Functional nonequivalence of structurally homologous domains of ...

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 22, Insue of August 5, pp. 16388-16395,1993 Printed in U.S.A.

Functional Nonequivalenceof Structurally Homologous Domains of Neurokinin- 1 and Neurokinin-2 Type Tachykinin Receptors* (Received for publication, January 14, 1993, and in revised form, March 29, 1993)

Paul BlountS and JamesE.Krauset From the Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, Missouri, 63110

The neurokinin-1 (NK-1)and neurokinin-2 (NK-2) receptors are both members of the tachykinin receptor family. Although both receptors bind peptide ligands synthesized from common precursors and activate inositol 1,4,6-triphosphate and cAMP responses, differences between these receptors have been observed in the extent and kinetics of agonist-induced responses. Here, to testif structurally homologous domains of the NK-1 and NK-2 receptors are functionally distinct, stably transfected Chinese hamster ovary (CHO) cell lines expressing receptors that had either their putative third cytoplasmic loop (C3) or carboxyl tail (CT) domains replaced with the equivalent domain of the other receptor werecompared with stably transfected CHO cell lines expressing wild-type receptors. Radioligand binding demonstrated that each of these chimeric receptors hadagonist binding affinities indistinguishable from wild-type receptors. However, not all chimeric receptors were equivalent intheir ability to stimulate inositol phospholipid turnover and cAMP production. The datasuggest that theNK-1 C3 and the NK-2 CT domains play important roles in G-protein activation that cannot be replaced by the analogous domain of the other receptor. The characterization of CHO cell linesexpressingtruncatedforms of both receptors supportedthe hypothesis that theCT domain of the NK-2, but not the NK-1, receptor plays a critical role in G-protein activation.The datasuggest a potential mechanism forthedifferences observed in response characteristics in tissues expressingNK- 1 and NK-2 receptors and demonstrate that the mechanisms whereby highly homologous receptors activate G-proteins canbe different.

Substance P (SP),’ neurokinin A (NKA), and neurokinin *This workwas supported inpart by Grants NS21937 and NS29343 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Neuropharmacology Training GrantNS07129 from the National Institutes of Health and was a Keck Fellow. Present address: Lab. of Molecular Biology, University of Wisconsin, Madison, WI 53706. 3 To whom correspondence should be addressed Dept. of Anatomy and Neurobiology, Washington University School of Medicine, Box 8108, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3416; Fax: 314-362-3446. The abbreviations used are: SP, substance P; NKA, neurokinin A; NPy, neuropeptide y; NPK, neuropeptide K, CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; IBMX,3-isobutyl-lmethyl-xanthine; NK-1, neurokinin-1; NK-2, neurokinin-2; C3, predicted third cytoplasmic loop; CT, predicted carboxyl-terminal cytoplasmic tail; IP, inositol phosphate; IP3,inositol 1,4,5-trisphosphate.

