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Functional Expression of a Novel Human Neurokinin-3 Receptor Homolog That Binds [3H]Senktide and [125I-Mephe7]Neurokinin B

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Functional Expression of a Novel Human Neurokinin-3 Receptor Homolog That Binds [3H]Senktide and [125I-Mephe7]Neurokinin B Proc. Natl. Acad. Sci. USA Vol. 94, pp. 310–315, January 1997 Pharmacology Functional expression of a novel human neurokinin-3 receptor homolog that binds [3H]senktide and [125I-MePhe7]neurokinin B, and is responsive to tachykinin peptide agonists J. E. KRAUSE*, P. T. STAVETEIG,J.NAVE MENTZER,S.K.SCHMIDT,J.B.TUCKER,R.M.BRODBECK, J.-Y. BU, AND V. V. KARPITSKIY Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8108, St. Louis, MO 63110 Communicated by Avram Goldstein, Stanford University, Stanford, CA, November 8, 1996 (received for review September 21, 1996) ABSTRACT In 1992, Xie et al. identified a cDNA sequence Due to the homology among receptors in the G-protein- in the expression cloning search for the k opioid receptor. coupled receptor superfamily, several putative receptor se- When the cDNA was expressed in Cos-7 cells, binding of opioid quences have been cloned in which the natural ligand has not compounds was observed to be of low affinity and without k, been identified (4). In addition, various genetic strategies have m,ordselectivity [Xie, G.-X., Miyajima, A. and Goldstein, A. resulted in the identification of receptor-like sequences in (1992) Proc. Natl. Acad. Sci. USA 89, 4124–4128]. This cDNA which no known ligand has been determined (e.g., see ref. 5). was highly homologous to the human neurokinin-3 (NK-3) The identification of these so-called ‘‘orphan’’ receptors has receptor sequence, and displayed lower homology to NK-1 and also come about in the search for other receptors using more NK-2 sequences. This sequence was stably expressed in Chi- directed methodologies. In 1992, Xie and coworkers attempted nese hamster ovary cells, which do not express neurokinin to characterize, using an affinity enrichment procedure, the k receptors naturally, and ligand binding and second messenger opioid receptor from a human placental cDNA library (6). characteristics were compared with a human NK-3 receptor. 3 They identified a cDNA clone, called ‘‘hKIR,’’ which encoded The NK-3 receptor homolog bound [ H]senktide with a Kd of a 440 residue protein consistent with the structure of a 7 39 nM, similar to that of the NK-3 receptor. The rank order transmembrane-containing G-protein-coupled receptor of tachykinin peptides competing for [3H]senktide binding at (hereafter called NK-3 receptor homolog). This sequence was the NK-3 receptor homolog was [MePhe7]neurokinin B > highly homologous to the NK-3 subfamily of tachykinin re- senktide > substance P 5 neurokinin A > neurokinin B. This cell line also bound [125I-MePhe7]neurokinin B; however, ceptors, and had a lower identity to NK-1 and NK-2 receptors neurokinin B was an effective competitor. Tachykinin peptides (7). The sequence, when expressed in Cos-7 cells, induced stimulated both inositol phospholipid hydrolysis and arachi- binding of the opiate alkaloid bremazocine with a Kd of 87 nM. 3 donic acid release at NK-3 and NK-3 receptor homolog cell [ H]Bremazocine binding was displaced similarly by m, d, and lines, with similar rank orders of potency of [MePhe7] k selective compounds, but not substantially by the nonselec- neurokinin B 5 neurokinin B 5 senktide > NKA 5 substance tive antagonists naloxone or naltrexone. This relatively low P. These results indicate that expression of the NK-3 receptor affinity for bremazocine, coupled with a lack of appropriate k homolog cDNA in the Chinese hamster ovary cell system ligand selectivity, cast doubt on it being an opioid receptor. induces the expression of a receptor site with many similar- Indeed, a G-protein-coupled receptor has since been charac- ities but certain key differences from that of the human NK-3 terized that had the pharmacology consistent with that of a k receptor. The results are discussed with reference to the opioid receptor (8). Moreover, Xie and coworkers did not existence of a novel human tachykinin receptor. detect binding of the tachykinin peptide agonist [3H]eledoisin in Cos-7 cells transfected with hKIR, but could detect specific Over the past several years, much information has become binding in cells transfected with the rat NK-3 receptor. Con- available on the primary structures and functions of G-protein- sequently, the receptor properties of this NK-3 receptor ho- coupled peptide hormone receptors due to advances in mo- molog were uncertain and as such it was considered to be an lecular cloning strategies and in the development of potent ‘‘orphan’’ receptor. To clarify the biological relevance of this nonpeptide antagonists. One exemplary system in which this NK-3 receptor homolog, we examined the radioligand binding has occurred is that of the tachykinin peptide family. It