Functional consequences of alteration of N-linkedglycosylation sites on the neurokinin 1 receptorMorris F. Tansky*, Charalabos Pothoulakis†, and Susan E. Leeman*‡
*Department of Pharmacology, Boston University School of Medicine, Boston, MA 02118; and †Beth Israel Deaconess Medical Center,Division of Gastroenterology, Gastrointestinal Neuropeptide Center, Harvard Medical School, Boston, MA 02215
Contributed by Susan E. Leeman, April 16, 2007 (sent for review January 31, 2007)
The neurokinin 1 receptor (NK1R), a G protein-coupled receptorinvolved in diverse functions including pain and inflammation, hastwo putative N-linked glycosylation sites, Asn-14 and Asn-18. Westudied the role of N-linked glycosylation in the functioning of theNK1R by constructing three receptor mutants: two single mutants(Asn 3 Gln-14 and Asn 3 Gln-18) and a double mutant, lackingboth glycosylation sites. Using a lentiviral transfection system, themutants were stably transfected into NCM 460 cells, a nontrans-formed human colonic epithelial cell line. We observed that themagnitude of glycosylation as estimated by changes in gel migra-tion depends on the number of glycosylation sites available, withthe wild-type receptor containing the greatest amount of glyco-sylation. All mutant receptors were able to bind to substance P andneurokinin A ligand with similar affinities; however, the doublemutant, nonglycosylated NK1R showed only half the Bmax of thewild-type NK1R. In terms of receptor function, the ablation of bothN-linked glycosylation sites did not have a profound effect on thereceptors’ abilities to activate the MAP kinase families (p42/p44,JNK, and p38), but did affect SP-induced IL-8 secretion. All mutantswere able to internalize, but the kinetics of internalization of thedouble mutant receptor was more rapid, when compared withwild-type NK1R. Therefore, glycosylation of NK1R may stabilize thereceptor in the plasma membrane. These results contribute to theongoing elucidation of the role of glycosylation in G protein-coupled receptors and the study of the neurokinin receptors inparticular.
G protein-coupled receptor � substance P
The neurokinin 1 receptor (NK1R) mediates a range ofproinflammatory and pain processes. For example, NK1R
levels are up-regulated in sites of joint inflammation, asthma,inflammatory bowel disease, acute pancreatitis, and abdominalcell adhesion formation (1–6). It is also speculated to be involvedin modulation of HIV infection (7). There is considerableinterest in NK1R as a therapeutic target, and antagonists arecurrently being tested for treatment of depression, emesis,asthma, and breast cancer.
The NK1R was identified, cloned, and characterized as amember of the rhodopsin family of the G protein-coupledreceptor (GPCR) superfamily (8–10). The NK1R has differentbinding affinities for its tachykinin ligands, which include sub-stance P (SP), neurokinin A (NKA), NKA-like peptides, andneurokinin B. SP, first identified by von Euler and Gaddum (11)in 1931, was isolated by Chang and Leeman (12) in 1970, andlater synthesized by Tregear et al. (13). SP was identified as asialogogic peptide, but has also been shown to function as aneurotransmitter, a neuromodulator, and a key mediator ofinflammatory processes (14). In nontransformed mucosal epi-thelial cells, SP can induce secretion of proinflammatory cyto-kines, such as IL-6, IL-8, and TNF-�, and this induction requiresactivation of the transcription factor NF-�B (15, 16). SP bindingto the NK1R elicits inositol production (17), mobilizes Ca2� (18),and activates the ERK1 and ERK2 members of the MAPKfamily (19). In fact, transactivation of the EGF receptor has beendemonstrated upon SP stimulation (20), leading to cell prolif-
eration that involves matrix metalloproteinases and TGF-�secretion (21).
Although SP is the preferred ligand for NK1R, several groupshave shown that NKA binds NK1R with high enough affinity toelicit a biologic response (22–24). More recently, it has beenfound that benzoylphenylalanine3-SP, a photoactivatable ligandfor the NK1R, covalently labeled the N-terminal region of theNK1R between residues 11 and 21 (25). Therefore, the bindingregion for SP encompasses the glycosylation region of the NK1R,and perturbations of the glycosylation sites not only provideinsight on the receptor itself, but also on the ligand–receptorcomplex.
