Departments of Surgery and Physiology, University of California San Francisco, San Francisco, California 94143-0660
Submitted 21 November 2002 ; accepted in final form 3 June 2003
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ABSTRACT |
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desensitization; endocytosis; tachykinins
In the present investigation we compared signaling of the NK1R and NK3R,
which mediate the effects of tachykinins in the enteric nervous system.
Although both receptors couple to phospholipase C- and undergo
agonist-induced endocytosis and recycling
(10,
11,
19,
27), a detailed comparison of
these receptors in the same cell has not been made. We have recently reported
that differences in the interaction of the NK1R and NK3R with
-arrestins
may contribute to disparate signaling
(27). The NK1R interacts with
-arrestins for prolonged periods, suggesting a high-affinity
interaction, whereas the NK3R interacts with
-arrestins transiently,
indicative of a low-affinity interaction. In view of the multiple roles on
-arrestins in receptor signaling and regulation, these differences may
explain differences in the function of the NK1R and NK3R.
-Arrestins 1 and 2 play a major role in regulating GPCRs (reviewed in
Refs. 18 and
21). They were discovered as
cofactors of receptor desensitization. Agonist occupation of many receptors
triggers the translocation of cytosolic G protein receptor kinases (GRKs) to
the plasma membrane, where they phosphorylate receptors
(16).
-Arrestins
similarly translocate to interact with phosphorylated receptors and disrupt
their association with heterotrimeric G proteins to induce rapid
desensitization of signal transduction
(1).
-Arrestins are also
adaptors for clathrin and adaptor protein 2 and are thereby required for
agonist-mediated endocytosis of some receptors
(7,
9,
12,
19,
20,
27).
-Arrestin-dependent
endocytosis of receptors has several functions. It contributes to
desensitization by depleting the cell surface of high-affinity receptors that
are available to interact with extracellular agonists. Endocytosis,
intracellular sorting, which entails receptor dephosphorylation and
dissociation of ligand and
-arrestins, and recycling back to the cell
surface are also required for resensitization
(30). Endocytosis is also a
prerequisite for lysosomal degradation and the downregulation of certain GPCRs
that follows chronic exposure to agonists
(15). Finally,
-arrestins are scaffolds that recruit and organize components of the MAP
kinase pathway into endosomes, thereby specifying the subcellular localization
and function of activated MAP kinases
(4,
5,
17). Thus differences in the
nature of the interactions of GPCRs with
-arrestins may account for
marked differences in signaling.
We investigated the colocalization of the NK1R and NK3R with
-arrestins 1 and 2, the principal forms of arrestins that interact with
GPCRs. Although
-arrestins are required for endocytosis of both
receptors (27) and for
coupling the NK1R to MAP kinases
(4), nothing is known about the
ability of the receptors to interact with different
-arrestins and how
this interaction alters receptor function. Our aims were to 1)
compare agonist-induced signaling and trafficking of NK1R and NK3R;
2) compare desensitization and resensitization of NK1R and NK3R;
3) evaluate the ability of NK1R and NK3R to interact with
-arrestins 1 and 2; and 4) identify the domains of the NK1R and
NK3R that account for differences in signaling and regulation.
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MATERIALS AND METHODS |
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Generation of NK1R/NK3R chimeric receptors. Expression vectors encoding rat NK1R and NK3R cDNA in pcDNA3.1 have been described previously (27, 28). Chimeric receptors were generated by replacing the COOH tail and intracellular loop 3 of the NK1R with equivalent domains of the NK3R, and vice versa (Fig. 1). The COOH tails (ct), starting at the end of the seventh transmembrane domain, were exchanged by enzymatic digestion and ligation. Plasmids were incubated with AccI and NotI, which cut respectively at the corresponding seventh transmembrane domain of NK1R and NK3R cDNA and inside the plasmid polylinker. Products were separated on agarose gels, and the tail of the NK3R was ligated to the digested NK1R plasmid by incubation with T4 ligase overnight at 14°C. A similar strategy was used to replace the COOH tail of the NK3R with that of the NK1R. Chimeric receptors in which the intracellular loop 3 (l3) of the NK1R was replaced by the equivalent domain of the NK3R, and vice versa, were generated by PCR. Primers were designed to amplify the intracellular loop 3 of NK3R or NK1R cDNA, with a flanked fragment on each primer corresponding to the NK1R or NK3R cDNA. This PCR product was used as a primer to amplify each part of the NK1R or NK3R by using NK1R or NK3R cDNA as template and another primer corresponding to the COOH- or NH2-terminal part containing restriction sites for subcloning into pcDNA3.1. Mixed chimeras were generated by replacing intracellular loop 3 and the COOH tail of the NK1R with equivalent domain of the NK3R, and vice versa. Constructs with the substituted loop 3 were used as templates, and the COOH tails were replaced by enzymatic digestion and ligation as described. All constructs were sequenced.