is now and response properties of this cDNA after stable expression widely appreciated that the major tachykinin peptides, sub- in Chinese hamster ovary (CHO) cells. The results indicate stance P (SP), neurokinin A (NKA), and neurokinin B (NKB), that this cDNA sequence encodes a receptor with agonist are the primary agonists at three distinct pharmacologically binding properties and activities similar to, but not identical and molecularly characterized receptor types, called neuroki- with, the previously characterized human NK-3 receptor. nin-1 (NK-1), NK-2, and NK-3 (1–3). These receptors appear to mediate the functions of the tachykinin peptides on diverse biological processes including smooth muscle contraction, EXPERIMENTAL PROCEDURES blood pressure regulation, neuronal communication, and en- Materials. Most of the reagents used have been described (9, docrine and exocrine gland secretions. These peptides also 10). Oligonucleotides were synthesized by the Washington appear to be involved in the regulation of certain immune and University Protein and Nucleic Acid Chemistry Laboratory. inflammatory states. The PCR II vector was from Invitrogen. Maloney murine leukemia RNase H2 reverse transcriptase, called SuperScript The publication costs of this article were defrayed in part by page charge II, was from GIBCOyBRL. NKB (Asp-Met-His-Asp-Phe-Phe- payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: NKB, neurokinin B; NKA, neurokinin A; SP, sub- Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA stance P; NK-3, neurokinin-3; CHO, Chinese hamster ovary. 0027-8424y97y94310-6$2.00y0 *To whom reprint requests should be addressed: e-mail: PNAS is available online at http:yywww.pnas.org. [email protected]. 310 Downloaded by guest on September 29, 2021 Pharmacology: Krause et al. Proc. Natl. Acad. Sci. USA 94 (1997) 311 Val-Gly-Leu-Met-NH2), senktide (succinyl-Asp-Phe-MePhe- buffered saline (TBS, 0.05 M TriszHCl buffer containing 120 7 Gly-Leu-Met-NH2), and [MePhe ]NKB (Asp-Met-His-Asp- mM NaCl, pH 7.4), scraped from the tissue culture dish and Phe-Phe-MePhe-Gly-Leu-Met-NH2) were from Bachem, and resuspended in TBS. The binding reaction in a total volume of four batches of [3H]senktide (66, 66, 69, and 74 Ciymmol; 1 100 ml was initiated by adding 80 ml of the cell suspension to Ci 5 37GBq) were from DuPontyNew England Nuclear. radioligand and test competitors in 20 ml TBS containing 1 [125I-MePhe7]NKB was prepared by oxidative iodination of mgyml BSA, 0.2 mgyml bacitracin, 20 mgyml leupeptin, and 20 [MePhe7]NKB with chloramine-T and Na125I, and the mono- mgyml chymostatin. After incubation at 48C for 2 hr, the iodo form was purified by high performance liquid chroma- reactions were terminated by rapid filtration over GFyC filters tography. Na125I (2175 Ciymmol) was from DuPontyNew that had been presoaked for 2 hr in TBS containing 2% England Nuclear. Klentaq and Klentaq long and accurate were BSAy0.1% Tween-20. The filters were rinsed 4 times with 4 ml obtained from W. Barnes (Washington University School of ice-cold TBS containing 0.01% SDS. Under these conditions Medicine) (11). [3H]senktide and [125I-MePhe7]NKB-specific binding Molecular Cloning and Sequence Analysis of NK-3 and amounted to 90–95% of the total binding and to less than 5% NK-3 Homolog cDNAs. The human NK-3 receptor cDNA was of the total radioactivity. Nonspecific absorbtion to filters cloned using a reverse transcriptase–polymerase chain reac- amounted to less than 0.02% of the total radioactivity and to tion (PCR) protocol with hypothalamic RNA as the message about 5–10% of the total binding. Competition binding exper- source. Human hypothalamic tissue was obtained at autopsy (2 iments were carried out with 4 nM [3H]senktide and with 0.3 hr from death) from an adult who died from cardiac arrest, and nM [125I-MePhe7]NKB. Saturation binding experiments were was provided by the Washington University Department of carried out over a concentration range of 0.1 to 270 Pathology. The cDNA was generated as two fragments, using nM[3H]senktide. Nonspecific binding was defined with 1000- 59-CCACCATGGCCACTCTCCCAGCAGCA-39 and 59-CC- fold excess of unlabeled senktide or with 1000-fold excess of ATGACGGCCATTGCGGTGGAC-39 as forward primers unlabeled [MePhe7]NKB. In competition binding experiments and 59-TTAAGAATATTCATCCACAGAGGT-39 and 59- the competitor concentration producing 50% inhibition (IC50) GGAAGGCAAGTAGAAATGCTAG-39 as reverse primers of the radioligand binding and the Hill coefficient (nH), values (12, 13). For the first primer, an optimal Kozak sequence were determined from Hill plots of log (ByBo 2 B) versus log CCACC (14) was inserted upstream of the initiator methio- of the competitor concentration, where Bo and B are specific nine to enhance 40s ribosomal subunit binding. The PCR binding in the absence and in the presence of the competitor, products were cloned into the PCR II vector and characterized respectively.
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