The neurokinin receptors contain putative N-linked glycosyl-ation sites on their extracellular amino termini: residues 14 and18 on NK1R, residues 11 and 19 on neurokinin-2 receptor, andresidues 23, 50, and 73 on neurokinin-3 receptor. The role ofthese receptor glycosylation sites is unclear. Based on theparticular receptor, carbohydrate moieties have been shown tobe involved in various receptor functions, including receptorstability and activation state (EGF receptor) (26); receptorfolding (luteinizing hormone receptor) (27); trafficking andexpression (human � opioid receptor, neurotensin receptor) (28,29); and ligand binding and signal transduction (parathyroidhormone receptor, somatostatin subtype 3 receptor) (30, 31). Itis apparent from these studies that glycosylation effects onGPCRs are empiric and specific to the receptor, requiringinvestigation in each receptor system.
Based on the evidence pointing to receptor glycosylation ascritical in ligand binding, internalization dynamics, and ligand-directed downstream signaling, as well as our previous worksuggesting that rat NK1R was highly glycosylated when ex-pressed in CHO cells (32), we sought to understand the role ofglycosylation in the NK1R. To do this, we created mutants ofNK1R that lack one or both of its N-linked glycosylation sites.
ResultsExtent of Glycosylation in Transfected NCM 460 Cells. An NK1Rimmunoblot was performed to analyze the extent of receptorglycosylation in the transfected colonic epithelial NCM 460 cells.The immunoblot shows a greater relative mass of the wild-typeNK1R as compared with the two single glycosylation mutantsand the double glycosylation mutant (Fig. 1A). The singleglycosylation mutants appear to migrate to the same extent aseach other and also indicate a relative mass that is greater thanthe double mutant. The wild-type and single glycosylationmutants tend to have a more diffuse banding pattern as com-pared with the double glycosylation mutant, which runs in a
Author contributions: M.F.T. and S.E.L. designed research; M.F.T. performed research;M.F.T. and C.P. contributed new reagents/analytic tools; M.F.T. analyzed data; and M.F.T.,C.P., and S.E.L. wrote the paper.
sharp band very close to the NK1R predicted peptide mass of�50 kDa. These results confirm earlier evidence that the nativeNK1R is glycosylated, and that the mutations at N14Q andN18Q, as expected, lead to a decrease in the level of receptorglycosylation. Furthermore, the various cell lines were shown toexpress equivalent NK1R transcript, as shown by quantitativeRT-PCR (Fig. 1B; � � 0.05, P � 0.05, null hypothesis accepted).
Binding Assays of NCM 460 Cells Transfected with NK1R. To observewhat effect receptor glycosylation has on ligand binding, weperformed competitive binding analysis of wild-type and glyco-sylation mutant NK1R expressed in NCM 460 cells. UnlabeledSP was used to compete with 125I Bolton Hunter-SP (125I-BH-SP) reagent in receptor binding assays, as detailed in Materialsand Methods. These assays (Fig. 2A) demonstrated no effect ofglycosylation on the Ki of SP in the presence of 125I-BH-SP (datawere normalized to wild-type values). The Ki of SP in all cell lineswas �1.0 nM, in agreement with published results of wild-typeNK1R (33–35). These results indicate that the various glycosyl-ation mutants have affinities for SP similar to that of thewild-type receptor. In contrast, the apparent Bmax depended on
whether there was an N-linked glycosylation site available. Theapparent Bmax of the single glycosylation mutants were similar tothat of the wild-type receptor, but the apparent Bmax of thedouble mutant was only 48% of wild type. This finding suggestedthat the number of receptors expressed on the surface of the cellstransfected with mutant, nonglycosylated receptor may bealtered.
As shown in Fig. 2B, confirming results from the competitionbinding assays above, 125I-BH-SP saturation analysis of wild-typeand glycosylation mutant receptors demonstrates no effect ofglycosylation on the KD of 125I-BH-SP. For saturation analysisexperiments, cells were treated with increasing concentrations of125I-BH-SP with and without 1 �M SP, representing nonspecificand total binding, respectively. The KD was �3 nM, which iswithin the range found in the literature, indicating that ligandaffinity is not affected by receptor glycosylation. However, thissaturation analysis demonstrates a dramatic reduction in theBmax of the double glycosylation mutant, when compared withwild-type receptor (Fig. 2B). Whereas, the single glycosylationmutants did not vary significantly from the wild-type receptor,the Bmax of the nonglycosylated receptor was reduced signifi-cantly (�2-fold). This result indicates that the amount of func-tional, fully nonglycosylated NK1R expressed in the plasmamembrane is almost half that of wild-type NK1R and suggeststhat glycosylation may play a role in NK1R stabilization anddynamics in the membrane.
