©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
C-terminal Truncation of the Neurokinin-2 Receptor Causes Enhanced and Sustained Agonist-induced Signaling
ROLE OF RECEPTOR PHOSPHORYLATION IN SIGNAL ATTENUATION (*)

Jacqueline Alblas , Ingrid van Etten , Azra Khanum , Wouter H. Moolenaar (§)

From the (1) Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The G protein-linked receptor for neurokinin A (NKA) couples to stimulation of phospholipase C and, in some cells, adenylyl cyclase. We have examined the function of the C-terminal cytoplasmic domain in receptor signaling and desensitization. We constructed C-terminal deletion mutants of the human NK-2 receptor (epitope tagged) to remove potential Ser/Thr phosphorylation sites, and expressed them in both mammalian and insect cells. When activated, truncated receptors mediate stronger and more prolonged phosphoinositide hydrolysis than wild-type receptor; however, the amplitude and kinetics of the NKA-induced rise in cytosolic Caremain unaltered. Protein kinase C (PKC)-activating phorbol ester abolishes wild-type receptor signaling but not mutant receptor signaling. Mutant receptors also mediate enhanced and prolonged cAMP generation, at least in part via PKC activation. When expressed in COS cells or Sf9 insect cells, the wild-type receptor is phosphorylated; receptor phosphorylation increases after addition of either NKA or phorbol ester. In contrast, mutant receptors are not phosphorylated by either treatment. Our results suggest that C-terminal Ser/Thr phosphorylation sites in the NK-2 receptor have a critical role in both homologous and heterologous desensitization. Removal of these phosphorylation sites results in a receptor that mediates sustained activation of signaling pathways and is insensitive to inhibition by PKC.


INTRODUCTION

Signal transduction via G protein-coupled receptors is characterized by rapid desensitization, i.e. attenuation of cellular responses upon prolonged or repeated agonist exposure (1, 2, 3) . The mechanisms of desensitization have been extensively studied in the -adrenergic receptors and the visual receptor rhodopsin. In those systems, signal attenuation occurs through receptor phosphorylation by various Ser/Thr kinases, in particular protein kinase A, protein kinase C (PKC),() and specific receptor kinases (4) . Much less is known about the desensitization mechanisms of PLC-coupled receptors, although the available evidence suggests that, again, phosphorylation may play a critical role (5, 6) .

We are interested to examine how signal transduction via PLC-coupled receptors and their cellular actions are altered when the putative ``desensitization domain'' of the receptor is deleted or mutated. One would expect that such desensitization-defective receptors, when activated, mediate prolonged rather than short-lived generation of second messengers. Prolonged signal generation is likely to affect long-term cellular responses such as cell growth and differentiation. Our approach makes use of the receptor for the decapeptide neurokinin A (NKA, also known as substance K; tachykinin receptor subtype, NK-2) (7, 8) . The neurokinins (tachykinins) are a family of neuropeptides involved in diverse physiological and pathological processes, ranging from inflammation to neurotransmission. Where tested, the neurokinin receptors couple to stimulation of PLC with subsequent mobilization of Cafrom intracellular stores (9, 10, 11, 12) . In some cell systems, there is additional coupling to stimulation of adenylyl cyclase, either directly via G(9) or secondary to Camobilization (10) , or arachidonate metabolism (13) . Like other G protein-coupled receptors, the neurokinin receptors undergo agonist-induced desensitization, but the molecular basis is not known, although C-terminal phosphorylation would be a plausible mechanism. However, a recent study (14) reports that the C-terminal domain of the NK-2 receptor is not involved in desensitization.

To address the possible role of the cytoplasmic tail in NK-2 receptor signaling and desensitization, we have constructed and selected mutant receptors that lack various parts of the C-terminal tail and yet remain coupled to PLC activation in an agonist-dependent manner. We expressed these mutant receptors in various cell systems, including Rat-1 fibroblasts, COS cells, and Sf9 insect cells, and examined how their signaling properties are altered. Our results indicate that truncated receptors mediate enhanced and prolonged activation of signaling pathways, apparently due to loss of regulatory phosphorylation sites in the C-terminal domain.