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Generation of transfected cells. Kirsten murine sarcoma
virus-transformed rat kidney epithelial cells (KNRK) were from the American
Type Culture Collection (CRL 1569; ATCC, Rockville, MD). The generation and
characterization of KNRK cells stably expressing NK1R or NK3R have been
described previously (19,
27,
28). KNRK cells were similarly
transfected with the cDNAs encoding chimeric receptors to generate stable cell
lines. Transfected cell lines were sorted by flow cytometry by using an
antibody to the FLAG epitope to generate stable lines expressing receptors at
similar levels (19,
27,
28). For some experiments,
cells were transiently transfected with NK1R, NK3R, or chimeric receptors.
This approach permitted comparisons of transfected and nontransfected cells by
microscopy. In addition, KNRK cell lines stably expressing NK1R, NK3R, or the
chimeric receptors were transiently transfected with -arrestin 1 or 2
tagged with green fluorescent protein (GFP)
(20,
27). KNRK cells were
transiently transfected with 5 µg/ml cDNA by lipofection
(19,
27,
28). The medium was removed,
and fresh medium 10% FCS was added for 48 h before experiments. Cells were
plated on glass coverslips for measurement of Ca2+ mobilization and
for microscopy, or on plastic wells for MAP kinase assays.
Microscopy and immunofluorescence. To examine endocytosis, cells
were incubated with 100 nM Alexa-SM-SP (NK1R agonist), Alexa-MP-NKB (NK3R
agonist), or Alexa-SP (which gave similar results to SM-SP) for 60 min at
4°C (for equilibrium binding), washed at 4°C, and either fixed
immediately or incubated in medium at 37°C for 15 min (for trafficking to
proceed) (19,
27,
28). Cells were fixed with 4%
paraformaldehyde in 100 mM PBS, pH 7.4, for 20 min at 4°C. Endogenous
-arrestin 1 or 2 was detected by using antibodies to
-arrestin 1
(1 µg/ml, overnight at 4°C) or
-arrestin 2 (1:50, overnight at
4°C) with secondary antibodies conjugated to FITC (1:200, 1 h at room
temperature). Cells were observed with a Zeiss Axiovert microscope, an MRC
1000 confocal microscope (Bio-Rad, Hercules, CA) with a kryptonargon laser,
and a Zeiss plan-Apochromat x100 oil-immersion objective (NA 1.4,
0.7).
Measurement of intracellular Ca2+. Intracellular Ca2+ concentration ([Ca2+]i) was measured in populations of cells expressing as described previously (19, 27, 28). Fluorescence was measured at 340 and 380 nm for excitation and 510 nm for emission, and the results were expressed as the ratio of the fluorescence at the two excitation wavelengths, which is proportional to the [Ca2+]i. For concentration-response analyses, cells were exposed once to graded concentrations of SM-SP or MP-NKB. To assess desensitization and resensitization, cells were preincubated with 10 nM SM-SP or MP-NKB or vehicle (control) for 10 min at 37°C. They were washed and challenged again with the same agonist at 0 or 30 min after washing. Desensitization was calculated as the percentage of the response to vehicle-treated cells.
MAP kinase assays. Cells were maintained in minimal essential
medium without serum overnight and incubated with 10 nM SM-SP or MP-NKB for
0-30 min at 37°C (4,
28). They were lysed in
boiling 20 mM Tris · HCl, pH 8, 10 mM EDTA, 0.3% SDS, and 67 mM DTT and
then passed through a 20G syringe needle. Lysates (20 µg of total protein)
were analyzed by 12% SDS-PAGE and transferred to polyvinylidene difluoride
membranes. Membranes were incubated with antibody to phosphorylated (p)ERK1/2
(1:1,000, overnight, 4°C), followed by goat anti-mouse IgG conjugated to
horse-radish peroxidase (1:30,000, 1h, room temperature). Proteins were
visualized by autoradiography after addition of the peroxidase substrate ECL
(enhanced chemiluminescence reagent; Amersham, Piscataway, NJ). Blots were
stripped in 2% SDS-1 mM -mercaptoethanol in 50 mM Tris · HCl, pH
6.8, 150 mM NaCl for 60 min, washed, and reprobed with antibody to total
ERK1/2 to ensure that equal levels of ERK1/2 were present at each time point.
