Departments of Surgery and Physiology, University of California San Francisco, San Francisco, California 94143-0660
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ABSTRACT |
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An understanding of the mechanisms that regulate signaling by
the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest
because of their role in inflammation and pain. By using activators and
inhibitors of protein kinase C (PKC) and NK1-R mutations of potential
PKC phosphorylation sites, we determined the role of PKC in
desensitization of responses to SP. Activation of PKC abolished
SP-induced Ca2+ mobilization in cells that express
wild-type NK1-R. This did not occur in cells expressing a
COOH-terminally truncated NK1-R (NK1-R324), which may correspond to
a naturally occurring variant, or a point mutant lacking eight
potential PKC phosphorylation sites within the COOH tail (NK1-R
Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392,
Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the
t1/2 of SP-induced Ca2+
mobilization was seven- and twofold greater in cells expressing NK1-R
324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the
t1/2 of SP-induced Ca2+
mobilization. Neither inhibition of PKC nor receptor mutation affected
desensitization of Ca2+ mobilization to repeated challenge
with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates
SP-induced Ca2+ mobilization by full-length NK1-R and does
not regulate a naturally occurring truncated variant. PKC does
not mediate desensitization to repeated stimulation or endocytosis of
the NK1-R.
substance P; tachykinins; downregulation; G protein-coupled receptors
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INTRODUCTION |
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BIOLOGICAL
RESPONSES TO AGONISTS of G protein-coupled receptors (GPCRs) for
neurotransmitters and hormones are closely regulated by mechanisms that
operate at the level of the ligands and their receptors (4,
24). These mechanisms prevent uncontrolled stimulation, which
may otherwise lead to disregulation and disease. At the level of the
ligand, the balance between release and uptake or degradation
determines the availability of agonists to interact with cell surface
receptors. At the level of the receptor, several mechanisms terminate
signal transduction. These mechanisms include homologous
desensitization to repeated application of the same agonist,
heterologous desensitization to repeated challenge with different
agonists, and receptor endocytosis. Desensitization is principally
mediated by uncoupling receptors from heterotrimeric G proteins. G
protein receptor kinases (GRKs) and second messenger kinases such as
protein kinase C (PKC) and protein kinase A play important roles in
desensitization (see Ref. 4 and references therein).
Agonists of many GPCRs induce translocation of GRKs and second
messenger kinases to the plasma membrane where they phosphorylate
activated receptors. -Arrestins also translocate to the plasma
membrane where they interact with GRK-phosphorylated receptors and
thereby uncouple them from G proteins to mediate desensitization.
-Arrestins also couple receptors to clathrin and thereby mediate
endocytosis, which contributes to desensitization by depleting GPCRs
from the plasma membrane (14, 19). These regulatory
mechanisms have been characterized in detail for only a few GPCRs, and
the relative importance of these processes differs from one receptor to
another (4). Although truncation of the intracellular COOH
tails and mutation of potential phosphorylation sites within these
regions can impair uncoupling and endocytosis of certain GPCRs
(2, 3, 5, 7, 18, 25, 34, 35, 38), the receptor domains
that are critical for these processes have not been fully characterized.
We investigated regulation of the neurokinin 1 receptor (NK1-R) for the
neuropeptide substance P (SP). An understanding of regulation of the
NK1-R is of interest because this receptor mediates neurogenic
inflammation, nociception, and the regulation of gastrointestinal secretion and motility (30). Biological responses to SP
are rapidly attenuated in the continued presence of agonists and
diminish to repeated challenge, indicating that the NK1-R rapidly
desensitizes (8, 16, 28). However, the mechanisms of this
desensitization are incompletely understood. GRKs and -arrestins may
mediate desensitization, because GRK-2/3 extensively phosphorylates the NK1-R (23), and SP induces translocation of GRK-2/3 and
-arrestins 1/2 from the cytosol to the plasma membrane where they
interact with the NK1-R (1, 28, 29). PKC also
participates, because activators of PKC induce phosphorylation of the
NK1-R and inhibit SP signaling (32, 40), and SP stimulates
membrane translocation of PKC
2 (1, 27).