EXPERIMENTAL PROCEDURES

Materials

The PKC inhibitor Ro31-8220 was obtained from Roche Research Center (Welwyn Garden City, United Kingdom). Endoglycosidase H was purchased from Boehringer Mannheim GmbH, indo-1 acetoxymethylester was from Molecular Probes Inc., [2-[I]iodohistidyl]neurokinin A (2000 Ci/mmol), myo-[H]inositol (80-120 Ci/mmol), L-[S]methionine/ L-[S]cysteine labeling mix (>1000 Ci/mmol), and P(10 mCi/ml) were from Amersham, as well as the ECL kit. Prot A-Sepharose (CL-4B) beads were from Pharmacia Biotech Inc., and peroxidase-conjugated antibodies were from Dako. All other chemicals were from Sigma.

Cell Culture

Rat-1 fibroblasts, human embryo kidney (HEK293) cells, and COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7.5% fetal bovine serum and antibiotics. Spodoptera frugiperda (Sf9) insect cells were obtained from the ATCC and were grown in Grace's insect medium supplemented with 10% fetal calf serum and antibiotics at 27 °C.

Construction of Mutant NK-2 Receptors and Recombinant Baculovirus

The cDNA of the human NK-2 receptor (kindly provided by Dr. R. Kris (12) ) was subcloned into a modified pMT2 vector (with altered multiple cloning site constructed by M. Gebbink) containing an epitope-tag sequence derived from the VSV-glycoprotein (15) directly preceded by a NotI site (construct designed and made by L. van der Voorn and G. Hateboer). Polymerase chain reaction fragments were generated up and until the desired last amino acid of the NK-2 receptor (amino acids 398, 338, 328, and 309, respectively), containing a NotI overhang for in frame cloning with the tag sequence. This resulted in C-terminal extension of full-length and mutant receptors with the sequence SGRPYTDIEMNRLGK. The constructs coding for wt and 328 were subcloned into the pVL1393 vector and cotransfected with lethal virus DNA (BaculoGold kit by PharMingen) into Sf9 cells. Recombinant virus was plaque-purified and amplified. Protein expression was determined by Western blot using monoclonal antibody P5D4 against the epitope tag.

DNA Transfections

Cells were transfected with 10 µg of purified recombinant pMT2 plasmid by the calcium phosphate precipitation method. For stable transfection of Rat-1 cells, 0.1 µg of pSVneo was included in the precipitate. The next day, cells were split 1:10 and selected in DMEM supplemented with 1 mg/ml G418. Resistant clones were isolated and screened for expression by radioligand binding assay and subsequently subcloned by single cell dilution. Comparison of tagged versus non-tagged NK-2 receptor expressed in Rat-1 cells revealed no differences in behavior of the receptors as measured by Camobilization (not shown). For stable transfection of HEK293 cells, 0.5 µg of pSV23.6, containing the cDNA for murine Na,K-ATPase that confers ouabain resistance (16) , was included in the precipitate. The HEK293 cells were selected with 1 µ M ouabain, and resistant clones were picked and screened for NK-2 receptor expression and subcloned in conditioned medium from untransfected HEK293 cells. COS cells were grown in 10-cm dishes and transfected with 10 µg of DNA by the DEAE-dextran method, trypsinized the next day, and divided over a 6-well plate. They were serum-starved overnight, labeled with Por S-labeled Met/Cys for 3 h, and stimulated with the agonists required.

Radioligand Binding

Cells were grown to confluency in 24-well plates, washed with Hepes-buffered DMEM, and incubated on ice with the same buffer containing 0.1% bovine serum albumin, 1 m M Mn, and 0.1 n M I-NKA for at least 1 h. Cells were then washed four times with ice-cold phosphate-buffered saline containing 1 m M Caand 1 m M Mg, dissolved in 0.1 N NaOH, and monitored for radiation. Nonspecific binding was measured in the presence of 0.5 µ M unlabeled NKA.

Inositol Phosphates

Cells were grown in 6-well plates to near confluency and labeled overnight in serum-free medium containing 2 µCi/ml myo-[H]inositol. Cells were washed with DMEM-Hepes and equilibrated for 2 h. After stimulation in the presence of 10 m M LiCl, inositol phosphates were extracted and isolated using AG 1-X8 anion exchange columns (formate form; Bio-Rad) as described (17) . Total inositol phosphates (IP) were eluted with 1 M ammonium formate, 0.1 M formic acid.

