The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with {beta}-arrestins

Fabien Schmidlin, Dirk Roosterman, and Nigel W. Bunnett

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with {beta}-arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of {beta}-arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only {beta}-arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with {beta}-arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca2+ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with {beta}-arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with {beta}-arrestin 1 and determine the rate of resensitization.

desensitization; endocytosis; tachykinins


MANY HORMONES AND NEUROTRANSMITTERS interact with multiple G protein-coupled receptors (GPCRs) that are often coexpressed by the same cell. For example. the tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) each interact with three neurokinin receptors (NK1R, NK2R, and NK3R), albeit with graded affinities (25). These neurokinin receptors are frequently coexpressed. For instance, intestinal myocytes express NK1R and NK2R (26, 29), and enteric neurons express NK1R and NK3R (11). The preprotachykinin A gene that encodes both SP and NKA is expressed by both extrinsic and intrinsic neurons within the intestinal tract. Thus both SP and NKA could regulate intestinal myocytes and neurons by activating two different receptors. The physiological consequences of one agonist simultaneously activating distinct receptors on the same cell are not fully understood. Therefore, it is of interest to directly compare signaling mechanisms of receptors that are coexpressed in the same cell.

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-{beta} 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 {beta}-arrestins may contribute to disparate signaling (27). The NK1R interacts with {beta}-arrestins for prolonged periods, suggesting a high-affinity interaction, whereas the NK3R interacts with {beta}-arrestins transiently, indicative of a low-affinity interaction. In view of the multiple roles on {beta}-arrestins in receptor signaling and regulation, these differences may explain differences in the function of the NK1R and NK3R.

{beta}-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). {beta}-Arrestins similarly translocate to interact with phosphorylated receptors and disrupt their association with heterotrimeric G proteins to induce rapid desensitization of signal transduction (1). {beta}-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). {beta}-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 {beta}-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, {beta}-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 {beta}-arrestins may account for marked differences in signaling.

We investigated the colocalization of the NK1R and NK3R with {beta}-arrestins 1 and 2, the principal forms of arrestins that interact with GPCRs. Although {beta}-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 {beta}-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 {beta}-arrestins 1 and 2; and 4) identify the domains of the NK1R and NK3R that account for differences in signaling and regulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. NK1R agonist [Sar9,Met(O2)11]-SP (SM-SP), NK3R agonist [MePhe7]-NKB (MP-NKB), and SP were from Phoenix Pharmaceuticals (St. Joseph, MO). Agonists were labeled with Alexa594 as described previously (7). The sources of other reagents have been described previously (19, 27, 28).

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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Fig. 1. Generation of chimeras of the rat neurokinin 1 (NK1R) and 3 receptors (NK3R). C, COOH; N, NH2; wt, wild type; l3, third intracellular loop; ct, COOH tail.

 

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 {beta}-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 {beta}-arrestin 1 or 2 was detected by using antibodies to {beta}-arrestin 1 (1 µg/ml, overnight at 4°C) or {beta}-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, {infty}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 {beta}-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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of cell lines expressing NK1R or NK3R. To confirm that NK1R and NK3R were functional, we compared agonist-induced receptor trafficking and mobilization of intracellular Ca2+. In cells expressing NK1R, the selective agonist Alexa-SM-SP was bound to the plasma membrane after 60-min incubation at 4°C (Fig. 2A). Washing and warming to 37°C for 15 min resulted in redistribution of label to perinuclear endosomes. In cells expressing NK3R, the selective agonist Alexa-MP-NKB similarly bound to the plasma membrane at 4°C and redistributed to perinuclear endosomes after 15 min at 37°C (Fig. 2B). In cells expressing NK1R, SM-SP increased [Ca2+]i in a concentration-dependent manner with an EC50 of ~1 nM (Fig. 3A). Similarly, MP-NKB increased [Ca2+]i in cells expressing the NK3R with an EC50 of ~1 nM (Fig. 3B). Thus wild-type NK1R and NK3R are appropriately located at the cell surface, internalize similarly, and mobilize intracellular Ca2+ similarly.



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Fig. 2. Binding and internalization of NK1R agonist Alexa-[Sar9,Met(O2)11]-substance P (SM-SP) and NK3R agonist Alexa-[MePhe7]-neurokinin B (MP-NKB) in cells expressing NK1R and chimeras (A) and NK3R and chimeras (B). Cells were incubated with agonists (100 nM) for 60 min at 4°C and fixed immediately (0 min) or washed and incubated for 15 min at 37°C. Note that agonists all bound to the cell surface and rapidly internalized. Scale bar = 10 µm.