Receptor Internalization in NCM 460 Cells. We next examinedreceptor dynamics by immunocytochemistry of NCM 460 cellsstably transfected with wild-type and nonglycosylated NK1R.Cells were treated with 10 nM SP for 0 min to 2 h, probed witha polyclonal antibody raised against the H83 epitope of theNK1R, and visualized by confocal microscopy. As shown in Fig.3, unstimulated cells have a ribbon-like NK1R immunoreactivity,reflecting a uniform NK1R distribution on the membranesurface of the cells. After SP stimulation, NK1R immunoreac-tivity becomes punctate, reflecting formation of vesicles andinternalization. Much of this punctate immunoreactivity resolvesby 2 h of continuous SP stimulation, indicating that receptorinternalization has reached a steady state.
Immunoreactivity of cells containing nonglycosylated NK1Rindicates punctate staining within 15 min of SP stimulationsimilar to the wild-type receptors, suggesting that NK1Rs lackingglycosylation are still able to internalize and recycle back to thesurface. Interestingly, the singly and doubly glycosylation mutant
50 kD
N14Qwt N18Q N14/18Q
35 kD
BA
Fig. 1. Extent of glycosylation and expression of wild-type and mutantreceptors. (A) NK1R immunoblot of transfected NCM 460 cell lysates shows thenonglycosylated mutant receptors migrate at a lower relative mass than thewild-type NK1R, reflecting a loss of carbohydrate moieties. (B) Real-time PCRcomparison of lentiviral mutants in NCM 460 cells, demonstrating equal NK1Rmessage for the NCM 460 cells transfected with the various glycosylationmutants (� � 0.05, P � 0.05). NCM 460 NK1R mRNA levels were normalized toTATA-box binding protein mRNA. The values are expressed as a percentage ofthe wild-type NK1R mRNA abundance.
A B
Fig. 2. Competition binding and 125I-BH-SP saturation analyses of wild-type and glycosylation receptor mutants in NCM 460 cells. These analyses demonstratethat there are no effects of glycosylation on the Ki of SP in the presence of 125I-BH-SP (A; competition binding analysis), but that there is a 48% decrease in theBmax of the nonglycosylated mutant when compared with the other mutants and wild-type (B; saturation analysis).
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receptors show more punctate staining at 45 min of continuousSP stimulation, suggesting that the absence of carbohydratemoieties may affect receptor recycling back to the surface, afterinternalization.
Kinetics of Receptor Internalization in NCM 460 Cells. Our confocaldata of receptor internalization and recycling showed that a fullydeglycosylated receptor may internalize faster, but return to thecell surface more slowly, than wild-type receptors. We sought tofurther elucidate these processes by using acid wash and radio-active quantification as detailed in Materials and Methods (36).We found that 24% more of the double glycosylation receptormutants (N14Q/N18Q) are internalized within 5 min, relative tothe amount of internalized wild-type receptors (Fig. 4), indicat-
ing that receptors lacking glycosylation may internalize fasterthan wild-type receptors. These results are consistent with ourconfocal microscopy observations. One explanation for theseresults may be that receptors continuously oscillate and are inequilibria among different states that may favor internalization;glycosylation may shift these equilibria to internalization-favored states. The lack of steric hindrance conferred by thecarbohydrate moieties may lower the threshold for conforma-tional changes of the receptor because of the energy of thesystem.
MAPK Family Signaling in NCM 460 Cells Transfected with NK1Rs. Wenext sought to characterize NK1R-induced signaling pathways inour various NK1R-expressing NCM 460 cell lines, becauseprevious work has shown that NK1R can activate the MAPKfamilies after 100 nM SP stimulation (20). Our cell lines con-taining the mutant NK1Rs were therefore compared afterSP-induced phosphorylation of the MAPK pathways p38, p42/p44, and JNK. As shown by immunoblot in Fig. 5, p38 andp42/p44 are phosphorylated within 5 min of SP incubation,whereas JNK phosphorylation occurs within 30 min in cells withwild-type and mutant NK1Rs. To ensure equivalent loading ofsamples, total p42/p44, total p38, and total JNK were alsoassayed; cells did not show an increase in total (phosphorylatedplus unphosphorylated) MAPK levels (Fig. 5 and data notshown).