However, the domains of the NK1-R that are important for uncoupling and
endocytosis are not fully defined. The COOH-terminal tail of the NK1-R
contains 26 serine and threonine residues that could be phosphorylated
by GRKs or PKC and which may interact with
-arrestins. Truncation of
the COOH tail to remove some of these sites diminishes SP-induced
desensitization and endocytosis of the NK1-R (5, 25, 33).
Since a naturally occurring variant of the NK1-R lacking most of the
COOH tail has been identified (9, 15, 22), an
understanding of the importance of the COOH tail in receptor regulation
is of considerable interest.
We examined the role of PKC in SP-induced uncoupling and endocytosis of the NK1-R by using activators and inhibitors of PKC and by receptor mutation to remove putative PKC phosphorylation sites with the COOH tail. Our aims were to 1) generate cell lines expressing truncation mutants of the NK1-R and point mutants of potential PKC sites; 2) determine the consequences of mutation for uncoupling and endocytosis of the NK1-R; and 3) investigate the effects of PKC activators or inhibitors on NK1-R signaling and endocytosis. Our results show that PKC plays an important role in attenuating SP-induced Ca2+ mobilization.
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MATERIALS AND METHODS |
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Reagents.
SP was from Phoenix Pharmaceutical (Belmont, CA).
[125I]SP (2,000 Ci/mmol) was from Amersham Pharmacia
Biotech (Uppsala, Sweden). Phorbol 12,13 dibutyrate (PDBu),
4-phorbol 12,13-didecanoate (PDD), GF-109203X, and
bisindolylmaleimide V were from Calbiochem (San Diego, CA). Fura 2-AM,
pluronic acid, and propidium iodide were from Molecular Probes (Eugene,
OR). The synthesis of cyanine 3.8-labeled SP (Cy3-SP) has been
described (10). The monoclonal M2 antibody directed
against the FLAG epitope (DYKDDDDK) was from International
Biotechnologies (New Haven, CT). Goat-anti-mouse IgG labeled with FITC
was from Caltag Laboratories (Burlingame, CA). GeneEditor in vitro
site-directed mutagenesis system was from Promega (Madison, WI). The
expression vector pcDNA3 was from Invitrogen (Carlsbad, CA).
Oligonucleotides were from Genemed Biotechnologies (San Francisco, CA).
Lipofectin, DMEM, and PBS were from Life Technologies (Gaithersburg,
MD). G418 was from Gemini Bio-Products (Calabasas, CA). Kirsten sarcoma
virus-transformed rat kidney epithelial cells (KNRK) were from American
Type Culture Collection (Rockville, MD). Other reagents were from Sigma
Chemical (St. Louis, MO).
Description of NK1-R mutants.
Of the 99 residues in the COOH tail of rat NK1-R, 26 are serine and
threonine. We truncated the NK1-R to remove portions of the
COOH tail (Fig. 1). NK1-R324, the most
truncated mutant that may correspond to a naturally occurring variant,
lacks 83 of the COOH-terminal residues, including all serine and
threonine. NK1-R
342 and NK1-R
354, respectively, lack 65 and 53 residues and possess only 3 and 7 serine and threonine
residues. The NK1-R COOH tail includes two serine/threonine
clusters, one in region 338-360 close to the transmembrane domain,
and the other near the COOH terminus in region 376-403.
NK1-R
342 lacks both clusters, and NK1-R
354 lacks the cluster
338-360. Point mutations of full-length NK1-R were made to disrupt
potential PKC phosphorylation sites (Fig. 1). Because phosphorylation
by PKC requires the presence of a basic residue near phosphorylated
serine and threonine, we considered potential phosphorylation sites to
be serine or threonine residues adjacent to, or one residue from,
arginine or lysine. We mutated 8 of the 26 serine and threonine
residues in the COOH tail. In NK1-RKC1, Ser-338 and Thr-339 were
mutated to alanine. In NK1-RKC2, we mutated Ser-352, which lies within
a stretch of 12 residues between NK1-R
342 and NK1-R
354. In
NK1-RKC3, Ser-387, -388, -390, -392, and -394 were mutated to alanine.