B (NKB) aremembers of a family of small peptides that have been called tachykinins because of their rapid contractile effects on gastrointestinal tissues. These peptides also have additional physiological effects in several other tissue types in the periphery and the centralnervous system (Helke et al., 1990; Nakanishi, 1991). Two additional NH2 terminally extended derivatives of NKA, neuropeptide K (NPK), and neuropeptide y (NPy), have also been isolated and have been shown to be functional (Helke et al., 1990). SP, NKA, NPK, and NPy are synthesized from common precursors and have been shown to be copackaged and cosecreted (MacDonald et al., 1989). The physiological effects of these tachykinins are mediated predominantly by two different receptors: neurokinin 1 (NK-1) and neurokinin 2 (NK-2) receptors, with NK-1 expressing a higher affinity for SP, and NK-2 expressing a higher affinity for NKA, NPK, and NPy. NKB synthesized is from an independent precursor molecule and mediates its actions predominantly through the neurokinin 3(NK-3) receptor. The best characterized second messenger system that mediates the effects of tachykinin peptides has been stimulation of inositol phosphate (IP) production. Increases or decreases in cAMP levels have also been observed upon SP stimulation in certain systems (Duffy and Powell, 1975; Yamashita et al., 1983; Narumi and Maki, 1978; Hanley et al., 1980) but not others (Quik et al., 1978; Watson, 1984; Hunter et al., 1985). Differences have been observed in the onset, duration, and intensity of SP versus NKA responses, with NKA and related peptides generally having more sustained effects (Falconeri Erspamer et al., 1980; Leeet al., 1982; Nawa et al., 1984). Because the NK-1 and NK-2 receptors have been shown to be differentially expressed in tissues (Quirion et al., 1988), these differences in response characteristics could either be attributed to fundamental differences in NK-1 and NK-2 receptor structure leading to different functionally properties, and/or differences in signal transduction systems present in the cell types in which these receptors are expressed. The cDNAs encoding the NK-1 and NK-2 receptors of several species, including rat (Sasai andNakanishi, 1989; Yokota et al., 1989; Hershey and Krause, 1990), have been cloned and characterized. Analysis of the predicted protein sequences has demonstrated that the NK-1 and NK-2 receptors are homologous, sharing 48% identity in rat (Yokota et al., 1989) and aremembers of the family of G-protein-coupled receptors (Masu et al., 1987). Results of expression studies of the rat NK-1 and NK-2 receptor in a common stably transfected host, Chinese hamster ovary (CHO) cells, has indicated that both inositol phospholipid hydrolysis and increases in cAMP formation occur both in cells and in membrane preparations, suggesting that each of these responses are a direct result of receptor stimulation (Nakajima et al., 1992). The CHO cell line has been used for these and similar studies because it expresses multiple G-proteins that couple to IP

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Functional Nonequivalence of N K - 1 and NK-2 Receptors metabolism (Ashkenazi et al., 1989). Our laboratory has also expressed the rat NK-1 and NK-2 receptors in CHO cells (Takeda et al., 1992) and noted a difference in magnitude of the CAMP, but not the IP3 response, thus supporting the hypothesis that agonist-induced IP production and cAMP formation occur independent of one another. However, in boththese studies, only quantitative, but not qualitative, differences were observed, suggesting that the two receptors are functionally similar. Hence, initial studies using transfected cell lines expressing tachykinin receptors gave little insight into themolecular basis of the differences in response kinetics observed in tissues. In thecurrent series of experiments, we have addressed the question of which cytoplasmic domains of these tachykinin receptors are responsible for the different second messenger responses. We have concentrated on the following twodifferent domains that have been shown to be functionally important in other systems: the putative third cytoplasmic loop (C3), which has been shown to be important for G-protein coupling in several systems, and the predicted carboxyl-terminal cytoplasmic tail (CT), which has been associated with phosphorylation-dependent receptor desensitization and/or down-regulation in some systems (for a recent review on functional domains of G-protein-coupled receptors, see Dohlman et al., 1991). Complimentary DNAs encoding chimeras and truncated forms of the NK-1 and NK-2 receptors were constructed, and the encoded proteins were stably expressed intransfected CHO cells. Surprisingly, experiments using these cell lines demonstrate that structurally homologous domains of the rat NK-1 and NK-2 receptor do not have functionally equivalent roles. Our studies suggest that theC3 domain of the NK-1 receptor plays an important role in Gprotein activation that cannot be recovered by insertion of the NK-2 C3 domain. We also provide evidence that the role of G-protein activation in theNK-2, but not theNK-1 receptor, is encoded, at least in part, within the CTdomain of this receptor. Hence, although the NK-1 and NK-2 receptors stimulate identical second messenger systems when expressed in CHO cells, the structuralrequirements for second messenger responses are not encoded in the same domains in the two receptors. These differences, perhaps in conjunction with the potential differences in effector and regulatory proteins expressed in host cell types, may define the basis for the diversity of responses observed in different tachykinin systems. EXPERIMENTALPROCEDURES