Furthermore, the observed MAPK phosphorylation is adownstream signaling event of the NK1R, because pretreatmentwith the NK1R antagonist CJ12,255 ablates p42/p44 and JNKphosphorylation induced by SP (Fig. 6, purple, SP alone; orange,antagonist plus SP) at 10 and 30 min of SP treatment for p42/44and pJNK activation, respectively. Therefore, the results showthat SP can activate the MAPK families p38, p42/p44, and JNK,and that this activation is specifically mediated by the NK1R.
IL-8 Production in NCM 460 Cells Transfected with NK1R. The NK1Rhas been shown to mediate SP activation of inflammatorypathways, including IL-8 production (15). Therefore, an IL-8secretion assay to functionally characterize the lentivirally in-troduced wild-type and mutant NK1R was undertaken. NCM
untreated
SP stimulation15 min
SP stimulation45 min
SP stimulation2 hours
wildtype N14Q N18Q N14/18Q
Fig. 3. NK1R immunostaining of NCM 460 cells transfected with wild-type and mutant NK1 receptors, after stimulation by SP for the indicated durations.Untreated cells containing wild-type or mutant receptors show NK1R immunoreactivity on the cell surface, whereas SP stimulation triggers NK1R internalization,visualized as punctate staining (closed arrows). After longer SP stimulation, the mutant receptors are still retained within the cell, in contrast to the wild-typereceptors that have returned to the cell surface (open arrows) by 45 min. Representative images of each cell line are shown. (Scale bar: 20 �m.)
**
Fig. 4. Internalization kinetics of NK-1R mutants. Cells preincubated with125I-BH-SP for 1 h at 4°C were warmed to 37°C for the indicated times. Cellswere acid-washed to remove surface-bound ligand and treated with alkalinewash to determine the amount of internalized ligand by a gamma counter.The double glycosylation receptor mutant displays a significant 24% increasein the relative amount of internalized receptor when compared with thewild-type receptor (� � 0.01, P � 0.01).
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460 cells transfected with wild-type NK1R showed increasedIL-8 production as a function of time after bolus SP treatment(0–8 h) and SP concentration (0.3 nM-30 �M). Four hours of100 nM SP treatment increased IL-8 secretion, which remainedelevated after 8 h. After 4 h of 100 nM SP treatment, NCM 460cells with wild-type NK1R showed a 13-fold increase in IL-8secretion as measured by ELISA, compared with untreatedtransfected cells, indicating that NK1R is functionally active inthese cells (Fig. 7). In contrast, the amount of IL-8 produced byunstimulated NCM 460 did not plateau but increased steadilyover time, indicating that the IL-8 secreting machinery ofunstimulated cells is not saturated. Furthermore, the NK1R isactivated by SP specifically, because a NK1R antagonist abro-gates this observed IL-8 production (data not shown). In con-trast, NCM 460 cells with the double nonglycosylated mutantNK1R demonstrated a pronounced diminution of IL-8 produc-tion after 100 nM SP stimulation for 4 h, showing an 85%decrease from IL-8 production (Fig. 7).
DiscussionOur evidence shows that glycosylation of the NK1R has func-tional consequences. We demonstrate here that NK1R glyco-sylation may not be necessary for ligand binding and downstreamMAPK signaling. However, nonglycosylated NK1R has signifi-cantly reduced cell surface levels of functional receptor. As well,nonglycosylated NK1Rs elicit reduced IL-8 secretion in responseto SP activation.