In NK1-RKC4, all of the 8 mutations of NK1-RKC1, NK1-RKC2, and NK1-RKC3
were introduced.
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Generation of NK1-R mutants.
All mutants were derived from rat NK1-R with the
NH2-terminal FLAG epitope. This construct was subcloned
into pcDNA3 to generate the wild-type NK1-R (NK1-Rwt). The FLAG epitope
does not affect signaling, desensitization, or trafficking of the NK1-R
(39). Truncation mutants were generated by introduction of
a stop codon after residues position 324, 342, and 354 to form
NK1-R324, NK1-R
342, and NK1-R
354 as described
(5). Potential PKC consensus sites in the COOH tail were
mutated using a GeneEditor site-directed mutagenesis kit. The
primers used to obtain the mutants were KC1, 5'-GGGCTGGAAATGAAAGCCGCCCGGTACCTCCAGACA-3',
which carries the mutations Ser-338, Thr-339/Ala (bold), and a
KpnI restriction site (underlined); KC2,
5'-CAGAGCAGCGTATACAAGGTCGCCCGCCTGGAGACCACC-3', Ser-352/Ala mutation (bold), AccI site (underlined); and
KC3, 5'-CTCACCTCCAACGGCGCCGCTCGAGCCAACGCCAAGGCCATGACAGAAAGCTCC-3', Ser-387, Ser-388, Ser-390, Ser-392, Thr-394/Ala (bold), XhoI
site (underlined). The mutant KC4 was obtained using all three primers and contained all the corresponding mutations. After transformation of
JM109 bacteria, clones were screened by restriction analysis and DNA sequencing.
Transfection and cell culture. KNRK cells expressing wild-type and truncated NK1-Rs were generated and characterized as described (13, 17, 29, 39). KNRK cells were transfected with cDNAs encoding the point mutants using Lipofectin. After 7-10 days, cells expressing high levels of receptor were sorted using flow cytometry (13, 29). A minimum of 24 clones was transferred in 24-well plates on coverslips and analyzed for expression by immunofluorescence using the FLAG M2 antibody and by binding of Cy3-SP. Selected clones were maintained in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.4 mg/ml G418. Clones were selected to express similar levels of NK1-R, as determined by binding experiments using radiolabeled SP, binding of Cy3-SP, and flow cytometry using the FLAG antibody.
Measurement of [Ca2+]i. NK1-R signaling was assessed by measuring SP-induced Ca2+ mobilization (16). Cells were incubated with 2.5 µM fura 2-AM and 0.2% pluronic acid for 20 min at 37°C, washed, and fluorescence was measured at 340- and 380-nm excitation and 510 nm emission in a spectrophotometer (F-2000; Hitachi Instruments, Irvine, CA). The ratio of the fluorescence at the two excitation wavelengths, which was proportional to the intracellular Ca2+ concentration [Ca2+]i, was calculated. For concentration-response analyses, cells were exposed to a single application of graded concentrations of SP. To determine the EC50, the sigmoidal shape of the dose-response curve was transformed by a logit/log representation where logit (response) = log[response/(1-response)] is plotted against log(dose). A linear regression was used to obtain the best fit, and the EC50 was calculated as the concentration for which the logit = 0, corresponding to the inflection point of the sigmoidal curve. To examine desensitization, cells were exposed to a first challenge of SP or vehicle (control) for 2 min, washed, and were then reexposed to a second challenge of SP 5 min after the first. Observations were from more than three experiments.
Binding and endocytosis of [125I]SP. The rate of internalization of the NK1-R was determined by binding assays with [125I]SP (17). Cells were incubated in Hanks' balanced salt solution with 0.1% BSA and 50 pM [125I]SP for 1 h at 4°C, washed, and incubated at 37°C for 0 to 30 min. Cells were washed with ice-cold PBS and incubated in 250 µl of ice-cold 0.2 M acetic acid containing 0.5 M NaCl (pH 2.5) on ice for 5 min to separate acid-labile (cell surface) from acid-resistant (internalized) label. Radioactivity was counted in the acid fraction, and the fraction internalized in the cells was first detached by lysing the cells with 0.5 N NaOH overnight at 4°C. Nonspecific binding was measured by preincubation of the cells with 1 µM SP and subtracted to obtain specific binding. Observations were in triplicate in n > 3 experiments.
