of 10 oocytes in a representative
experiment.
H]SR48968 were performed
on membrane preparations of oocytes in at least two independent
experiments. Each receptor preparation showed a single class of
saturable binding sites with K in the range from
0.5 to 1.0 nM. The K values were
comparable to the K value of 2.7 ± 0.3
nM for NK2 in COS cells(13) . The maximum binding
capacity B varied from batch to batch of oocytes
in the range of 70-160 fmol/mg of total membrane protein.Agonist-induced Desensitization of NK2
Receptor
Restimulation of oocytes expressing wild-type NK2
with 1 µM NKA 5 min after the first stimulation failed to
elicit chloride channel activity ( Fig. 2and Fig. 3).
This lack of response lasted for more than 30 min and even up to
1-3 h in some batches. Quite strikingly, this lack of response
was observed only when NKA or NPK was used as a ligand at a 0.1 or 1
µM concentration. In contrast, second stimulation by
either GR64349 or N(4-10) produced a chloride channel activity
comparable to the first stimulation in amplitude but slightly greater
in latency of response by an average of 2-3 s. Moreover, up to
four repeated stimulations by 1 µM GR64349 at 5-min
intervals gave responses, whereas a single stimulation with NKA at a
concentration 10-fold lower (0.1 µM) caused
desensitization. Identical results were obtained with oocytes
expressing the mutant receptor F248A. When oocytes were co-injected
with cRNA for NK2 and interleukin 8 receptor (IL8RA), the NKA-induced
desensitization of NK2 did not affect the ability of IL8RA to function
in response to stimulation with 1 µM interleukin 8 (data
not shown) indicating NK2 receptor desensitization rather than
depletion of inositol triphosphate-sensitive intracellullar
Ca stores. When oocytes expressing NK2 were injected
with 1 pmol of heparin 30 min prior to 1 µM NKA
stimulation, the desensitization was clearly attenuated in 70% of the
oocytes tested with an amplitude for the second response equal to
10-20% of the initial response (Fig. 5). Treatment of the
oocytes expressing NK2 with either the protein kinase inhibitor
staurosporine (1 µM) or the specific PKC inhibitor
Ro-31-8220 (5 µM), before and during ligand
application, had no effect on NKA-induced desensitization.
Surprisingly, incubation of the oocytes with staurosporine, but not
with Ro-31-8220, restored the ability of the NK2 receptor to
desensitize in response to either GR64349 or N(4-10) ( Fig. 6and Fig. 7).
A Point Mutation in the Third Intracellular Loop
Prevents NK2 Desensitization
A phenylalanine to serine
mutation at position 248 in the third intracellular loop of NK2 has no
noticeable effect on [H]SR48968 binding affinity (K
= 0.62 ± 0.18 nM compared to 0.54 ± 0.1 nM for wild-type NK2).
Receptor signal transduction by NK2 or NK2 F248S was identical as
assayed by activation of chloride currents or Ca efflux ( Fig. 3and Fig. 4). Moreover, stimulation
of receptors by NKA, NPK, N(4-10), or GR64349 gave responses of
the same amplitude. However, the ability of NK2 F248S to desensitize on
repeated application of NKA at 5-min intervals was totally impaired.
When oocytes expressing the mutant F248S were treated with the protein
kinase inhibitor staurosporine (1 µM) or Ro-31-8220
(5 µM) before and during stimulation with agonists, the
ability of the receptor to desensitize in response to NKA was restored.
However, and in contrast to the wild-type NK2, the F248S mutant
receptor produced unaltered responses to repeated exposures to 1
µM GR64349 regardless of the presence or absence of either
Ro-31-8220 or staurosporine ( Fig. 6and Fig. 7).
The mutant F248A in which phenylalanine at position 248 was replaced by
alanine was indistinguishable from wild-type NK2 in regard to
functional activation or desensitization (data not shown). We created
the mutant F248E, which substitutes a glutamic acid residue at position
248, thus mimicking a phosphorylated serine. Mutant F248E was
functionally active but resistant to desensitization by either NKA or
GR64349 (data not shown).
Truncation of NK2 Carboxyl Terminus Impairs
Desensitization
Progressive truncation of the COOH-terminal
tail of NK2 in mutants 62 and 87 produced functional
receptors with current responses to NKA progressively diminished
compared to the wild-type NK2 (Fig. 8), suggesting some loss of
activity. However, and in contrast to NK2, the responses to a second
application of NKA were 5-10% and 25-35% of the initial
response for 62 and 87, respectively, indicating a resistance
to agonist-induced desensitization.