 


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Fig. 3. Effects of graded concentrations of NK1R agonist (SM-SP) and NK3R agonist (MP-NKB) on intracellular Ca2+ concentration ([Ca2+]i) in cells expressing NK1R and chimeras (A) and NK3R and chimeras (B). Values are means ± SE. Note that all receptors were functional with comparable potencies.

 

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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Fig. 4. Effects of 10 nM NK1R agonist (SM-SP) and NK3R agonist (MP-NKB) on [Ca2+]i in cells expressing NK1R and chimeras (A) and NK3R and chimeras (B). Note the prolonged increase in [Ca2+]i in cells expressing wild-type NK1R and the transient response in cells expressing NK3R. Replacement of loop 3 and the COOH tail of the NK1R with the same domains of the NK3R attenuated the response, whereas substitution of these domains of the NK3R had little effect.

 


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Fig. 5. Effects of 10 nM NK1R agonist (SM-SP) and NK3R agonist (MP-NKB) on the duration of the elevated [Ca2+]i in cells expressing NK1R and chimeras (A) and NK3R and chimeras (B). Results are expressed as the time that [Ca2+]i remained elevated over basal. Replacement of loop 3 and the COOH tail of the NK1R with the same domains of the NK3R attenuated the response, whereas substitution of these domains of the NK3R had little effect. *P < 0.05 compared with wild-type NK1R (in A) and NK3R (in B). #P < 0.05 compared with wild-type NK1R. Values are means ± SE; n = 3.

 

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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Fig. 6. Time course of desensitization and resensitization of Ca2+responses to the NK1R agonist (SM-SP) and NK3R (MP-NKB) in cells expressing NK1R and its chimeras (A) and NK3R and its chimeras (B). Cells were incubated with 10 nM agonist or vehicle for 10 min, washed, and challenged immediately (0 min) or after 30 min. Results are expressed as the percent response to the second challenge compared with vehicle-treated controls. Note the slow resensitization of NK1R and the rapid resensitization of NK3R. Replacement of loop 3 and, to a lesser extent, the COOH tail of NK1R with NK3R accelerated resensitization of NK1R. Conversely, replacement of these domains of the NK3R slowed resensitization. *P < 0.05 compared with wild-type NK1R (A) and NK3R (B). #P < 0.05 compared with wild-type NK1R. Values are means ± SE; n = 3.

 

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 {beta}-arrestins 1 and 2. {beta}-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 {beta}-arrestins, and receptor recycling (8, 28). Thus differences in the association of {beta}-arrestins with the NK1R and NK3R may contribute to differences in the rates of resensitization of these receptors. {beta}-Arrestins are cytosolic proteins that translocate to the plasma membrane to interact with agonist-occupied receptors. To evaluate the association of {beta}-arrestins with the NK1R and NK3R, we evaluated the translocation of immunoreactive {beta}-arrestins 1 and 2 to the plasma membrane in cells treated with Alexa-SM-SP or Alexa-MP-NKB. In the unstimulated state, {beta}-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 {beta}-arrestin 1 (Fig. 7B) and {beta}-arrestin 2 (Fig. 7C) from the cytosol to the plasma membrane. In contrast, in cells expressing the NK3R, only {beta}-arrestin 2 translocated to the plasma membrane, and {beta}-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 {beta}-arrestin 1 or 2 (not shown, but see Ref. 20). This sequestration of {beta}-arrestins to endosomes resulted in a marked depletion of {beta}-arrestins from the cytosol. In marked contrast, after 10 min at 37°C, Alexa-MP-NKB was detected in endosomes that only occasionally contained {beta}-arrestin 2, whereas {beta}-arrestin 2 was mostly present in the cytosol (Fig. 7D). Thus the NK1R colocalizes with both {beta}-arrestins 1 and 2 at the cell surface and in endosomes for prolonged periods, but the NK3R colocalizes only with {beta}-arrestin 2 at the cell surface, and {beta}-arrestin 2 rapidly returns to the cytosol.