CaMeasurements

Rat-1 cells were plated on rectangular glass coverslips and grown to 80% confluency. The cells were loaded with 5 µ M indo-1 acetoxymethylester in serum-free Hepes-buffered DMEM for 20-30 min, washed with HBS (10 m M Hepes, pH 7.4, 140 m M NaCl, 5 m M KCl, 1 m M CaCl, 1 m M MgCl, 10 m M glucose), and incubated at 37 °C in HBS in quartz cuvettes. Infected Sf9 cells were harvested 24 h postinfection, gently washed in HBS (pH 6.5), loaded in HBS containing 2 µ M indo-1 acetoxymethylester for 45 min, and transferred to quartz cuvettes (10cells/ml) kept at 27 °C. Fluorescence was measured with 355 nm excitation and 405 nm emission wavelength as described (18) .

cAMP Measurements

Confluent serum-starved cells were stimulated in the presence of 1 m M isobutylmethylxanthine and assayed for cAMP content using a [H]cAMP assay system (Amersham Corp.) according to the manufacturer's instructions.

Infection and Labeling of Sf9 Insect Cells

Insect Sf9 cells were infected with recombinant baculovirus for 30 min in Grace's insect medium without fetal calf serum in a suspension of 25 10cells/ml and then plated in Grace's medium with fetal calf serum in 10-cm dishes. After 40 h of infection, cells were radiolabeled by incubating them in synthetic Sf9 medium (50 m M PIPES, pH 6.5, 55 m M KCl, 10 m M MgCl, 5 m M CaCl, 5 m M NaHCO, and 100 m M sucrose) containing 750 µCi/ml Pfor 3 h. Immunoprecipitation of wt and mutated NK-2 receptors was carried out as described for mammalian cells (see below). The immunoprecipitates were dissolved in sample buffer containing 6 M urea, separated on an 11% gel, transferred to nitrocellulose, and probed with the P5D4 antibody followed by the ECL reaction.

Immunoprecipitations

Cells labeled with either S-labeled Met/Cys or Pwere lysed in Nonidet P-40-lysis buffer (0.5% Nonidet P-40, 50 m M Tris, pH 7.4, 150 m M NaCl, 5 m M MgCl, 1 m M EGTA, 0.1 m M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin; phosphatase inhibitors, in the case of P labeling, were 0.1 m M NaVO, 10 m M NaF, and 10 m M NaPO). Lysates were precleared twice with normal mouse serum precoupled to fixed Staphylococcus aureus or to Sepharose-protein A beads. Specific precipitation was performed with the P4D5 monoclonal antibody recognizing the tag. P5D4 monoclonal antibody was used as culture supernatant from the hybridoma (15) . Precipitates were washed six times in the same buffer. For endoglycosidase H digestion, immunocomplexes were resuspended in 25 µl of citrate buffer (50 m M sodium citrate, pH 5.5, 0.2% SDS) with or without 0.5 milliunits endoglycosidase H and incubated for 1 h at room temperature. SDS-sample buffer containing 6 M urea was added; samples were not boiled to avoid protein aggregation and were immediately loaded on a polyacrylamide gel. The amount of immunoprecipitated material in each lane was assessed by Western blotting. The blots were probed with P5D4 and peroxidase-conjugated swine anti-mouse immunoglobulin, subjected to the ECL procedure, briefly washed, dried, and exposed to film.


RESULTS

Construction of Truncated Receptors

To examine the importance of the cytoplasmic tail in NK-2 receptor signaling and desensitization, we set out to generate mutant receptor cDNAs. The predicted secondary structure of the 398-amino acid human NKA receptor (NK-2 receptor) is shown in Fig. 1 A. Several consensus sites for post-translational modifications such as glycosylation, palmitoylation, and phosphorylation are indicated. There are 25 intracellular serine/threonine residues, 13 of which occur in motifs similar to known consensus sequences for PKC, cAMP-dependent protein kinase, or members of the ARK family (19, 20) . We chose to delete 60, 70, or 89 amino acids from the C-terminal tail (Fig. 1 A; truncation sites indicated by arrows), yielding three distinct mutant receptors termed 338, 328, and 309 lacking 18, 17, and 15 Ser/Thr residues, respectively. All receptor constructs were extended at their C terminus with a 15-amino acid epitope tag derived from the VSV-glycoprotein (Ref. 15, see also ``Experimental Procedures''), as illustrated in Fig. 1 B. The cDNA constructs were cloned into the pMT2 expression vector and transfected into COS cells and Rat-1 fibroblasts.