Autoradiograms were photographed using a digital camera, and images were
analyzed using Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA).
Phosphorylation was assessed by histogram analysis of band density (mean
density x no. of pixels) and fold phosphorylation over the basal level
was calculated.
Statistical analysis. All observations were in n > 3 experiments. Results are expressed as means ± SE. Differences between multiple groups were analyzed with one-way analysis of variance and the Student-Newman-Keuls test, with P < 0.05 considered to be significant.
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RESULTS |
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Desensitization and resensitization of agonist-induced Ca2+ mobilization. We compared the extent of agonist-stimulated desensitization and resensitization of Ca2+ mobilization in cells expressing NK1R or NK3R. First, we compared the attenuation of the agonist-induced elevation of [Ca2+]i in the continued presence of agonist. In cells expressing the NK1R, 10 nM SM-SP increased [Ca2+]i, and the response returned to baseline levels within 119.8 ± 7.6 s (Figs. 4 and 5). In cells expressing the NK3R, 10 nM MP-NKB stimulated a similar increase in [Ca2+]i. However, the response to the NK3R agonist returned to the baseline after only 47.3 ± 4.7s(P < 0.05 compared with NK1R). The more prolonged increase in [Ca2+]i after activation of the NK1R suggests that this receptor preferentially couples to channels at the plasma membrane to allow entry of Ca2+ from the extracellular fluid. In support of this possibility, the duration of the response was similar for the NK1R (51.1 ± 2.5 s) and NK3R (50.3 ± 4.7 s) when studied in the absence of extracellular Ca2+ (not shown).
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Second, we compared desensitization and resensitization to repeated challenge with agonist. Cells were exposed to 10 nM SM-SP, 10 nM MP-NKB, or vehicle (control) for 10 min, washed, and then challenged again with 10 nM of the same agonist immediately (0 min) or after 30 min. Both NK1R and NK3R desensitized to a similar extent at 0 min (Fig. 6, A and B). However, after 30 min, the NK1R response was only 37.5 ± 8.1%, whereas the NK3R response was 91.5 ± 5.2% of vehicle-treated cells. Thus the NK1R and NK3R show similar kinetics of desensitization, but the NK3R resensitizes at least twofold more than the NK1R at 30 min.
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Receptor domains that mediate differences in Ca2+ signaling and resensitization of NK1R and NK3R. Although the NK1R and NK3R showed similar rates of agonist-induced endocytosis, there were distinct differences in the duration of increased [Ca2+]i and in the rate of receptor resensitization. Domains in the third intracellular loop of GPCRs determine receptor interaction with heterotrimeric G proteins, and the intracellular COOH tail often contains numerous Ser and Thr residues that can be phosphorylated during desensitization and that must be dephosphorylated for resensitization to occur. To identify the domains of the NK1R that were responsible for the more sustained increase in [Ca2+]i and the slower rate of receptor resensitization, we exchanged the third intracellular loop and COOH tail of the NK1R with equivalent domains of the NK3R, both individually and together, and vice versa.
Given that chimeric receptors can exhibit defects in signaling and trafficking, we first evaluated whether chimeras of the NK1R and NK3R were appropriately localized at the cell surface and undergo similar agonist-induced trafficking and signaling. In cells expressing NK1R, NK1R/NK3Rl3, NK1R/NK3Rct, and NK1R/NK3Rl3ct, Alexa-SM-SP bound to the plasma membrane at 4°C and internalized to perinuclear endosomes after 15 min at 37°C (Fig. 2A). In cells expressing NK3R, NK3R/NK1Rl3, and NK3R/NK1Rct, Alexa-MP-NKB similarly bound to the plasma membrane and then internalized (Fig. 2B). In cells that were transiently transfected to express high levels of NK3R/NK1Rl3ct, we detected a very low level of binding of Alexa-MP-NKB at the cell surface. The intensity of the signal was too low for further analysis. These results suggest that the construct is capable of binding agonist. However, in repeated attempts to generate cell lines that stably expressed the construct, we were unable to detect binding of Alexa-MP-NKB. Thus expression of the chimeric receptor is completely lost in stable cell lines, suggesting a misfolding or degradation of the protein by the cell. These cells were not studied further. In cells expressing NK1R, NK1R/NK3Rl3, NK1R/NK3Rct, and NK1R/NK3Rl3ct, SM-SP stimulated Ca2+ mobilization with similar efficacy and potency, although the EC50 value in the NK1R/NK3Rct line was approximately threefold higher than for the wild-type receptor (Fig. 3A). MP-NKB similarly stimulated Ca2+ mobilization in cells expressing NK3R, NK3R/NK1Rl3, and NK3R/NK1Rct. Thus these chimeric receptors are appropriately localized and signal and traffic normally.