MatericlL+"y~-[~H]Inositol(specific activity = 20 Ci/mmol) was from American Radiolabeled Chemicals, Dowex 1-8X ion-exchange resin (100-200 mesh formate form) was from Bio-Rad, and high salt capacity Tru Count was from In/Us Service corporation. The cAMP radioimmunoassay kits were from Amersham Corp. Tachykinin radioligands were prepared and purified by high performance liquid chromatography as described (MacDonald et al., 1989; Takeda et al., 1991). The cDNAs encoding wild-type (Hershey, 1991; Takeda et al., 1992), chimeric, and truncated receptors were ligated into the pMZ expression vector (Takeda et al., 1991; Matzuk, et al., 1987). Other reagents were the highest purity available from Sigma or Fisher Scientific and have been described (Takeda et al., 1990,1991; Hershey and Krause, 1990; Hershey et al., 1991). Generation of Chimeric and Truncated Receptors-All cDNAs encoding chimeras were generated by PCR mutagenesis (Innis et al., 1990). Approximately 20 cycles with an oligonucleotide annealing temperature a t least 5 "C below the lowest predicted melting temperature was used with a starting DNA concentration (cDNA in supercoiled pBLUESCRIPT) of 10-100 ng. For chimeras of the putative third cytoplasmic loop (C3) domain, oligonucleotides 24-25 bases in length that were exact hybridization matches for at least 1 2 adjacent nucleotides, together with primers for the 5' and 3' end of the cDNA, were used in PCR reactions to generate three cDNA fragments for each receptor: a fragment encoding from the 5' start codon to theC3