Figure 1: Schematic representation of the human NK-2 receptor. A, predicted secondary structure. The standard one-letter amino acid code is used. Bold circles indicate cytoplasmic Ser/Thr residues; arrows indicate sites of truncation in the C-terminal tail; shaded Asn residues in the N terminus are consensus sequences for N-linked glycosylation; the potential myristoylation site (Cys-323) is marked with a zigzag; B, constructs of wt and deletion mutant NK-2 receptors. Transmembrane ( TM) domains are shaded; the epitope tag sequence is indicated with a flag. The proline ( P) in the tag sequence does not belong to the original VSV-glycoprotein epitope as described (15).



Expression of Wild-type and Truncated NK-2 Receptors in COS Cells

Expression of the NK-2 receptor constructs was examined by COS cell transfections. At 48 h after transfection, the cells were metabolically labeled with S-labeled Met/Cys for 3 h followed by immunoprecipitation and SDS-PAGE analysis of precipitated proteins (Fig. 2). Proteins of various molecular masses are detected, ranging from about 44 kDa for wt receptor to 35 kDa for 309. It is seen that each transfected cDNA translates into a doublet of protein bands, differing about 6 kDa in size, as well as a band of roughly twice the predicted mass. After treatment of the immunoprecipitates with endoglycosidase H to remove high mannose N-linked sugars, the higher molecular weight forms shift back to the lower molecular weight form ( lanes marked with +). Lower endoglycosidase H doses yielded a partial digestion resulting in a third protein product of intermediate size (not shown). Thus, the upper band represents a double glycosylated form of the receptor, the lower band represents the non-glycosylated form, and the middle band (poorly visible in Fig. 2 ) contains one N-linked glycan. The endoglycosidase H-sensitive proteins in the upper region of the gel presumably represent receptor aggregates (dimers).


Figure 2: Expression of wt and mutant NK-2 receptors in COS cells. COS cells transiently transfected with the constructs depicted in Fig. 1 B were metabolically labeled at 48 h after transfection with S-labeled Met/Cys for 3 h and then lysed; NK-2 receptors were immunoprecipitated with anti-tag monoclonal antibody P5D4. Immunoprecipitates were treated with endoglycosidase H (+) (Endo H) as indicated and analyzed by SDS-PAGE and autoradiography as described under ``Experimental Procedures.''



Coupling of the receptors to phospholipase C was measured by the accumulation of IP. Agonist stimulation of transfected COS cells showed a 2.5-3-fold increase in IPformation with all constructs except for the 309 mutant (results not shown). This indicates that mutant 309 is not functional; therefore, this mutant was not used in further experiments.

Expression of Wild-type and Truncated NK-2 Receptors in Rat-1 Cells

To investigate the signaling properties of the wt and mutant NK-2 receptors, stable transfectants of Rat-1 cells were generated. Clones were selected on the basis of I-NKA binding. Agonist displacement analysis (Fig. 3) reveals that truncated and wild-type receptors have similar affinities for NKA. The ICvalue for displacement by unlabeled NKA is estimated at 3-6 n M, in agreement with reported affinity values for endogenous and transfected NK-2 receptors (10, 21, 22, 23) . Analysis of these data (24) yields receptor densities of about 8,000/cell for wt receptor-expressing cells, 11,000/cell for 328, and 26,000/cell for 338. In our further signaling studies, we focused mainly on wt and 328 receptors, given their comparable expression levels in Rat-1 cells. Yet, where tested, mutant 338 behaved qualitatively similar to 328.