To identify domains of the NK1R that are responsible for the prolonged Ca2+ response, we measured the duration of the elevated [Ca2+]i to 10 nM SM-SP in NK1R/NK3R chimeric receptors. The duration above baseline was as follows: NK1R, 119.8 ± 7.6 s; NK1R/NK3Rl3, 47.1 ± 10.3 s; NK1R/NK3Rct, 90.8 ± 11.0 s; and NK1R/NK3Rl3ct, 49.8 ± 7.8 s (Figs. 4 and 5). In comparison, the duration of the Ca2+ response to 10 nM MP-NKB in cells expressing the NK3R was 47.3 ± 4.7 s. Thus replacement of the third intracellular loop and, to a lesser extent, the COOH tail of the NK1R with that of the NK3R attenuates the sustained elevation in [Ca2+]i. Conversely, substitution of the COOH tail of the NK3R with comparable domains of the NK1R had little effect on the duration of the Ca2+ response: NK3R, 47.3 ± 4.7 s; NK3R/NK1Rl3, 46.4 ± 4.5 s; and NK3R/NK1Rct, 53.5 ± 2.3 s (Figs. 4 and 5). Thus loop 3 and the COOH tail of the NK1R are required for the sustained elevation in [Ca2+]i that is probably mediated by influx of extracellular Ca2+. The domains of the NK3R that are required for the transient elevation in [Ca2+]i remain to be determined.
To identify domains of the NK1R that are responsible for the rapid resensitization, we compared the extent of resensitization of the NK1R/NK3R chimeras after exposure to 10 nM SM-SP for 10 min. When the interval between two challenges with SM-SP was 0 min, the extent of desensitization was similar for cells expressing all receptors. Replacement of loop 3 of the NK1R with that of the NK3R markedly accelerated resensitization after 30 min to that of the NK3R: NK1R, 37.5 ± 8.1% of vehicle-treated cells; NK1R/NK3Rl3, 107.1 ± 2.6%; NK3R, and 91.5 ± 5.2% (Fig. 6). Although substitution of the COOH tail also accelerated resensitization of the NK1R, the effect of the loop 3 substitution was greater. Thus loop 3 and, to a lesser extent, the COOH tail of the NK1R contribute to the slow resensitization of this receptor. Conversely, the rapid resensitization of the NK3R was slowed by substitution of loop 3 and the COOH tail of the NK1R (Fig. 6). Therefore, the third intracellular loop and COOH tail of the NK1R and NK3R determine the rates of resensitization of these receptors.
Colocalization of NK1R and NK3R with -arrestins 1 and
2.
-Arrestins mediate desensitization and endocytosis of many
GPCRs, including the NK1R and NK3R
(27). Resensitization of the
NK1R involves receptor endocytosis, intracellular sorting that may involve
dissociation of ligand, receptor dephosphorylation, dissociation of
-arrestins, and receptor recycling
(8,
28). Thus differences in the
association of
-arrestins with the NK1R and NK3R may contribute to
differences in the rates of resensitization of these receptors.
-Arrestins are cytosolic proteins that translocate to the plasma
membrane to interact with agonist-occupied receptors. To evaluate the
association of
-arrestins with the NK1R and NK3R, we evaluated the
translocation of immunoreactive
-arrestins 1 and 2 to the plasma
membrane in cells treated with Alexa-SM-SP or Alexa-MP-NKB. In the
unstimulated state,
-arrestins 1 and 2 were localized to the cytosol
with no detectable presence at the plasma membrane
(Fig. 7A). In cells
expressing the NK1R, Alexa-SM-SP bound to the plasma membrane after 60 min at
4°C and induced the translocation of both
-arrestin 1
(Fig. 7B) and
-arrestin 2 (Fig.
7C) from the cytosol to the plasma membrane. In contrast,
in cells expressing the NK3R, only
-arrestin 2 translocated to the
plasma membrane, and
-arrestin 1 remained in the cytosol
(Fig. 7, B and
C). After incubation at 37°C for 5-30 min at
37°C, Alexa-SM-SP was detected in endosomes containing
-arrestin 1
or 2 (not shown, but see Ref.
20). This sequestration of
-arrestins to endosomes resulted in a marked depletion of
-arrestins from the cytosol. In marked contrast, after 10 min at
37°C, Alexa-MP-NKB was detected in endosomes that only occasionally
contained
-arrestin 2, whereas
-arrestin 2 was mostly present in
the cytosol (Fig. 7D).