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domain, a fragment encoding only the C3 domain, and a fragment encoding from the end of the C3 domain to the carboxyl end of the protein including the stop codon. The C3 domain was defined to extend from amino acid 218 to 249 and 219 to 251 for the NK-1 and NK-2 receptor, respectively. Appropriate fragments were combined, and, because the priming oligonucleotides of the C3 junctions were designed to overlap at least 12 nucleotides, cDNAs encoding fulllength chimeric receptors (shown schematically in Fig. L4) were generated. The junction siteswere in regions that are highly conserved between these two receptors, helping to assure the exchange of homologous structural domains. For chimeras of the predicted carboxyl-terminal CT domain, sets of oligonucleotides 18 base pairs in length were used as primers for the CTjunction. The generation and transfection of the NK-1 receptor with the NK-2 CT domain was performed by Hershey (1991). The two sets of oligonucleotides used to generate the two CT chimeras overlapped 12 conserved nucleotides (encoding 4 amino acids, Arg-Cys-Cys-Pro). The junctions for the CT chimeras were a t amino acid positions 324-325for the NK-1 receptor and 326-327 for the NK-2 receptor. The oligonucleotides used as primers of the extreme 5' and 3' ends hadadditional sequence at theirends that contained a consensus site for Hind111 and BamHI restriction enzymes, respectively. These sites allowed for the digestion, and subsequently, the directional subcloning of the final PCR products into pBLUESCRIPT. All cDNAs encoding chimeric receptors were fully sequenced by the Sanger dideoxy method to assure that the integrity of the entire coding sequence was maintained through PCR amplifications. The cDNA encoding the truncated NK1 receptor was also generated using PCR mutagenesis. As previously described (Hershey, 1991), an oligonucleotide 33 bases in length that would encode a stop codon at amino acid 325 of the NK-1 receptor was used as a 3' primer. Using this primer in conjunction with a 5' primer, a cDNA encoding only the first 324 amino acids was generated. The final cDNA was subcloned and subjected to full sequence analysis as above. Truncations of the NK-2 receptor were generated by subcloning the 3' end of the cDNA encoding the NK-2 receptor (the AccI site in the cDNA to the BamHI site in the polylinker) into M13mp19, generating single-stranded phage, and using a oligonucleotide-directed in vitro mutagenesis system from Amersham (version 2.1) to generate stop codons in the desired locations (amino acids 327, 347, and 367; see Fig. 1B). TheM13 insert was subjected to full sequence analysis as above and subcloned into pBLUESCRIPT containing the remaining NK-2 cDNA, using the restriction enzyme sites Sac1 in the insert and BamH I in the polylinker. The resulting clones were characterized by restriction enzyme analysis. All clones were subcloned into the pM' expression vector (Matzuk et al., 1987); the truncated NK-2 receptors were subcloned into this vector using a polylinker inserted into thenormal BamHI insertion site (Sachais et al., 1993). The pM' vector contains a Harvey murine sarcoma virus long terminal repeat that drives expression of the cloned cDNA and also contains a neomycin resistance gene under the transcriptional control of an SV40 early promoter. Cell Growth, Transfection, and Selection-CHO cells weregrown in minimal essential medium-a containing 10% fetal bovine serum. One day prior to transfection, cells were plated at a density of 3 x lo5 cells/lO-cm tissue culture dish. Cells were transfected with 10 pg of the appropriate cDNA clone in the pMZexpression vector (Matzuk et al., 1987) using a calcium phosphate transfection procedure (Graham and van der Eb, 1973) with modifications (Hershey, 1991; Takeda et al., 1992) and were grown in the presence of 0.8 mg active G418/ml. Six to 10 days later, colonies were isolated, propagated, and screened by ligand binding. Ligand Binding Assays-Cells were rinsed with cold phosphatebufferedsaline (PBS), scraped from the tissue culture flask, and resuspended in PBS at a concentration of approximately 2.5 x lo5 cells/ml. Saturation binding experiments were performed with ligand added in one-quarter volume 5 x binding buffer (5 X binding buffer: PBS, 1 mg/ml bovine serum albumin, 0.2 mg/ml bacitracin, 20 pg/ ml leupeptin, 20 pg/ml chymostatin) incubated for 2 h at 4 "C. Eight agonist concentrations, ranging from 8 X 10"' to 6 X lo+' M lZ5ITyr"-SP for NK-1 receptors or 5 X lo-" to 1.2 X IOv8M lZ5I-Np7 for NK-2 receptors, were used. Separation of free from bound ligand was by rapid filtration. Nonspecific binding was determined by addition of 1pM unlabeled peptide. Data was analyzed by the computer program Ligand (Munson and Rodbard, 1980) and all data best fit a single site model. Cell Stimulation and cAMP Assays-Similar to the paradigm previously described (Takeda etal., 19921, cells were rinsed, scraped, and resuspended in minimal essential medium-a (without serum) at a

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Functional Nonequivalence of NK-1 and NK-2 Receptors