Fluorescence microscopy. To localize the NK1-R and examine endocytosis, we used Cy3-SP (20, 29). Cells were incubated in DMEM with 0.1% BSA containing 10-100 nM Cy3-SP for 1 h at 4°C for equilibrium binding. They were washed in DMEM-BSA at 4°C and either fixed immediately or incubated in SP-free medium at 37°C for various times to permit receptor endocytosis and trafficking to proceed. Cells were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min at 4°C, washed, and mounted. Cells were observed with an MRC 1000 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis. Results are expressed as means ± SE and are compared with Student's t-test or ANOVA and Student-Newman-Keuls test, with P < 0.05 considered significant.
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RESULTS |
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Truncated and mutant receptors were functional.
We verified that all cell lines expressed functional receptors by
measuring SP-induced Ca2+ mobilization. SP stimulated a
similar rapid increase in [Ca2+]i in cell
lines expressing wild-type, truncated, and point mutant NK1-Rs with
comparable efficacies (not shown). In cells expressing NK1-Rwt, SP
induced a Ca2+ response with an EC50 of 0.6 nM
(Fig. 2). In cells expressing the
truncated receptors, EC50 was 0.2, 0.1, and 0.2 nM for the 324,
342, and
354, respectively (Fig. 2A). We
obtained similar results with the point mutants for which the
EC50 of KC1, KC2, KC3, and KC4 were 0.2, 0.1, 0.1, and 0.1 nM, respectively (Fig. 2B). Thus SP stimulates
Ca2+ mobilization in cells expressing truncated and point
mutant NK1-Rs, which lack serine and threonine residues in their COOH
tails, with three- to sixfold higher potency than in cells expressing NK1-Rwt.
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PKC inhibits SP-induced
Ca2+ mobilization.
Activation of PKC with phorbol esters strongly inhibits signaling by
several GPCRs (18, 34, 35, 37, 38, 40). To determine the
role of PKC in regulating the NK1-R, we preincubated cells with 1 µM
PDBu for 10 min, which activated the classical and novel subtypes of
PKC, and then challenged cells with SP at concentrations close to the
EC50 (0.1-0.3 nM), which similarly increased
[Ca2+]i. In cells expressing NK1-Rwt, PDBu
abolished SP-induced Ca2+ mobilization (Fig.
3A and Fig.
4). This effect of PDBu was prevented by preincubation with 0.1 µM GF-109203X for 20 min, which inhibits PKC. Preincubation with GF-109203X alone did not affect the magnitude of SP-induced Ca2+ mobilization. The inactive enantiomers
PDD and bisindolylmaleimide V had no effect (not shown). Thus in cells
expressing NK1-Rwt, activation of PKC prevents SP-induced
Ca2+ mobilization.
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PKC partially mediates desensitization of
SP-induced
Ca2+ mobilization.
Homologous desensitization of GPCRs is manifested by the attenuation of
responses in the continued presence of agonists and by diminished
responses to repeated application of the same agonist (4).
To determine the role of PKC in these processes, we examined the
duration of SP-induced Ca2+ mobilization and
desensitization of Ca2+ transients to repeated challenge
with SP in cells expressing wild-type and mutated NK1-R in the presence
or absence of PKC inhibitors. We compared cells expressing NK1-Rwt with
those expressing NK1-R324 or KC4, since these mutants exhibited the
most marked differences in responsiveness to PDBu compared with
NK1-Rwt.
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PKC does not regulate endocytosis of the
NK1-R.
Agonist-induced endocytosis of GPCRs may contribute to desensitization
by depleting receptors from the plasma membrane. For several GPCRs,
serine and threonine residues in the COOH tail are required for
endocytosis (2, 21). We have previously reported that
truncation of the NK1-R at 324 and 342, but not at 354, inhibits
SP-induced endocytosis of the NK1-R and that tyrosine residues in the
COOH tail may contribute to endocytic motifs (5).