The presence of N-glycosylation sites in the N-terminal do-main is a common characteristic of GPCRs, although oligosac-charide function in GPCR function varies with different systems,
and therefore the role of glycosylation must be determinedempirically. For example, studies with N-glycosylation of the ratangiotensin II receptor (37) showed that receptors with all threedefective N-glycosylation sites are not expressed on the plasmamembrane, but instead are accumulated in the endoplasmicreticulum; interestingly, the preservation of Asn-176 in thesecond extracellular loop enabled surface expression similar towild-type receptors. Glycosylation-deficient receptors displayedwild-type KD values for its ligand sarcosine, whereas levels ofinositol phosphate production upon activation were unchanged.In contrast, site-directed mutagenesis of two glycosylation sitesat the extracellular domain of the follicle-stimulating hormonereceptor showed that one intact site is sufficient for receptorexpression at the cell surface and ligand binding with highaffinity (38). This group also reported that removal of sixN-glycosylation sites on rat lutropin receptor had no effect onbinding to human chorionic gonadotropin, although glycosyla-tion may be involved in proper receptor folding (39). Similarly,gonadotropin-releasing hormone (GnRH) receptor mutants inwhich the consensus N-linked glycosylation sites were ablatedshowed unchanged ligand affinity and were able to stimulatedownstream signaling pathways. However, the glycosylation-defective GnRH receptors displayed decreased Bmax values at39–46% of wild-type depending on mutation site, implyingdecreased receptor expression and/or stability at the plasmamembrane (40).
Fig. 5. Comparison of SP-induced phosphorylation of MAPK pathways p38, p42/p44, and JNK in cell lines transfected with NK1R mutants. Immunoblot showsthe duration of SP-mediated activation of p38, p42/p44, and JNK in all cell lines. p38 and p42/p44 are phosphorylated within 5 min of SP incubation, whereasJNK phosphorylation occurs within 30 min. Wild-type and mutant receptors exhibit similar phosphorylation patterns. Total p42/p44 (shown), total p38, and totalJNK were also assayed to assure loading equivalency. Representative immunoblots from independent experiments are shown.
– + – +– – + +
– + – +– – + +
– + – +– – + +
– + – +– – + +10nM SP
1µM CJ 12,255
p42/p4410min SP
pJNK30min SP
Total MAPK10min SP
Wt NK1R N14Q/N18Q NK1RN18Q NK1RN14Q NK1R
– + – +– – + +
– + – +– – + +
– + – +– – + +
– + – +– – + +10nM SP
1µ
p42/p4410min SP
pJNK30min SP
Total MAPK10min SP
Wt NK1R N14Q/N18Q NK1RN18Q NK1RN14Q NK1R
Fig. 6. Immunoblots demonstrating SP-specific effect of MAPK phosphory-lation, using NK1R antagonist CJ12,255. Pretreatment of cells with the NK1Rantagonist ablates p42/p44 and JNK phosphorylation induced by SP (purple:SP alone; orange: antagonist plus SP) at the indicated times, suggesting thatSP acts solely through NK1R to activate the MAPK family.
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Fig. 7. Transfected NCM 460 cells were stimulated for 4 h with 100 nM SP,demonstrating by ELISA a pronounced diminution of IL-8 production in theN14Q/N18Q mutant cell line lacking both glycosylation sites. This reductionwas significant when compared with cells containing the wild-type and singlemutant receptors (� � 0.01, P � 0.001).
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In other systems, GPCRs do not require N-glycosylation formembrane targeting, ligand binding, or downstream signaling.For example, glycosylation-defective �1-adrenergic, H2 hista-mine, and M2-muscarinic receptors do not have altered cellsurface expression and/or stability. Further, site-directed mu-tagenesis at two of three consensus sites of the histamine H2receptor show that lack of glycosylation does not alter thereceptor’s ability to bind ligand, nor does it affect cAMPactivation and intracellular calcium accumulation. Immuno-staining and binding experiments localized the glycosylation-defective receptors to the plasma membrane, implying thatN-glycosylation is not required for intracellular targeting of theH2 receptor (41).
The present studies of a glycosylation-defective NK1R mayindicate that N-glycosylation is necessary for stable receptorexpression in the plasma membrane, because the glycosylation-defective receptors show reduced functional cell surface expres-sion and more rapid internalization upon ligand binding. Inter-estingly, previous work on the extensively glycosylated EGFRshows that receptor self-dimers have 5-fold more kinase activitythan monomeric receptors (independent from ligand binding);this self-dimerization and thus kinase activity highly depends onglycosylation (26). Fernandes et al. (26) suggest that glycosyla-tion confers a ‘‘kinase-active’’ conformation that enables self-dimerization. As in the present studies with NK1R, lack of theoligosaccharides might contribute to receptor destabilizationand degradation. The above studies do not, however, rule out thepossibility of impaired receptor export, regulation of whichrequires endoplasmic reticulum chaperones, accessory proteins,and receptor activity modifying proteins, which can dimerizewith GPCRs and modulate receptor folding.