Mutant Receptors Mediate Sustained Inositol Phosphate Accumulation

To characterize PLC activation by wt and truncated receptors, we measured NKA-induced inositol phosphate formation in the various Rat-1 transfectants. Stimulation of wt receptor with 1 µ M NKA for 5 min resulted in an approximate 3-fold increase in IP(Fig. 4 A). Brief pretreatment of the cells with the PKC-activating phorbol ester TPA (100 ng/ml) completely inhibited NKA-induced IPformation in wt receptor-expressing cells without affecting control IPlevels. Conversely, when PKC activity was inhibited by the selective PKC inhibitor Ro31-8220 (3 µ M), NKA-induced IPformation was consistently elevated when compared with control cells (Fig. 4 A); similar results were obtained after down-regulation of PKC by prolonged TPA treatment (100 ng/ml for 24 h). These results strongly suggest that NKA-induced PLC activation via wt NK-2 receptors is subject to feedback inhibition by PKC.


Figure 4: Agonist-induced phosphoinositide hydrolysis in transfected Rat-1 cells. A, confluent cells were labeled with myo-[H]inositol for 20 h and stimulated for 5 min in the presence of Liwith either buffer (control) or NKA (1 µ M). Where indicated, cells were pretreated with 100 ng/ml TPA for 5 min or with the PKC inhibitor Ro31-8220 (3 µ M) for 10 min. B, time course of NKA-induced inositol phosphate formation. [H]Inositol-labeled cells were stimulated with NKA for the indicated periods of time and assayed for inositol phosphate formation. Each data point represents mean ± S.E. C, duration of NKA-induced PLC activity. myo-[H]Inositol-labeled cells were stimulated with NKA at time point zero. LiCl (10 m M) was added at 0, 30, or 60 min after NKA addition, and inositol phosphates were allowed to accumulate for 30 min. Incubations were terminated, and inositol phosphate levels were determined as described under ``Experimental Procedures.'' Data represent the amount of IP accumulated during a 30-min incubation with Li. C, control.



In cells expressing mutant receptor 328, the relative increase in IPaccumulated during 5 min of agonist stimulation was consistently higher than observed with wt receptor (Fig. 4 A). Strikingly, in 328-expressing cells, NKA-induced phosphoinositide breakdown was completely insensitive to inhibition by TPA (Fig. 4 A). Furthermore, neither the PKC inhibitor Ro31-8220 (Fig. 4 A) nor prolonged TPA treatment (not shown) had any potentiating effect on NKA-induced IPformation via truncated NK-2 receptor. Collectively, these results suggest that (feedback) inhibition of NK-2 receptor signaling by PKC is mediated by phosphorylation of the receptor's C-terminal tail.

The kinetics of receptor-mediated PLC activation by NKA are shown in Fig. 4 B. The response to wt receptor leveled off after about 5 min, whereas activation of 328 gave a steadily increasing formation of inositol phosphates. To examine the duration of PLC activation in further detail, we measured IPaccumulation during 30-min Lipulses at various times after agonist addition (17) . After 1 h of stimulation, the mutant receptor still mediated a 5-fold increase in IPfollowing a Lipulse, whereas the PLC activation by wt receptor was almost back to control levels (Fig. 4 C). Thus, signal termination as observed with wt receptor is not attributable to depletion of agonist-sensitive phosphoinositide pools but rather reflects receptor signaling shut-off (homologous desensitization), a phenomenon not observed with truncated receptor.

Taken together, these results indicate that deletion mutant receptor mediates enhanced and sustained activation of PLC as compared with wt receptor.

CaMobilization by NK-2 Receptors Expressed in Rat-1 and Sf9 Cells

Does sustained PLC activation lead to altered Casignaling kinetics? Fig. 5 A shows typical time courses of NKA-induced Camobilization in stably transfected Rat-1 and human 293 cells, expressing either wt or 328. No significant differences are observed in the amplitude and duration of the NKA-induced Catransient mediated by either wt or mutant receptor in both cell types.