Moreover, in cells expressing NK1-R324, SP does not cause
translocation of
-arrestin to the plasma membrane, suggesting that
NK1-R
324 does not interact with
-arrestins, which may explain the
impaired endocytosis and desensitization (11). To evaluate
the importance of serine and threonine residues in the COOH tail and of
the role of PKC, we studied SP-induced endocytosis of NK1-Rwt and point
mutants lacking serine and threonine residues in the COOH tail in the
presence of the PKC inhibitor GF-109203X.
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DISCUSSION |
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Acute activation of PKC abolished SP-induced Ca2+
mobilization by NK1-Rwt. In marked contrast, PKC activators did not
affect signaling by truncated NK1-R324 or by NK1-RKC4 lacking eight potential PKC sites in the COOH tail. Inhibition of PKC prolonged SP-induced Ca2+ mobilization by 35% in cells expressing
NK1-Rwt but had no effect in cells expressing NK1-R
324 or NK1-RKC4.
SP-induced Ca2+ mobilization was prolonged by sevenfold in
NK1-R
324 cells and by twofold in NK1-RKC4 cells, compared with cells
expressing NK1-Rwt. However, desensitization of Ca2+
responses to repeated application of SP and SP-induced endocytosis were
unaffected by NK1-R truncation, deletion of potential PKC sites, or by
administration of PKC inhibitors. Thus our results show that PKC plays
a major role in determining the magnitude and duration of SP-induced
Ca2+ mobilization by NK1-Rwt, and serine and threonine
residues within the COOH tail are necessary for this regulation. PKC
does not contribute to homologous desensitization or to SP-induced
endocytosis of the NK1-R. Importantly, PKC does not regulate signaling
by a naturally occurring truncated variant, which is likely to be of
functional importance in tissues that predominantly express this receptor.
PKC regulates the magnitude and duration of
SP-induced
Ca2+ mobilization.
Receptor phosphorylation and -arrestin-mediated uncoupling from
heterotrimeric G proteins principally mediate desensitization of GPCRs.
Homologous desensitization is manifested by attenuation of the
magnitude and duration of responses after a single challenge with
agonist and diminished responsiveness to repeated challenge. Several
observations from the present study suggest that PKC contributes to
attenuation of the magnitude and duration of SP-induced
Ca2+ mobilization. First, in cells expressing NK1-Rwt, PDBu
abolished SP-induced Ca2+ mobilization. This effect was due
to activation of PKC, since it was reversed by GF-109203X, which
selectively inhibits the classical and novel subtypes of PKC, and an
inactive enantiomer of PDBu had no effect. Second, GF-109203X increased
the duration of the signal by 35% in cells expressing NK1-Rwt. Third,
deletion of potential PKC sites in the COOH tail of the NK1-R (notably in NK1-R
324 and KC4) reversed the inhibitory effect of PDBu and prolonged the Ca2+ response to SP by two- to sevenfold,
compared with NK1-Rwt. Finally, deletion of these sites increased the
potency of SP signaling by three- to sixfold. This increase in potency
may indicate diminished desensitization of Ca2+ signaling
or increased affinity for SP. Alternatively, NK1-Rwt may be
constitutively phosphorylated on one or more potential PKC sites, the
removal of which would lead to more efficient coupling. However,
constitutive phosphorylation of the NK1-R has not been observed
(32, 40). In support of the conclusion that PKC regulates NK1-R, others have shown that activation of PKC inhibits SP signaling in transfected cells and in acinar cells from the parotid gland that
naturally expresses the NK1-R (37, 40). Moreover,
activation of PKC inhibits agonist-induced signaling of other GPCRs,
including protease-activated receptor 2, gastrin-releasing peptide
receptor, thromboxane A2 receptor, purinergic
P2Y2 receptor, and angiotensin II type 1A receptor
(6, 18, 34, 35, 38, 41). Proteinase-activated receptor 2 resembles NK1-R since mutation of serine and threonine residues in the
COOH tail increases the magnitude and duration of signaling and
reverses the inhibitory effects of PDBu (6, 12).