concentration of 1 X lo6 cells/ml. Aliquots of the cell suspension (400 A pl) were preincubated at 37 “C for 5 to 15 min, after which onewild-type NK-1 Receptor quarter volume 5 X binding buffer (as above with protease inhibitors, but with media rather than PBS) with or without agonist was added. NKlNKZC3 For experiments using 3-isobutyl-1-methyl-xanthine (IBMX), preincubations with 1 mM IBMX were performed for 20 min at 37 “Cprior NKI/NK20 to agonist addition as above. After the indicated incubation time, cells were lysed by addition of 0.5 volume of 3 M trichloroacetic acid. Cell lysates were centrifuged for 5 min, and the supernatants were extracted five to six times with 2 X volume diethyl ether. After wild-type NK-2Rccepta removal of residual ether by incubation at 65 “C, the extracts were dried and one-eighth of the sample (representing 5 X lo4 cells) was NKmlC3 subjected to a cAMP radioimmunoassay. Cell Stimulation and ZP Assays-The accumulation of total 13H] NKWJKlCl’ IPS was measured in the presence of LiCl to inhibit IP, monophosphatase by a modification (Raddatz et al.)’ of the method of Berridge 8 et al. (1983). Prior to confluence, cells were labeled with myo-13H] inositol (5 pCi/ml) for 16 h. Cells were rinsed with PBS several times wild-lype N K - I Receptor and briefly treated with 2 ml of 3 mM EDTA in PBS. Cells were then triturated from the flask with a 1-ml pipettor, sedimented in a clinical NKI-A325 centrifuge, and washed with Delbecco’sPBS containing 0.9 mM CaCl, 1 mg/mI glucose, and 20 mM HEPES, pH = 7.4. After sedimenting for the second time, the cells were resuspended in Delbecco’s PBS containing 0.9 mM CaCl, 1 mg/ml glucose, 20 mM HEPES, and 20 mM Licl, pH = 7.4, at a concentration of 3.5 X lo5 cells/ml. LiCl at wild-type NK-2 Rcaptor 20 mM has been determined empirically t o be sufficient to prevent NK2-A367 IPl degradation in these cells. Aliquots of the cell suspension (400 d ) were preincubated at 37 “Cfor 15 min, after which one-quarter volume NKZ-A347 5 X binding buffer (complete with protease inhibitors listed above, but made with Delbecco’s PBS with Ca2+,glucose, HEPES, and Li’ NK2-A321 as above) with or without agonist was added. After the appropriate incubation time, cells were lysed by addition of 3 volumes chloroform/ FIG. 1. Schematic representations of tachykinin receptor methanol (1:2),vortexed, and incubated on ice for 30-120 min, after which 0.5 ml ofwater and 1ml of chloroform were added, and samples constructs usedin this study.A , wild-type and chimeric NK-1 and were vortexed briefly and incubated at room temperature for 30 min. NK-2 receptors. B, wild-type and truncated NK-1 and NK-2 recepThe aqueous fraction was recovered and incubated at 50 “C for 10- tors. Receptors are illustrated with the seven predicted transmem15 min to remove residual chloroform. Anion-exchange resin (0.5 ml, brane regions depicted as boxes and the putative palmitoylation site approximately 50% weight/volume slurry in water) was added to the depicted as a dark line extending from the receptor just after the 7‘h extracts, vortexed, and the resin allowed to settle. The supernatant transmembrane domain. The C3 domain is contained between the 5th was discarded, and theresin was washed five times with 3 ml of water and Bth predicted transmembrane regions, and the CT domain, as containing 5 mM myo-inositol. After the last wash, the supernatant defined here, begins after the putative palmitoylation site. The was discarded and [3H]IPs were eluted in 2 ml of 1.5 M ammonium cDNAs encoding chimeric receptors were constructed, sequenced, and formate in 100 mM formic acid. The eluent was added to 5 ml of high subcloned into anexpression vector as described under “Experimental salt capacity Tru Count scintillation fluid, and radioactivity was Procedures.” Stable CHO cell lines expressing wild-type, chimeric, or truncated receptors were established, as described under “Experimendetermined in a scintillation counter. tal Procedures,” and used for the present studies. RESULTS