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Fig. 7. A: localization of {beta}-arrestins 1 ({beta}-ARR1) and 2 ({beta}-ARR2) in unstimulated Kirsten murine sarcoma virus-transformed rat kidney epithelial (KNRK) cells. B and C: localization of {beta}-ARR1 (B) and {beta}-ARR2 (C) with Alexa-SM-SP and Alexa-MP-NKB in cells expressing wild-type NK1R and NK3R. Cells were incubated with 100 nM agonists for 60 min at 4°C and fixed, and {beta}-ARR1 and {beta}-ARR2 were localized with specific antibodies. The same cells are shown in each column. Note that NK1R agonist triggered membrane translocation of {beta}-ARR1 and {beta}-ARR2 (arrowheads), but NK3R agonist induced membrane translocation of {beta}-ARR2 but not {beta}-ARR1, which remained in the cytosol (yellow arrows). D: localization of {beta}-ARR2 and Alexa-MP-NKB in cells expressing wild-type NK3R after 10 min at 37°C. Note that Alexa-MP-NKB was mostly present in endosomes that did not contain {beta}-ARR2 (white arrows) and that there was colocalization only rarely (yellow arrows). {beta}-ARR2 was mostly in the cytosol after 10 min. Scale bars = 10 µm.

 

To confirm the colocalization of NK1R and NK3R with {beta}-arrestins 1 and 2 at the plasma membrane or in endosomes, we studied cells expressing {beta}-arrestin 1 or 2 tagged with GFP. {beta}-Arrestin-GFP has been widely used to investigate interactions with GPCRs (2, 20). This approach for detection of {beta}-arrestins is highly specific, because it avoids the use of antibodies, and is sensitive because of the overexpression of {beta}-arrestins tagged with GFP. In unstimulated cells, {beta}-arrestin 1-GFP and {beta}-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 {beta}-arrestin 1-GFP and {beta}-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 {beta}-arrestin 1-GFP and {beta}-arrestin 2-GFP in endosomes, and there was marked depletion of both isoforms of {beta}-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 {beta}-arrestin 2-GFP to the plasma membrane (Fig. 8C). In contrast, {beta}-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 {beta}-arrestin 1-GFP and {beta}-arrestin 2-GFP were most prominently located in the cytosol (Fig. 8, B and D). These results confirm the findings obtained with {beta}-arrestin antibodies, showing that NK1R associates with {beta}-arrestins 1 and 2, whereas NK3R preferentially associates with {beta}-arrestin 2. The overexpression of {beta}-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 {beta}-arrestin 1.



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Fig. 8. Localization of {beta}-ARR1-green fluorescent protein (GFP) (A and B) and {beta}-ARR2-GFP (C and D) with Alexa-SP and Alexa-MP-NKB in cells expressing wild-type NK1R and NK3R. Cells were incubated with 100 nM agonists for 60 min at 4°C, washed, incubated at 37°C for 0 min (A and C) or 10 min (B and D), and fixed. {beta}-ARR1 and {beta}-ARR2 were localized using GFP. The same cells are shown in each column. At 0 min in NK1R wild-type cells, note that NK1R agonist triggered membrane translocation of {beta}-ARR1-GFP (A) and {beta}-ARR2-GFP (C) to the plasma membrane (arrowheads). At 0 min in NK3R wild-type cells, the NK3R agonist stimulated membrane translocation of {beta}-ARR2-GFP (C, arrowheads), but {beta}-ARR1-GFP is mostly cytosolic (A, yellow arrows), with only low levels at the cell surface (arrowheads). At 10 min in NK1R wild-type cells, note that NK1R agonist colocalized with {beta}-ARR1-GFP (B) and {beta}-ARR2-GFP (D) in prominent endosomes (white arrows), which depletes the cytosol of {beta}-ARRs. At 10 min in NK3R wild-type cells, the NK3R agonists is in endosomes, but {beta}-ARR1-GFP (B) and {beta}-ARR2-GFP (D) are mostly in the cytosol (yellow arrows). Scale bar = 10 µm.

 

To identify the domains of the NK1R that are required for colocalization with {beta}-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 {beta}-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 {beta}-arrestin 2 (Fig. 9B). This latter result is expected, because the NK3R can also recruit {beta}-arrestin 2 (Fig. 7). Thus the third intracellular loop and COOH tail of the NK1R are required for recruitment of {beta}-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 {beta}-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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Fig. 9. Localization of {beta}-ARR1 (A) and {beta}-ARR2 (B) with Alexa-SM-SP in cells expressing wild-type NK1R and its chimeras. Cells were incubated with 100 nM agonist for 60 min at 4°C and fixed, and {beta}-ARR1 and {beta}-ARR2 were localized with specific antibodies. Note that replacement of loop 3 and the COOH tail of the NK1R with equivalent domains of the NK3R markedly impedes the membrane translocation of {beta}-ARR1 but not {beta}-ARR2. Scale bar = 10 µm.