We also sought to monitor receptor-mediated Camobilization in insect Sf9 cells expressing either wt or 328 receptor. Previous work has indicated that certain mammalian or avian receptors can effectively couple to endogenous G proteins in Sf9 cells (21, 25) . At 24 h after infection with recombinant baculovirus, cells were loaded with indo-1 acetoxymethylester, and agonist-induced changes in intracellular Caconcentration were measured. As shown in Fig. 5B, NKA evokes a rapid Catransient in Sf9 cells expressing either wt receptor or 328 in the presence of EGTA, indicative of receptor-mediated Carelease from intracellular stores. As in Rat-1 cells, the NK-2 receptor expressed in Sf9 cells appears to be sensitive to inhibition by PKC, since addition of TPA for 15 min blocks NKA-induced Camobilization. In contrast, the Catransient mediated by mutant 328 remains unaffected after TPA addition.


Figure 5: NKA-induced Ca mobilization in Rat-1 and Sf9 cells expressing NK-2 receptors. A, NKA-induced Camobilization in stable transfectants of Rat-1 ( upper panel) or HEK293 ( lower panel) expressing wt NK-2 receptor or 328. Cells were loaded with indo-1 acetoxymethylester, and agonist-induced calcium signaling was monitored in the presence of 3 m M EGTA; arrowheads indicate time point of NKA addition. The concentration of NKA was 1 µ M in all experiments. B, Sf9 cells infected for 24 h with either wt ( upper panel) or 328 ( lower panel) receptor recombinant virus were loaded with indo-1 acetoxymethylester and stimulated with 1 µ M NKA ( arrow) in the presence of EGTA (3 m M). TPA (100 ng/ml) was added for 15 min.



These results indicate that there is no direct relationship between the duration of PLC activation and that of the resulting Casignal. Thus, termination of the Catransient is not regulated at the level of receptor desensitization; instead, the rate-limiting step is likely at the level of IPmetabolism and/or IPreceptor desensitization.

Truncated Receptors Mediate Increased cAMP Formation in a PKC-dependent Manner

In Rat-1 cells expressing wt receptor, NKA induces a rather small increase in cAMP content (Fig. 6 A). However, it is seen that stimulation of mutant receptors results in a much more sustained cAMP production. Various mechanisms can account for receptor-mediated cAMP production: (i) direct activation of adenylyl cyclase (AC) via G, (ii) indirect activation of AC via production of eicosanoids acting on their own G protein-coupled receptors, (iii) activation of one or more AC isoforms by Cacalmodulin, i.e. secondary to Camobilization, and (iv) activation of an AC isoform by PKC.

As shown in Fig. 6 B, inhibition of PKC by Ro31-8220 or long-term (24 h) treatment with TPA strongly inhibited NKA-induced cAMP formation but did not affect the response to isoproterenol, which acts on the G-coupled -adrenergic receptor. Intracellular Castores were depleted with either thapsigargin (26) or ionomycin in the absence or presence of EGTA. Neither of these treatments affected NKA-induced cAMP formation (Fig. 6 B), nor did the cyclo-oxygenase inhibitor indomethacin (20 µ M) (not shown), arguing against regulation by intracellular Caor prostaglandin production.


Figure 6: Agonist-induced cAMP accumulation in Rat-1 transfectants. A, time course of NKA-stimulated cAMP formation in two independently selected clones expressing wt, two clones expressing 328, and one clone expressing 338 receptor. B, effect of modulation of intracellular Calevels by thapsigargin or ionomycin and modulation of PKC activity by TPA (100 ng/ml; 5 min or 24 h) on NKA-induced (1 µ M, 5 min) cAMP formation in 328 cells; Ro31-8220 was added 10 min prior to NKA. Depletion of intracellular Castores was achieved by addition of thapsigargin 5 min prior to NKA addition; ionomycin (1 µ M) was added 30 s before NKA. Data points represent mean ± S.E. C, nonstimulated cells (control).



Together, our results are consistent with enhanced cAMP production being mediated, either directly or indirectly, by PKC. The recent cloning and expression of adenylyl cyclases have shown that type II AC is stimulated by phorbol ester, presumably via direct PKC phosphorylation of the enzyme (27) . However, we were unable to raise cAMP levels in Rat-1 cells by addition of TPA alone. We therefore conclude that in addition to PKC, other as yet unidentified mechanisms participate in NKA-induced activation of AC.