high affinity agonist binding sites and similar magnitude IP Comparison of Wild-type versus Chimeric NK-1 and NK-2 responses, t h e cell line expressing wild-type NK-2 receptors Receptors Suggests a Nonequiualence of Structurally Homolo- had a cAMP response three times greater than the cell line gous Domains-Fig. lA shows a schematic representation of expressing wild-type NK-1 receptors, suggesting a quantitawild-type and chimeric receptor proteins predicted to be en- tive difference inthe functional propertiesof these two recepcodedbycDNAs that weregenerated as describedunder tors (Fig. 2 and summarized in Table I). This difference is “Experimental Procedures.’’ Each cDNA was subcloned into apparently not due to a more rapid degradation of ligand pM2, an expression vector (Matzuk et al., 1987), and trans- because restimulation of either cell line witha second doseof et fected intoCHO cells. As described previously (Takeda al., agonist failed to elicita second response (Takedaet al., 1992; 1992) and under “Experimental Procedures,” stably transHershey, 1991). Also consistent with previous studies (Naselected by resistance to G418 kajimaet al., 1992; Takeda et al., 19921, clonalcelllines fected clonal cell lines were (Geneticin), and screenedbyagonistbindingusing lz5I- expressing wild-type receptors, and each group of chimeric Tyr”SP or 1261-NPrfor NK-1 or NK-2 receptors, respecreceptors that had detectable second messenger responses, a subsequent showed a positive correlation between the level tively. Fromthe estimation of binding sites from of high affinity chosen to be assayed fora more detailed receptor expression and magnitude of second messenger rescreen, cell lines were examination of ligand binding properties by Scatchard analysponses, and ECL;s values did not change with changes in the sis (1957) and for agonist-induced cAMP increases and IP number of agonist binding sites. Hence, the cell lines expressproduction as described under “Experimental Procedures.” ing wild-type receptors that were chosen for this study had Wild-type NK-1 a n d NK-2 receptor expressing CHO cell binding and functional properties similarto those previously linescontainingapproximatelythesamenumber of high described for NK-1 and NK-2 receptors expressed in trans(3.6 x lo5 and 3.4 x lo5 sites/cell, affinitybindingsites fected C H O cells. respectively) were chosen as standards for comparison. ConFor each chimera, two to four of the highest expressing sistent with our previous studies (Takeda et al., 1992), we clonalcelllineswereanalyzedforsecondmessengerrefound that although these cell lines had a similar number of sponses. 100 nM SP or 1 g~ NKA were used for stimulation R. Raddatz, P. Blount, R. M. Snider, and J. E. Krause, manuscript experiments because these concentrations have been shown to be saturating (Takeda et al., 1992; Hershey, 1991). All in preparation.

of NU-1 and NK-2 Receptors

Functional Nonequiualence

25

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-

-

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FIG. 2. Analysis of second messenger responses of wild-type and chimeric NK-1 and NK-2 receptors. Levels of cAMP (leftpanels) and IPS (right panels) were measured subsequent to stimulation with either 100 nM SP for the wild-type NK-1 (open squares), NKl/NK2.C3 (filled triangles), and NKl/NKP.CT (filledcircles) receptors (top panels) or 1 p~ NKA for the wildtype NK-2 (open squares), NK2/ NKl.C3 (filled triangles) and NK2/ NK1.CT (filkd circles) receptors (bottom panels) as described under “Experimental Procedures.” The inset of the bottom left panel shows the data for the NK2/ NK1.CT receptor on a more appropriate scale for the y axis. cAMP levels are presented in fmol/5 X lo4 cells; values for IPS are presented as percent total cell labeling which ranged from 8.4 X lo3 to 2.7 X lo4 counts/min/l.4 X lo6 cells. Shown are the average and S.D. or range of two to four independent experiments.