 


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Fig. 10. Localization of {beta}-ARR1 with Alexa-MP-NKB in cells expressing wild-type NK3R and its chimeras. Cells were incubated with 100 nM agonist for 60 min at 4°C and fixed, and {beta}-ARR1 was localized with specific antibodies. Note that replacement of loop 3 and the COOH tail of the NK3R with equivalent domains of the NK1R did not confer membrane translocation of {beta}-AAR1. Scale bar = 10 µm.

 

We took advantage of the specificity and sensitivity of {beta}-arrestin-GFP to examine the localization of chimeric receptors with {beta}-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 {beta}-arrestin 1-GFP to the plasma membrane (Fig. 11A) but had no effect on the capacity of the NK1R to recruit {beta}-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 {beta}-arrestin 1-GFP (Fig. 11A) and {beta}-arrestin 2-GFP (Fig. 11C). In the case of NK1R/NK3Rl3ct, {beta}-arrestin 1-GFP (Fig. 11B) and {beta}-arrestin 2-GFP (Fig. 11D) colocalized with agonists in endosomes after 10 min at 37°C. However, {beta}-arrestin 1-GFP was also prominently detected in the cytosol, especially in cells expressing {beta}-arrestin 1-GFP at a high level. In NK3R/NK1Rct cells, there was minimal colocalization of {beta}-arrestin 1-GFP or {beta}-arrestin 2-GFP with agonists in endosomes (Fig. 11, B and D). In general, these results are consistent with those obtained using antibodies to {beta}-arrestins. The results suggest that the third intracellular loop and COOH tail of the NK1R are required for prominent colocalization of the NK1R with {beta}-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 {beta}-arrestin 2 at the plasma membrane. However, this domain is not adequate for association of the NK3R with {beta}-arrestin 1 or 2 in endosomes, suggesting a requirement for additional domains.



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Fig. 11. Localization of {beta}-ARR1-GFP (A and B) and {beta}-ARR2-GFP (C and D) with Alexa-SP and Alexa-MP-NKB in cells expressing NK1R/NK3Rl3ct and NK3R/NK1Rct. Cells were incubated with 100 nM agonists for 60 min at 4°C, washed, incubated at 37°C for 0 min (A and C) or 10 min (B and D), and fixed. {beta}-ARR1 and {beta}-ARR2 were localized using GFP. The same cells are shown in each column. At 0 min in NK1R/NK3Rl3ct cells, note that {beta}-ARR1-GFP (A) showed some localization at the plasma membrane (arrowheads) but that there was prominent cytosolic labeling (yellow arrows), whereas {beta}-ARR2-GFP (C) was mostly at the plasma membrane (arrowheads). At 0 min in NK3R/NK1Rct cells, note that {beta}-ARR1-GFP (A) and {beta}-ARR2-GFP (C) were at the plasma membrane (arrow-heads). At 10 min in NK1R/NK3Rl3ct cells, note that NK1R agonist colocalized with {beta}-ARR1-GFP (B) and {beta}-ARR2-GFP (D) in some endosomes (white arrows), but that there were also substantial amounts of {beta}-ARR1-GFP and {beta}-ARR2-GFP in the cytosol (yellow arrows). At 10 min in NK3R/NK1Rct cells, the NK3R agonist was prominent in endosomes (white arrows), but {beta}-ARR1-GFP (B) and {beta}-ARR2-GFP (D) were mostly cytosolic (yellow arrows). Scale bar = 10 µm.

 

Coupling of NK1R and NK3R to MAP kinase activation. {beta}-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). {beta}-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 {beta}-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 {beta}-arrestins 1 and 2, and the NK3R, which interacts only with {beta}-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 {beta}-arrestin 2. Thus the NK1R and NK3R can efficiently activate ERK1/2 despite differences in their colocalization with {beta}-arrestins.