-dependent Cl
currents elicited
by NKA in oocytes injected with wild-type NK2 or deletion mutants
62 and 87. Conditions are as in the legend of Fig. 3.
efflux assay, an
indirect receptor activation assay measuring intracellular
Ca
mobilization following phospholipase C activation
in oocytes (11) (Fig. 4). This indicates that the F248S
mutant is as active as the wild-type receptor in stimulating second
messenger pathway. The potent ligand GR64349 is a full agonist at NK2
in guinea pig trachea (14) as well as in Chinese hamster ovary
cells stably transfected with the human NK2 receptor (15) or in
transiently transfected COS cells (data not shown). In all these
systems, the ratios of agonist concentration for half-maximal
activation (EC
) to NK2 receptor binding affinity of NKA
and GR64349 are identical. Thus, the observed difference in
desensitization cannot be explained by partial agonism of ligands or
partial activity of mutant F248S.
adrenergic receptors
causing different conformational changes and different degrees of
G
and G
coupling(16) . The existence of
divergent conformational requirements for angiotensin II receptor
internalization and signaling has been recently proposed (17) .
Taken together with our results, this clearly indicates that G
protein-coupled receptors may have multiple active conformers
displaying different specificity for agonist binding and distinct
functional activities.
-adrenergic receptor kinase 1 and 2 in
vitro(18) , and attenuation of agonist-induced
desensitization of NK1 by truncation of the COOH terminus was
demonstrated(19) . Similar observations were reported for
adrenergic receptor (20) or rhodopsin in
vivo(21) . Desensitization of the NK2 receptor in oocytes
was recently reported(22) , but the mechanism of
desensitization remained unclear. In this work, two lines of evidence
suggest a mechanism of desensitization by phosphorylation of the NK2
receptor.
62 and 87), removing serine and threonine sites of
phosphorylation, produced active receptors displaying attenuated
desensitization. Thus, the COOH terminus of NK2 plays a role in acute
desensitization(24) . This is in contrast to Josiah et al.(22) that indicated no activity for NK2 81 mutant and
complete desensitization for NK2 62 mutant. These discrepancies
remain unexplained. The difference in loss of desensitization of the
62 mutant compared to the 87 mutant suggests that the
COOH-terminal 62 residues play only a minimal role and that the
COOH-terminal region proximal to the seventh transmembrane domain is
determinant for NK2 receptor desensitization. We cannot exclude,
however, that the increased loss of desensitization for the 87
mutant may be reflective of its reduced activity compared to wild type
NK2 (Fig. 8).
1 µM), in contrast to
the 2-adrenergic receptor where desensitization was completely
abolished by 1 µM heparin(23) . Therefore, we
cannot exclude that the observed impairment of NK2 desensitization by
heparin may be mediated by inhibition of PKC or other signaling
pathways. In the absence of a specific inhibitor of G protein receptor
kinase, it is difficult to probe the exact role of these kinases in
regulation of NK2 desensitization by phosphorylation. Thus, we cannot
exclude a mechanism involving other protein kinases(24) .VK . . .
). However, in G protein-coupled receptor systems, receptor
phosphorylation causes signal attenuation, whereas here we observe the
opposite effect. The differences observed between F248A and F248S, or
F248E mutations combined with the effect of staurosporine and
Ro-31-8220 implies an agonist-induced phosphorylation event. Our
findings are consistent with the hypothesis that agonist-induced
conformational changes in the third cytoplasmic loop exposes
Ser
or other sites on the cytoplasmic face of the
receptor to a kinase, probably PKC. The resulting phosphorylation
alters G protein receptor kinase recognition or activation, thus
preventing agonist-induced desensitization. In support of this
hypothesis, mutant F248E, which substitutes an acidic residue at
position 248 and resembles phosphorylated serine both sterically and
electronically, was defective in desensitization. Interestingly, in the
case of the mutant F248S, this mechanism functions for receptor
interaction with NKA but not GR64349, whereas in the case of wild-type
NK2 the differential effect on desensitization is observed with GR64349
and not NKA. This further demonstrates the existence of multiple
induced active conformations of G protein-coupled receptors and the key
role of the position 248 in modulating these conformations.
))-NKA(4-10); PKC,
protein kinase C.
We thank Dr. K. Lundstroem, S. Jemelin, and F. Talabot
for the construction of mutants F248S, F248E, and F248A, Dr. S. Valera
for help with the electrophysiology, Drs. A. North and M. Edgerton for
critical reading of the manuscript, and Dr. J. Knowles for enthusiastic
support during these studies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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G. Turcatti, K. Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel, and A. Chollet Probing the Structure and Function of the Tachykinin Neurokinin-2 Receptor through Biosynthetic Incorporation of Fluorescent Amino Acids at Specific Sites J. Biol. Chem., August 16, 1996; 271(33): 19991 - 19998. [Abstract] [Full Text] [PDF]
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T. Palanche, B. Ilien, S. Zoffmann, M.-P. Reck, B. Bucher, S. J. Edelstein, and J.-L. Galzi The Neurokinin A Receptor Activates Calcium and cAMP Responses through Distinct Conformational States J. Biol. Chem., September 7, 2001; 276(37): 34853 - 34861. [Abstract] [Full Text] [PDF]
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