Receptor Phosphorylation in COS Cells and Sf9 Insect Cells

The possible link between signal attenuation and receptor phosphorylation was examined in COS cells expressing wt or truncated NK-2 receptor; expression levels in transfected Rat-1 cells turned out to be too low for reliable receptor phosphorylation experiments. Immunoprecipitated wt NK-2 receptor from P-labeled COS cells showed basal phosphorylation of the wt receptor bands (both unglycosylated and glycosylated forms) (Fig. 7 A). Stimulation with NKA for 20 min resulted in increased receptor phosphorylation, as is also seen after treatment with TPA. In contrast, mutant receptor showed little basal phosphorylation, which did not increase after addition of either NKA or TPA.

We also examined receptor phosphorylation in baculovirus-infected Sf9 cells expressing wt or 328 NKA receptor. Radiolabeling experiments confirmed that surface expression of wt and mutant receptors was about equal (not shown). Fig. 7 B shows that receptor phosphorylation was significantly increased following addition of either NKA or TPA. In contrast, mutant receptor 328 showed only minor basal phosphorylation levels, which were not increased after NKA or TPA treatment. Western blot analysis confirmed that equal amounts of receptor protein were immunoprecipitated.


Figure 7: Phosphorylation of NK-2 receptors expressed in COS cells and insect Sf9 cells. A, COS cells were transfected with wt or mutant 338 NK-2 receptor. Transfected cultures were serum starved overnight, labeled for 3 h with 200 µCi/well P, stimulated with NKA (1 µ M) or TPA (100 ng/ml) for 20 min, and then lysed and immunoprecipitated with antibody P5D4. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography as described under ``Experimental Procedures.'' B, receptor phosphorylation in baculovirus-infected Sf9 cells. Cells were infected and labeled as described under ``Experimental Procedures,'' stimulated, and immunoprecipitated as in Fig. 7 A. The receptor immunoprecipitates were analyzed by SDS-PAGE, transferred to nitrocellulose, and probed with P5D4 antibody followed by the ECL reaction to visualize the amount of receptor protein (lower part of the autoradiograph). The same blot was then briefly washed, dried, and exposed to film to detect P-labeled proteins. C, non-stimulated cells (control).



Together with the signal transduction data, these results demonstrate that there is concordance between agonist- and phorbol ester-induced receptor phosphorylation and signal attenuation.


DISCUSSION

Desensitization of G protein-coupled receptors is a complex process involving various molecular mechanisms. Deletion or mutation of putative phosphorylation sites in G protein-coupled receptors has indicated the importance of receptor phosphorylation in mediating desensitization (28, 29, 30, 31) . In particular, agonist-induced Ser/Thr phosphorylation of the cytoplasmic tail has been implicated in receptor desensitization. We have used the NK-2 receptor as a model system for investigating the role of the C-terminal domain in receptor signaling, desensitization, and phosphorylation. Our major findings can be summarized as follows: (i) C-terminal deletion of 60 or 70 residues results in a receptor that is still functional but fails to undergo desensitization in response to agonist; (ii) truncated receptors are resistant to inhibition by PKC-activating phorbol ester; and (iii) wild-type receptor, but not truncated receptor, is phosphorylated upon addition of either NKA or phorbol ester. From these results, we conclude that C-terminal tail phosphorylation is responsible for both homologous and heterologous desensitization of the human NK-2 receptor.

That truncated receptors (328 and 338) are desensitization defective is indicated by their sustained stimulation of both PLC and AC as compared with the transient activation observed with wt receptor. It is noteworthy that despite sustained PLC activation, the kinetics of NKA-induced Casignal from intracellular stores (reflecting IPaction) were the same for wt and mutant receptors. This indicates that the rate-limiting step for termination of Casignaling occurs downstream of the receptor, most likely at the level of IPcatabolism and/or IPreceptor regulation.

Generation of cAMP by deletion mutants follows a pattern similar to IPformation. All our results point to a pivotal role of PKC in stimulating AC activity, although PKC-activating phorbol ester alone is incapable of raising cAMP levels in Rat-1 cells; hence, additional mechanisms are likely to participate in AC activation by (truncated) NK-2 receptors. In transfected Chinese hamster ovary cells, the neurokinin receptors also couple to stimulation of both PLC and AC, although it is not clear whether AC activation is PKC dependent as it is in Rat-1 cells (9) . In another study on transfected Chinese hamster ovary cells, the NK-2 receptor reportedly mediates enhanced cAMP generation as a secondary response to eicosanoid production (13) . However, our results argue against a role for eicosanoids in NKA-induced cAMP formation. Whatever the exact mechanisms of enhanced cAMP generation, the data show that at least two distinct effectors, PLC and AC, are constitutively activated by truncated receptors, indicating the importance of the C-terminal tail in controlling signal termination.