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Fig. 12. Effects of 10 nM NK1R agonist (SM-SP) and NK3R agonist (MP-NKB) on phosphorylation of ERK1/2 in cells expressing NK1R, NK3R, and NK1R/NK3Rl3ct. Cells were exposed to 10 nM SM-SP or MP-NKB for indicated times, and cell extracts were analyzed by Western blotting with antibodies to total and phosphorylated (phospho) ERK1/2. A: blots of representative experiments. B: fold phosphorylation over basal. Values are means ± SE of n = 3 experiments. Note the similar extent of ERK activation in each cell line. Levels of total ERK1/2 for NK1R wild-type cells were lower than for other cell lines because of a shorter film exposure, although 2 bands were detected after a longer exposure.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many cells express several receptors for the same agonists, and it is therefore of interest to compare signaling by these receptors. We compared signaling of the NK1R and NK3R, which are often coexpressed. Our results show that NK1R agonists induce prolonged increases in [Ca2+]i that slowly resensitize to repeated challenge. In contrast, NK3R agonists induce brief increases in [Ca2+]i that rapidly resensitize. By studying chimeric receptors, we have shown that domains in intracellular loop 3 and the COOH tail of the NK1R, and to some extent the NK3R, determine the duration of the increase in [Ca2+]i and the rate of receptor resensitization. In view of the critical roles of {beta}-arrestins 1 and 2 in receptor signaling, we compared the capacity of NK1R and NK3R to colocalize with both isoforms of {beta}-arrestin by evaluating agonist-induced translocation of {beta}-arrestins from the cytosol to the plasma membrane. This analysis shows that the NK1R colocalizes with both isoforms of {beta}-arrestins, whereas the NK3R colocalizes predominantly with {beta}-arrestin 2. Domains in loop 3 and the COOH tail of the NK1R confer capacity to colocalize with {beta}-arrestin 1. {beta}-Arrestins mediate desensitization, endocytosis, and MAP kinase signaling of many GPCRs. However, despite differences in colocalization with the {beta}-arrestin isoforms, both NK1R and NK3R showed similar desensitization, endocytosis, and MAP kinase signaling. Thus NK1R and NK3R exhibit marked differences in the rate of resensitization and in their capacity to colocalize with isoforms of {beta}-arrestin that are determined by domains in intracellular loop 3 and the COOH tail.

Colocalization of NK1R and NK3R with {beta}-arrestins 1 and 2. We evaluated the capacity of NK1R and NK3R to interact with {beta}-arrestins 1 and 2 by examining agonist-induced trafficking of {beta}-arrestin isoforms by immunofluorescence, using specific antibodies and GFP-tagged {beta}-arrestins. Similar approaches have been used to evaluate interaction of other GPCRs with {beta}-arrestins (20, 24). Although this approach does not directly assess the physical interaction of GPCRs with {beta}-arrestins, we have previously shown that the NK1R interacts with {beta}-arrestins by immunoprecipitating {beta}-arrestins and immunoblotting for the NK1R (18). It is likely that the NK3R also physically interacts with {beta}-arrestins, but this was not studied. Consequently, we refer to colocalization of receptors with {beta}-arrestins and infer interaction. In NK1R cells, SM-SP induced rapid translocation of both {beta}-arrestins 1 and 2 from the cytosol to the plasma membrane. In contrast, MP-NKB induced membrane translocation of {beta}-arrestin 2. There was minimal translocation of {beta}-arrestin 1-GFP and no detectable membrane translocation of endogenous immunoreactive {beta}-arrestin 1, a difference attributable to sensitivity and the higher expression of {beta}-arrestin 1-GFP in transfected cells. These results suggest that the NK1R can interact with both isoforms of {beta}-arrestin but that the NK3R can interact preferentially with {beta}-arrestin 2. In support of our results, we and others have previously shown in transfected cells that NK1R agonists induce membrane translocation of {beta}-arrestins 1 and 2 tagged with GFP (2, 19, 20, 24). Additionally, SP and SM-SP also induce membrane translocation of immunoreactive {beta}-arrestin 1/2 in enteric neurons (19). We also observed marked colocalization of the NK1R with {beta}-arrestin 1-GFP and {beta}-arrestin 2-GFP in endosomes with depletion of cytosolic {beta}-arrestins. In support of this result are other reports of colocalization of NK1R and {beta}-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 {beta}-arrestin 1 or 2, localized using antibodies or GFP, which is supported by other reports (27). Thus {beta}-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 {beta}-arrestins 1 and 2, the results suggest a high-affinity interaction of the NK1R with {beta}-arrestins 1 and 2 but a low-affinity interaction between the NK3R and {beta}-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 {beta}-arrestins 1 and 2 with high affinity and internalize with {beta}-arrestins in endosomes (24). In contrast, the NK3R belongs to the "class A" GPCRs, including {beta}2- and {beta}1b-adrenergic, µ-opioid, endothelin A, and dopamine D1A receptors, which transiently interact preferentially with {beta}-arrestin 2 (24). These receptors form low-affinity, unstable interactions with {beta}-arrestins, dissociate from {beta}-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 {beta}-arrestins, suggesting that domains in the COOH tails specify interaction with {beta}-arrestins (24). The ability of {beta}-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 {alpha}2b-adrenergic receptor also determine its interaction with {beta}-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 {beta}-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 {beta}-arrestin 1 at the plasma membrane and in endosomes. There was no detectable colocalization of NK1R with immunoreactive {beta}-arrestin 1 at the plasma membrane, but there was a low level of colocalization with {beta}-arrestin 1-GFP at the plasma membrane and in endosomes, possibly due to overexpression of {beta}-arrestin 1-GFP in transfected cells. These substitutions did not affect colocalization with {beta}-arrestin 2, which is expected because both NK1R and NK3R interact with {beta}-arrestin 2. Thus domains in loop 3 and the COOH tail of the NK1R are important for colocalization with {beta}-arrestin 1. It is likely that these domains are also important for colocalization with {beta}-arrestin 2. However, this possibility could not be evaluated by studying chimeric receptors because both NK1R and NK3R interacted with {beta}-arrestin 2. Presently, we do not have an unequivocal explanation for these findings. However, {beta}-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 {beta}-arrestin 2 (23, 24). Replacement of the COOH tail of the NK3R with that of the NK1R conferred association of the NK3R/NK1Rct with {beta}-arrestin 1-GFP at the cell surface, suggesting that this domain is important for interaction with {beta}-arrestin 1. There was no detectable colocalization of NK3R/NK1Rct at the cell surface with endogenous {beta}-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 {beta}-arrestin 1-GFP or {beta}-arrestin 2-GFP in endosomes, suggesting that additional motifs are necessary for prolonged association.