Thus the NK1R colocalizes with both
-arrestins 1 and 2 at the cell
surface and in endosomes for prolonged periods, but the NK3R colocalizes only
with
-arrestin 2 at the cell surface, and
-arrestin 2 rapidly
returns to the cytosol.
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To confirm the colocalization of NK1R and NK3R with -arrestins 1 and
2 at the plasma membrane or in endosomes, we studied cells expressing
-arrestin 1 or 2 tagged with GFP.
-Arrestin-GFP has been widely
used to investigate interactions with GPCRs
(2,
20). This approach for
detection of
-arrestins is highly specific, because it avoids the use of
antibodies, and is sensitive because of the overexpression of
-arrestins
tagged with GFP. In unstimulated cells,
-arrestin 1-GFP and
-arrestin 2-GFP were localized to the cytosol (not shown, but see Ref.
20). In cells expressing NK1R,
Alexa-SP bound to the plasma membrane after 60 min at 4°C and stimulated
translocation of both
-arrestin 1-GFP and
-arrestin 2-GFP from the
cytosol to the plasma membrane (Fig. 8,
A and C). After 10 min at 37°C, Alexa-SP was
colocalized with
-arrestin 1-GFP and
-arrestin 2-GFP in endosomes,
and there was marked depletion of both isoforms of
-arrestin from the
cytosol (Fig. 8, B and
D). In cells expressing NK3R, Alexa-MP-NKB bound to the
plasma membrane at 4°C and stimulated translocation of
-arrestin
2-GFP to the plasma membrane (Fig.
8C). In contrast,
-arrestin 1-GFP mostly remained
within the cytosol, and only low levels were detected at the plasma membrane
(Fig. 8A). After 10
min at 37°C, Alexa-MP-NKB was internalized into endosomes, whereas
-arrestin 1-GFP and
-arrestin 2-GFP were most prominently located
in the cytosol (Fig. 8, B and
D). These results confirm the findings obtained with
-arrestin antibodies, showing that NK1R associates with
-arrestins
1 and 2, whereas NK3R preferentially associates with
-arrestin 2. The
overexpression of
-arrestin 1-GFP may account for our ability to detect
limited colocalization with NK3R at the plasma membrane, whereas we could not
detect colocalization of endogenous immunoreactive
-arrestin 1.
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To identify the domains of the NK1R that are required for colocalization
with -arrestin 1, we studied the chimeric receptors. Substitution of
loop 3 or the COOH tail of the NK1R with equivalent domains of the NK3R
diminished the capacity of these receptors to recruit
-arrestin 1 to the
plasma membrane, whereas replacement of both domains abolished the recruitment
(Fig. 9A). In
contrast, these substitutions had no effect on the capacity of the NK1R to
recruit
-arrestin 2 (Fig.
9B). This latter result is expected, because the NK3R can
also recruit
-arrestin 2 (Fig.
7). Thus the third intracellular loop and COOH tail of the NK1R
are required for recruitment of
-arrestin 1 to the cell surface.
Substitution of loop 3 or the COOH tail alone of the NK3R with that of the
NK1R did not reproducibly induce membrane translocation of
-arrestin 1
to the plasma membrane (Fig.
10), indicating that these domains alone are not sufficient to
confer this property to the NK3R.
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We took advantage of the specificity and sensitivity of -arrestin-GFP
to examine the localization of chimeric receptors with
-arrestins 1 and
2 at the cell surface and in endosomes. Substitution of loop 3 plus the COOH
tail of the NK1R with equivalent domains of the NK3R diminished, but did not
completely abolish, the capacity of the NK1R to recruit
-arrestin 1-GFP
to the plasma membrane (Fig.
11A) but had no effect on the capacity of the NK1R to
recruit
-arrestin 2-GFP (Fig.
11C). Substitution of the COOH tail of the NK3R with the
equivalent domain of the NK1R resulted in membrane translocation of
-arrestin 1-GFP (Fig.
11A) and
-arrestin 2-GFP
(Fig. 11C). In the
case of NK1R/NK3Rl3ct,
-arrestin 1-GFP
(Fig. 11B) and
-arrestin 2-GFP (Fig.