Ligand affinities for SP and NKA (data not shown) ofglycosylation-defective receptors are similar to that of the wild-type receptor, which suggests that for the NK1R, N-glycosylationis not involved in ligand binding, because the nonglycosylatedreceptor is able to attain proper folding to bind ligand. As well,glycosylation-deficient receptors are able to activate variousMAPKs similarly to wild-type receptors, leading to NF-�Bnuclear translocation (data not shown) and IL-8 production. Itis somewhat unexpected, however, that we observe a substantialreduction in IL-8 secretion in cells containing the glycosylation-deficient receptors, because proinflammatory cytokine produc-tion has been shown to be downstream of and dependent onprotein kinase pathways. This finding may reflect the sensitivityof IL-8 production to small changes in the timing of effectorphosphorylation, which, in turn, are amplified throughout theactivation cascades. For example, it has been previously shownthat IL-8 secretion is sensitive to the synergistic effects of theERKs, JNK, and the p38 MAPK cascades, such that a subtlechange in one pathway dramatically affects IL-8 induction (42).It is also possible that the NK1R-induced IL-8 secretion ob-served here depends on protein kinases outside of the scope ofthis present study.
It is clear from the study of various GPCRs that glycosylationcan have a wide range of effects on receptor function, and furtherstudy of the receptor mutants created here will enhance ourknowledge on the role of NK1R in inflammation associated withseveral disease states. As observed here, the decreased expres-sion of glycosylation-deficient NK1R at the plasma membranemay prove to be a critical point of therapeutic manipulation.
Materials and MethodsCreation of Lentiviral Vector with NK1R. Plasmids containing thewild-type human NK1R were obtained from N. Bunnett (Uni-versity of California, San Francisco, CA). Using restrictionenzyme digest, the NK1R sequence was extracted and ligatedinto PC3 DNA cloning vector (Stratagene, La Jolla, CA). Then,using splicing by overlap extension (43), single glycosylation
mutants of the NK1R were made, changing Asn3 Gln-14, andAsn 3 Gln-18, to create the N14Q and N18Q mutants, respec-tively. As well, a double N14/18Q glycosylation mutant wascreated. These three mutant plasmids, and the wild-type NK1Rin the PC3 cloning vector, were used as templates for a modifiedPCR, in which HotStart Ultra Pfu polymerase (Stratagene) andprimers with a Topo-directional four-base sequence and a Kozaksequence were used to create Topo-directional fragments. Theblunt-end fragments containing the NK1R sequence were in-corporated into the Lenti6/V5-D-TOPO vector (Invitrogen,Carlsbad CA). These constructs were verified by sequencing,purified, and transfected into 293FT cells with Lipofectamine2000 (Invitrogen). The virus-containing supernatant was har-vested, titered, and used to create stable NCM 460 cell lines.
Real-Time Quantitative RT-PCR. Total RNA was isolated fromNCM 460 cells with an RNeasy mini kit (Qiagen, Valencia, CA)and reverse-transcribed with the One-Step RT-PCR kit(PerkinElmer, Shelton, CT). The resulting DNA was used forreal-time PCRs on a 96-well plate using either NK1R-specificTaqman master mix from Applied Biosystems (Foster City, CA)or a human TATA-box binding protein Taqman master mix forendogenous control and analyzed in a GeneAmp 5700 detectionsystem (ABI/PerkinElmer, Boston, MA) (44).
ELISA. The secretion of IL-8 in NCM-460 cells was measured byusing an ELISA kit developed by R & D Systems (Minneapolis,MN). Cells were grown to 80% confluence in 24-well plates andserum-starved overnight (�18 h). Media were then replacedwith fresh serum-free media containing peptide agonist, antag-onist, or both and incubated for the indicated amount of time.The media were then analyzed for IL-8 levels as detailed by theR & D Systems protocol.