PKC does not mediate desensitization of the
NK1-R to repeated stimulation.
Although PKC can regulate the magnitude and duration of SP-induced
Ca2+ mobilization, we found no evidence that PKC mediates
desensitization to repeated challenge with SP. Exposure of NK1-Rwt
cells to graded concentrations of SP inhibited responses to a second
challenge 5 min later, and this desensitization was unaffected by
GF-109203X and is thus not mediated by PKC. Furthermore, mutation of
potential PKC sites in NK1-R324 and KC4 did not prevent
desensitization. In support of our results, PDBu phosphorylates the
NK1-R at sites that are different from those that are phosphorylated by
SP, and PKC inhibitors do not affect SP-stimulated phosphorylation
(32, 40). SP-induced phosphorylation of the NK1-R has been
observed in KNRK cells used in the present study (Ref. 40
and Bunnett, unpublished observations), although the sites of
phosphorylation remain to be determined. The most likely mechanism of
homologous desensitization is GRK-stimulated phosphorylation of the
NK1-R and interaction with
-arrestins. In favor of this hypothesis, GRK-2/3 strongly phosphorylate the NK1-R at unknown sites
(23), and SP triggers the rapid translocation of
GRK-2/3 and
-arrestins 1/2 from the cytosol to the
plasma membrane of transfected cells and neurons that naturally express
the NK1-R (1, 28, 29). Second messenger kinases play
variable roles in desensitization of other GPCRs. Thus PKC does not
mediate desensitization of the gastrin-releasing peptide receptor
(41), but protein kinase A can desensitize the
2-adrenergic receptor (31).
PKC does not mediate SP-induced
endocytosis of the NK1-R.
Truncation of the NK1-R inhibits SP-induced endocytosis of the NK1-R,
indicating that COOH-terminal domains are necessary for trafficking
(5). However, acute administration of GF-109203X did not
affect endocytosis of NK1-Rwt, and endocytosis of SP proceeded at the
same rate in cells expressing NK1-R mutants lacking potential PKC sites
within the COOH tail compared with NK1-Rwt. Therefore, PKC does not
participate in SP-induced endocytosis of the NK1-R. In support of our
results, PKC does not regulate endocytosis of the purinergic
P2Y2 receptor (18). In addition to their role in uncoupling GPCRs from heterotrimeric G proteins, -arrestins also
serve to couple receptors to clathrin for endocytosis (14, 19). Expression of dominant negative mutants of
-arrestin
strongly inhibits SP-induced endocytosis of the NK1-R
(13), and NK1-R
324 does not interact with
-arrestins, which may explain the diminished endocytosis
(11). Thus SP-induced endocytosis of the NK1-R proceeds by
a
-arrestin-dependent mechanism that requires GRK-mediated phosphorylation of the NK1-R rather than by PKC-induced phosphorylation.
Physiological implications.
Desensitization of signal transduction is important in preventing the
continued stimulation of cells in an uncontrolled manner. Defects in
the mechanisms that terminate signaling by SP, such as deletion of
neutral endopeptidase that degrades extracellular SP, result in
exaggerated neurogenic inflammation (26, 36). Our results
show that PKC regulates the duration and magnitude of SP-induced
Ca2+ mobilization and may thereby contribute to
desensitization of signaling. Thus defects in PKC expression may also
contribute to uncontrolled SP signaling. Although GRKs and
-arrestins are the most likely mediators of desensitization and
endocytosis, their specific roles in desensitization remain to be determined.
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ACKNOWLEDGEMENTS |
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We thank Michelle Lovett for technical assistance, Paul Dazin for assistance with flow cytometry, and Dr. Eileen F. Grady for helpful comments.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39957.
Address for reprint requests and other correspondence: N. W. Bunnett, Univ. of California San Francisco, 521 Parnassus Ave., C-317, San Francisco, CA 94143-0660 (E-mail: nigelb{at}itsa.ucsf.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 September 2000; accepted in final form 6 December 2000.
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