11D) colocalized with agonists in endosomes after 10 min
at 37°C. However,
-arrestin 1-GFP was also prominently detected in
the cytosol, especially in cells expressing
-arrestin 1-GFP at a high
level. In NK3R/NK1Rct cells, there was minimal colocalization of
-arrestin 1-GFP or
-arrestin 2-GFP with agonists in endosomes
(Fig. 11, B and
D). In general, these results are consistent with those
obtained using antibodies to
-arrestins. The results suggest that the
third intracellular loop and COOH tail of the NK1R are required for prominent
colocalization of the NK1R with
-arrestin 1 at the plasma membrane and
in endosomes, because replacement with domains of the NK3R diminishes (but
does not abolish) this association. The COOH tail of the NK1R also confers on
the NK3R the ability to associate with
-arrestin 2 at the plasma
membrane. However, this domain is not adequate for association of the NK3R
with
-arrestin 1 or 2 in endosomes, suggesting a requirement for
additional domains.
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Coupling of NK1R and NK3R to MAP kinase activation.
-Arrestins 1 and 2 serve as scaffolds that can couple certain receptors
to components of the MAP kinase pathway such as Src and Raf
(4,
5,
17).
-Arrestin couples
the NK1R to Src and thereby facilitates SP-induced activation of ERK1/2. Thus
differences in the association of the NK1R and NK3R with
-arrestins 1
and 2 may result in differences in the ability of these receptors to activate
MAP kinases. We compared the capacity of the NK1R, which interacts with both
-arrestins 1 and 2, and the NK3R, which interacts only with
-arrestin 2, to activate ERK1/2. In cells expressing the NK1R or NK3R,
10 nM SM-SP or MP-NKB, respectively, induced a prompt increase in
phosphorylation of pERK1/2 to a similar extent and with similar kinetics
(Fig. 12). Moreover, SM-SP
similarly stimulated phosphorylation of ERK1/2 in cells expressing
NK1R/NK3Rl3ct, which interacts only with
-arrestin 2. Thus the NK1R and
NK3R can efficiently activate ERK1/2 despite differences in their
colocalization with
-arrestins.
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DISCUSSION |
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Colocalization of NK1R and NK3R with -arrestins 1 and
2. We evaluated the capacity of NK1R and NK3R to interact with
-arrestins 1 and 2 by examining agonist-induced trafficking of
-arrestin isoforms by immunofluorescence, using specific antibodies and
GFP-tagged
-arrestins. Similar approaches have been used to evaluate
interaction of other GPCRs with
-arrestins
(20,
24). Although this approach
does not directly assess the physical interaction of GPCRs with
-arrestins, we have previously shown that the NK1R interacts with
-arrestins by immunoprecipitating
-arrestins and immunoblotting
for the NK1R (18). It is
likely that the NK3R also physically interacts with
-arrestins, but this
was not studied. Consequently, we refer to colocalization of receptors with
-arrestins and infer interaction. In NK1R cells, SM-SP induced rapid
translocation of both
-arrestins 1 and 2 from the cytosol to the plasma
membrane. In contrast, MP-NKB induced membrane translocation of
-arrestin 2. There was minimal translocation of
-arrestin 1-GFP
and no detectable membrane translocation of endogenous immunoreactive
-arrestin 1, a difference attributable to sensitivity and the higher
expression of
-arrestin 1-GFP in transfected cells. These results
suggest that the NK1R can interact with both isoforms of
-arrestin but
that the NK3R can interact preferentially with
-arrestin 2. In support
of our results, we and others have previously shown in transfected cells that
NK1R agonists induce membrane translocation of
-arrestins 1 and 2 tagged
with GFP (2,
19,
20,
24). Additionally, SP and
SM-SP also induce membrane translocation of immunoreactive
-arrestin 1/2
in enteric neurons (19). We
also observed marked colocalization of the NK1R with
-arrestin 1-GFP and
-arrestin 2-GFP in endosomes with depletion of cytosolic
-arrestins. In support of this result are other reports of
colocalization of NK1R and
-arrestin 1 and 2 endosomes for many hours
(2,
19,
24,
27). Although the NK3R rapidly
internalized, we detected very little colocalization of the NK3R in endosomes
with
-arrestin 1 or 2, localized using antibodies or GFP, which is
supported by other reports
(27). Thus
-arrestin 1
transiently colocalizes with NK3R in endosomes and rapidly returns to the
cytosol. Although we did not measure the affinity of interaction of the NK1R
and NK3R with
-arrestins 1 and 2, the results suggest a high-affinity
interaction of the NK1R with
-arrestins 1 and 2 but a low-affinity
interaction between the NK3R and
-arrestin 2. Thus the NK1R belongs to
"class B" GPCRs, including angiotensin II type 1A, neurotensin-1,
vasopressin V2, and thyroid-releasing hormone receptors, which
interact with
-arrestins 1 and 2 with high affinity and internalize with
-arrestins in endosomes
(24). In contrast, the NK3R
belongs to the "class A" GPCRs, including
2- and
1b-adrenergic, µ-opioid, endothelin A, and dopamine
D1A receptors, which transiently interact preferentially with
-arrestin 2 (24). These
receptors form low-affinity, unstable interactions with
-arrestins,
dissociate from
-arrestins near the plasma membrane, and are largely
excluded from endosomes.