In a recent study on desensitization of endothelin and NK-2 receptors, Cyr et al. (14) reported that a truncated human NK-2 receptor (336), nearly identical to the 338 construct used in the present study, behaves like wild-type receptor; the authors conclude that the C-terminal tail of the NK-2 receptor has no apparent role in desensitization. The discrepancy with the present study remains unexplained. However, direct comparison should be made with caution because the expression systems used were very different ( Xenopus oocytes versus mammalian cells).

Our results strongly suggest that receptor phosphorylation occurs at the C-terminal tail and is causally related to the observed receptor desensitization. Basal and agonist-induced phosphorylation of wt and mutant receptors is readily detected in COS cells and baculovirus-infected Sf9 cells. In both systems, the unstimulated wt receptor exists in a phosphorylated state. It remains to be seen whether this represents a normal physiological state, since our efforts to measure receptor phosphorylation at ``normal'' expression levels (as in Rat-1 transfectants) were unsuccessful. In COS cells and Sf9 cells, both NKA and phorbol ester induce a significant increase in basal phosphorylation of wt but not mutant receptors. We also find that the deletion mutants become resistant to inhibition by PKC and that inhibition or down-regulation of PKC results in enhanced NKA-induced PLC activity in stable transfectants. From these results, we conclude that by phosphorylating one or more C-terminal Ser/Thr residues, PKC mediates (feedback) inhibition of receptor signaling and thus participates in both homologous and heterologous desensitization.

The apparent involvement of PKC does not, of course, exclude the involvement of specific receptor kinases in mediating receptor phosphorylation and desensitization. The deleted part of the NK-2 receptor C terminus contains several consensus phosphorylation sites for both PKC (three sites) and ARK (six sites). In fact, the related NK-1 receptor can be phosphorylated by the -adrenergic receptor kinases ARK1 and ARK2 (32) , at least in vitro. The visual receptor rhodopsin is a target for both PKC and a specific rhodopsin kinase in a ligand-dependent manner (33) . Signal attenuation by the PLC-coupled thrombin receptor is also mediated, at least in part, by ARK (34) . Furthermore, the PLC-coupled receptor for platelet-activating factor may be a target for ARK (6) . Future studies should reveal whether the ARKs, which recognize only the ligand-activated form of the receptor, are also involved in NK-2 receptor phosphorylation and desensitization.

In conclusion, our results indicate a close link between C-terminal tail phosphorylation and signal attenuation of the human NK-2 receptor. Phosphorylation somehow leads to decreased coupling efficiency to the relevant G protein(s). The exact phosphorylation sites and the protein kinase(s) responsible remain to be identified in further experiments. Another outstanding question is whether sustained PLC stimulation mediated by truncated NK-2 receptors leads to altered long-term cell behavior, such as cell cycle progression and induction of mitogenesis. We hope to address these questions in further studies.


FOOTNOTES

*
This work was supported by the Netherlands Organization for Scientific Research (NWO) and by the Dutch Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 31-20-512-1971; Fax: 31-20-512-1989.

The abbreviations used are: PKC, protein kinase C; PLC, phospholipase C; TPA, 12- O-tetradecanoylphorbol-13-acetate; NKA, neurokinin A; AC, adenylyl cyclase; ARK, -adrenergic receptor kinase; VSV, vesicular stomatitus virus; IP, inositol 1,4,5-trisphosphate; IP, total inositol phosphates; PIPES, 1,4-piperazinediethanesulfonic acid; wt, wild type; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; I-NKA, [2-[I]iodohistidyl]-neurokinin A.


ACKNOWLEDGEMENTS

We thank R. Kris for providing the full-length cDNA of the human NKA receptor and M. Gebbink, L. van der Voorn, and G. Hateboer for expression vectors.


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