(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,
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.
The NK2 ()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) .
When expressed in Xenopus oocytes, the NK2 receptor causes release of Ca 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.
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
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.
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)-NKA(4-10) (N(4-10)) (12) are
shorter forms of NKA that have high selectivity and potency at NK2.
Figure 2:
Ca-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.
Figure 3:
Agonist-induced
Ca-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.
Functional activity of NK2 and mutant
F248S was further evaluated by Ca 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).
Figure 4:
Effects of NK2 agonists on 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.
To characterize NK2 and mutant receptors expressed
in Xenopus oocytes, equilibrium binding studies with the
specific NK2 antagonist [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.
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-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.
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 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.
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 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.
The substance P receptor (NK1) was shown to
be a substrate of -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.
First, truncations in the COOH-terminal part of the
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).
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 (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) .
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 ( . . . SVK . . .
). 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.
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.