Exchange of the COOH tails of class A and B receptors reverses their
affinities for -arrestins, suggesting that domains in the COOH tails
specify interaction with
-arrestins
(24). The ability of
-arrestin 2 to remain associated with these receptors, including the
NK1R, depends on the presence of a cluster of Ser and Thr residues in the COOH
tail that may be phosphorylated and interact with arrestins
(23). Domains in the third
intracellular loop of the
2b-adrenergic receptor also
determine its interaction with
-arrestin 2
(6). Therefore, we compared
chimeric receptors with exchanged COOH tails and third intracellular loops to
identify domains that may determine the colocalization of NK1R and NK3R with
-arrestin isoforms. Replacement of intracellular loop 3 and the COOH
tail, either alone or together, of the NK1R with the equivalent domains of the
NK3R markedly diminished the capacity of the NK1R to interact with
-arrestin 1 at the plasma membrane and in endosomes. There was no
detectable colocalization of NK1R with immunoreactive
-arrestin 1 at the
plasma membrane, but there was a low level of colocalization with
-arrestin 1-GFP at the plasma membrane and in endosomes, possibly due to
overexpression of
-arrestin 1-GFP in transfected cells. These
substitutions did not affect colocalization with
-arrestin 2, which is
expected because both NK1R and NK3R interact with
-arrestin 2. Thus
domains in loop 3 and the COOH tail of the NK1R are important for
colocalization with
-arrestin 1. It is likely that these domains are
also important for colocalization with
-arrestin 2. However, this
possibility could not be evaluated by studying chimeric receptors because both
NK1R and NK3R interacted with
-arrestin 2. Presently, we do not have an
unequivocal explanation for these findings. However,
-arrestins interact
with GRK phosphorylated receptor, and intracellular loop 3 and the COOH tail
of the NK1R contain numerous Ser and Thr residues that may be phosphorylated
after agonist binding. In support of this suggestion, GRK2 and 3 strongly
phosphorylate the NK1R (14),
and Ser and Thr residues in the COOH tail specify the high-affinity
interaction of the neurotensin-1 receptor, oxytocin receptor, angiotensin II
type 1A receptor, and NK1R with
-arrestin 2
(23,
24). Replacement of the COOH
tail of the NK3R with that of the NK1R conferred association of the
NK3R/NK1Rct with
-arrestin 1-GFP at the cell surface, suggesting that
this domain is important for interaction with
-arrestin 1. There was no
detectable colocalization of NK3R/NK1Rct at the cell surface with endogenous
-arrestin 1, possibly because of a lower sensitivity of detection.
Surprisingly, replacement of the COOH tail of the NK3R with an equivalent NK1R
domain did not confer colocalization with
-arrestin 1-GFP or
-arrestin 2-GFP in endosomes, suggesting that additional motifs are
necessary for prolonged association.
Implications of differential interactions of NK1R and NK3R with
-arrestins 1 and 2.
-Arrestins are multi-functional
proteins that participate in desensitization, endocytosis, and mitogenic
signaling of many GPCRs (18,
21). Thus differences in the
colocalization of NK1R and NK3R with
-arrestin isoforms could have
important functional implications. Although we have previously shown that
-arrestins are required for endocytosis of NK1R and NK3R
(19,
27), both receptors
internalized with similar kinetics. Moreover, both NK1R and NK3R underwent
homologous desensitization to a similar degree. We have also previously
reported that
-arrestins are scaffolds that couple Src to the NK1R and
facilitate activation of ERK1/2
(4). However, both NK1R and
NK3R coupled to activation of ERK1/2 with similar kinetics. Thus
colocalization with
-arrestin 2 alone is sufficient for desensitization,
endocytosis, and mitogenic signaling of the NK3R, whereas the NK1R may utilize
both
-arrestins 1 and 2 for these processes.