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clonal cell lines analyzed within each group gave consistent patterns of second messenger responses suggesting that the phenomena observed are not due to the chromosomal insertion site of the transfected DNA. In addition, basal levels of IPS andcAMP were indistinguishable from the parental and wild-type receptor expressing cell lines and were similar to previously published levels (Takeda et al., 1992). Data obtained from one fully analyzed clone from each group, chosen because of its level of expression of high affinity binding sites, are presented in Fig. 2 and summarized in Table I. All cell lines expressing chimeric receptors also expressed binding sites with affinities for the appropriate agonist that were indistinguishable from wild-type, demonstrating the expression of potentially functional receptors (Table I); all data fit well a model for a single population of high affinity agonist binding sites. Analysis of second messenger responses of these cell lines expressing chimeric receptors demonstrated that not all of these engineered receptors were as effective at stimulating second messenger responses as wild-type receptors (Fig. 2 and summarized in Table I). For example, although expressing 30% more high affinity agonist binding sites than wild-type receptor, a clone expressing NK-1 receptor with the NK-2 C3 domain (referred toas NKl/NKZ.C3) had no detectable cAMP response and a substantiallysmaller IPS response than wild-type receptor. By contrast, the clonal cell lines expressing the complimentary chimera, NKZ/NKl.C3, appeared to have responses similar to the wild-type NK-2 receptor. Although the NKZ/NKl.C3 clonal cell line shown in Fig. 2 and summarized in Table I expressed only 40% as many binding sites as wild-type NK-2 receptor, it had a cAMP response 74%, and an IPS response 140% that of wild-type NK-2 receptor. Hershey (1991)previously demonstrated that the NK-1 receptor with the NK-2 receptor C T domain (NKl/NKZ.CT) stably expressed in CHO cells had agonist-dependent increases in

30

(mln)

IPS, asdetermined by a IPSmass radioreceptor assay, similar to that of cell lines expressing the wild-type NK-1 receptor. Consistent with, and extending these results, we found that relative to wild-type this and other NKl/NKZ.CTexpressing cell lines had slightly increased IPS responses and substantially increased cAMP responses (data of one cell line is shown in Fig. 2 and summarized in Table I). However, all three clonal cell lines characterized from this group expressed more high affinity agonist binding sites than did the wild-type control; therefore, the increase in responsiveness observed in these cell lines may either reflect a change in receptor properties or simply the expression of more functional receptors. Nonetheless, analysis of cell lines expressing the complimentary chimera, NKZ/NKl.CT, suggested that thisreceptor was severely dysfunctional in its cAMP response, even though the expression level of binding sites was twice that for cell lines expressing wild-type NK2 receptors and IPS responses were similar to wild-type. Increasing the concentration of the agonist NKA to 10 p M , or stimulating cells with 1 ” NP7, a different NK-2 receptor agonist, did not increase the cAMP response for the NKZ/NKl.CT clones (not shown), suggesting that thedecreased responsiveness is not due to a large change in E G Oof the ligand, nor is it ligand-specific. Collectively, these data indicate that structurally homologous domains of the rat NK-1 and NK-2 receptor do not have functionally equivalent roles in transfected CHO cells. Comparison of WiM-type and Truncated NK-1 and NK-2 Receptors Suggests That the CT Domain of the NK-2 Receptor Plays a Role in G-protein Activation-The data presented thus far demonstrate the nonequivalence of the C3 and CT domains in the NK-1 and NK-2 receptors, and implicate the NK-1 receptor C3 domain and theNK-2 receptor CT domain in G-protein activation. An alternative hypothesis is that the NK-1 CTand/or NK-2 C3 domains inhibit receptor respon-

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Functional Nonequivalence of NK-1 and NK-2 Receptors

TABLEI Summary of radioligand bindingand second messenger response characteristics of wild-type, chimeric, and truncated NK-1 and NK-2 receptors

previously by Hershey (1991) who found that stably transfected CHO cells expressing either the wild-type NK-1 or NKl-A325 receptor displayed similar agonist-induced mass IP, responses. By contrast, a relatively small deletion of the Bindinf Respond CT domain of the NK-2 receptor (NK2-A367; 23 amino acids Cell type Binding deleted) substantially decreased the magnitude of second mesIPS Kd sites senger responses, especially CAMP, in a clonal cell line exrelotive pressing 2.6 times as many high affinity agonist binding sites receptor expressed nhf fold increase to W t as thewild-type NK-2 receptor. Consistent with these obserNK-1 wild-type 0.4 f 0.2 1.0 5.7 f 1.7 6.0 f 0.3 vations, a larger CT deletion (NK2-A347) resulted in expresNKl/NK2.C3 0.4 f 0.3 1.3