Immunoblot. The cell lysates were run on 4–12% SDS/PAGE gels,transferred, and immobilized on nitrocellulose or PVDF mem-branes using a modified Laemmli and Tobin method (45, 46).Cell lysates were boiled for 10 min and loaded on NuPAGE gels(Invitrogen) and run at 150 V for 2 h using Mops buffer (50 mMTris base/50 mM Mops/1 mM EDTA/3.5 mM SDS, pH 7.7). Thegels were transferred at 30 V for 4 h in NuPAGE transfer buffer(25 mM Bis-Tris/1 mM EDTA/25 mM Bicine/15% methanol).Membranes were blocked overnight at 4°C in 5% skim milk or5% BSA and washed with 0.05% Tween-20 in Tris-bufferedsaline, pH 7.4. The membranes were then incubated with theappropriate primary antibodies. NK1R was visualized by usingrabbit anti-NK1R antibodies (H-83; Santa Cruz Biotechnology,Santa Cruz CA) and HRP–labeled secondary antibodies, anddetected by SuperSignal Chemiluminescent Substrate (Pierce,Rockford, IL).
MAP kinase activation was assessed by stimulation of NCM460 cells cultured in complete medium, and when 75% conflu-ent, incubated for 18 h in 0% FBS medium. The cells werestimulated with 10 or 100 nM SP or NKA or 20 ng/ml EGF forthe stated times. The cells were processed for immunoblot asdescribed above, using phosphospecific antibodies (0.2 �g/ml)directed against ERK1 or ERK2 to detect MAPK activation,anti-JNK rabbit antibody or anti-p38 rabbit antibody (CellSignaling Technology, Beverly, MA).
Binding Kinetics. For competitive binding analysis of the NK1R inthe various cell lines, a fixed concentration of 125I-BH-SP (62.5pM) and a variable concentration of SP (1 pM—1 �M) wereincubated with a fixed number of cells (5 � 104) for 2 h at 4°Cin Krebs–Ringer’s solution–Hepes buffer (20 mM Hepes/1 mMMgCl2/5 mM KCl/120 mM NaCl, pH 7.4) supplemented with 6mg/ml glucose and 0.6 mg/ml BSA. For saturation analysisexperiments, cells were treated with increasing concentrations of
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125I-BH-SP with and without 1 �M SP, representing nonspecificand total binding, respectively.
Quantitative Receptor Internalization. NCM 460 cells plated in24-well plates were washed three times with 4°C Hanks’ bufferedsaline solution (HBSS) with 0.1% BSA at 80% confluence. Thecells were blocked for 1 h with 4°C HBSS plus 0.1% BSA. Afterblocking, the cells were treated with either 62 pM 125I-BH-SP or 62pM 125I-BH-SP and 1 �M SP for 1 h at 4°C. After incubation at 37°Cfor the specified time (0–45 min), the cells were treated with 0.2 Macetic acid/0.5 M NaCl for 10 min, which constituted the acid-washfraction or surface receptors. The cells were washed three timeswith chilled PBS and then treated with 0.5 M NaOH for 30 min atroom temperature. The base-wash fraction represented primarilythe internalized receptor. The acid and base solutions containingthe lysed cells were individually read for 10 min in a 1470 gammacounter (Wallac, Gaithersburg, MD). The amounts of ligand inboth the acid wash (surface-bound ligand) and alkaline wash(internalized ligand) were then determined by radioactive count;the data are represented as a ratio of internalized to total ligand(total is the sum of acid and alkaline washes). The labeled SP was
prepared by using 125I Bolton-Hunter reagent (PerkinElmer,Waltham MA) and SP (Bachem, King of Prussia, PA) in aconjugation reaction (47).
Immunofluorescence. Cells were treated with 10 nM SP for 0 minto 2 h, fixed with 4% formaldehyde, probed with a polyclonalantibody raised against the H83 epitope of the NK1R and arhodamine-conjugated secondary antibody, and finally visual-ized by confocal microscopy.
Statistical Analyses. Results were analyzed by using Statview forWindows (SAS Institute, Cary, NC) and KaleidaGraph (Synergy,Reading, PA). The data were subjected to a Student’s–Newman–Keuls’ test to determine significance.
We thank Dr. A. Stucchi (Boston University School of Medicine) forNK1R antagonist, C. Song for technical assistance, C.P.’s laboratory fortechnical assistance and helpful discussion (particularly Dr. D. Zhao),and Dr. N. Bunnett for the wild-type NK1R plasmid. This work wasfunded in part by National Institutes of Health Grants 5R21NS04322-02and R0-1 DK 47343 and the Russek Foundation.
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