Differences in affinity may determine the duration with which NK1R and NK3R
interact with -arrestins, with important consequences for receptor
resensitization. Once internalized, both NK1R and NK3R recycle to the plasma
membrane (10,
11). In the case of the NK1R,
resensitization requires receptor endocytosis, dissociation of agonist and
-arrestins in endosomes, and receptor recycling
(8,
28). The prolonged,
high-affinity colocalization of NK1R with both
-arrestins 1 and 2 may
account for the slow resensitization of this receptor. In contrast, the
transient and low-affinity colocalization of the NK3R with only
-arrestin 2 may explain the rapid resensitization of this receptor. In
support of these conclusions, replacement of loop 3 and the COOH tail of the
NK1R with domains of the NK3R prevented colocalization with
-arrestin 1
and accelerated resensitization of the NK1R to that of the NK3R. The affinity
of interaction between other GPCRs and
-arrestins similarly dictates the
kinetics of resensitization
(22).
Differences in interaction with -arrestins may also explain the
consequences of selectively activating the NK1R on desensitization and
endocytosis of the NK3R (27).
In cells coexpressing NK1R and NK3R, NK1R agonists induce sequestration of
-arrestins in NK1R endosomes, thereby depleting cytosolic
-arrestins and impeding homologous desensitization and endocytosis of
the NK3R. The present results suggest that the NK1R would sequester both
isoforms of
-arrestin. In contrast, NK3R activation would deplete only
-arrestin 2 from the cytosol, leaving
-arrestin 1 to interact with
the NK1R to mediate desensitization and endocytosis. Differences in
interaction with
-arrestins also explains the ability of the vasopressin
V2 receptor to inhibit endocytosis of the
2-adrenergic receptor
(13).
Ca2+ signaling of the NK1R and NK3R. We found
that NK1R and NK3R couple to elevated [Ca2+]i with
markedly different kinetics. Activation of the NK1R caused a prolonged
elevation of [Ca2+]i due to a rapid mobilization of
intracellular Ca2+ and a more sustained entry of Ca2+
from extracellular fluid. In contrast, agonists of the NK3R induced a
transient increase in [Ca2+]i, because it was unable to
couple to entry of extracellular Ca2+. Substitution of loop 3 and,
to a lesser extent, the COOH tail of the NK1R with the same domains of the
NK3R prevented the entry of extracellular Ca2+, suggesting that
domains in loop 3 are important for entry of extracellular Ca2+.
The mechanisms of these effects remain to be determined. One possibility is
that the NK1R and NK3R activate different kinases that regulate channel
activity. Activation of Ca2+ channels by GPCRs may involve
phosphorylation of the channel through second messenger kinases such as
protein kinase A or C. The -adrenergic receptor can directly interact
with L-type Ca2+ channels to induce activation
(3). Thus differences in the
ability of NK1R and NK3R to interact with Ca2+ channels in the
plasma membrane may also account for the observed differences. Further
experimentation is required to elucidate these mechanisms.
Physiological consequences of differences in signaling of NK1R and
NK3R. The NK1R and NK3R are coexpressed by certain cells, such as enteric
neurons, and would be coactivated by release of tachykinins from intrinsic or
extrinsic nerves of the intestine
(11). The released
tachykinins, mainly SP and NKA, would be expected to interact with both
receptors, albeit with different affinity. Activation of the NK1R with
high-affinity agonists (SP) would result in membrane translocation of
-arrestins 1 and 2 to mediate desensitization, endocytosis, and MAP
kinase signaling. Activation of the NK1R would sequester
-arrestins 1
and 2 in endosomes and thereby cause retention of the NK3R at the cell
surface, where it would be resistant to desensitization and internalization
(27). Such regulation could
permit cells to respond to tachykinins at a time when the NK1R was
desensitized and internalized. This state may persist until
-arrestins
return to the cytosol, when the NK3R could interact with
-arrestin 2,
uncouple and internalize, and the NK1R would have recycled. Rapid
resensitization of the NK3R would permit cells to quickly respond again to
stimulation. Together, these findings emphasize the importance of using
antagonists of both NK1R and NK3R to fully suppress tachykinin signaling in
the enteric nervous system.
Interaction of a GPCR with -arrestins could affect signaling of many
other receptors. Thus the high-affinity interaction of a class B GPCR with
both isoforms of
-arrestin would also affect
-arrestin-dependent
signaling of class A low-affinity GPCRs. In view of the roles of
-arrestins in uncoupling, endocytosis, and mitogenic signaling,
regulation of the interactions between GPCRs and
-arrestins would be
expected to have far-reaching functional implications. It is thus of interest
to determine the spectrum of GPCRs and
-arrestins that are coexpressed
in functionally important cell types, as well as the relative levels of
expression of these important regulatory proteins.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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