Implications of differential interactions of NK1R and NK3R with {beta}-arrestins 1 and 2. {beta}-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 {beta}-arrestin isoforms could have important functional implications. Although we have previously shown that {beta}-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 {beta}-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 {beta}-arrestin 2 alone is sufficient for desensitization, endocytosis, and mitogenic signaling of the NK3R, whereas the NK1R may utilize both {beta}-arrestins 1 and 2 for these processes.

Differences in affinity may determine the duration with which NK1R and NK3R interact with {beta}-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 {beta}-arrestins in endosomes, and receptor recycling (8, 28). The prolonged, high-affinity colocalization of NK1R with both {beta}-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 {beta}-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 {beta}-arrestin 1 and accelerated resensitization of the NK1R to that of the NK3R. The affinity of interaction between other GPCRs and {beta}-arrestins similarly dictates the kinetics of resensitization (22).

Differences in interaction with {beta}-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 {beta}-arrestins in NK1R endosomes, thereby depleting cytosolic {beta}-arrestins and impeding homologous desensitization and endocytosis of the NK3R. The present results suggest that the NK1R would sequester both isoforms of {beta}-arrestin. In contrast, NK3R activation would deplete only {beta}-arrestin 2 from the cytosol, leaving {beta}-arrestin 1 to interact with the NK1R to mediate desensitization and endocytosis. Differences in interaction with {beta}-arrestins also explains the ability of the vasopressin V2 receptor to inhibit endocytosis of the {beta}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 {beta}-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 {beta}-arrestins 1 and 2 to mediate desensitization, endocytosis, and MAP kinase signaling. Activation of the NK1R would sequester {beta}-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 {beta}-arrestins return to the cytosol, when the NK3R could interact with {beta}-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 {beta}-arrestins could affect signaling of many other receptors. Thus the high-affinity interaction of a class B GPCR with both isoforms of {beta}-arrestin would also affect {beta}-arrestin-dependent signaling of class A low-affinity GPCRs. In view of the roles of {beta}-arrestins in uncoupling, endocytosis, and mitogenic signaling, regulation of the interactions between GPCRs and {beta}-arrestins would be expected to have far-reaching functional implications. It is thus of interest to determine the spectrum of GPCRs and {beta}-arrestins that are coexpressed in functionally important cell types, as well as the relative levels of expression of these important regulatory proteins.


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39957 (to N. Bunnett), Deutsche Forschungsgemeinschaft Grant ROO2290/1-1 (to D. Roosterman), and Institut National de la Santé et de la Recherche Médicale (to F. Schmidlin).


    ACKNOWLEDGMENTS
 
We thank Dr R. J. Lefkowitz for the {beta}-arrestin antibody, Paul Dazin for assistance with flow cytometry, and Dr. E. F. Grady for helpful discussions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. W. Bunnett, Depts. of Surgery and Physiology, Univ. of California, San Francisco, 521 Parnassus Ave., 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.


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