Proc. Natl. Acad. Sci. USA Vol. 87, pp. 246-250, January 1990 Neurobiology

y-Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors (substance P/receptor subtypes/autoradiography/binding sites)

THAN-VINH DAM*tt, YASUO TAKEDA§, JAMES E. KRAUSE§, EMANUEL ESCHERt, AND RlMI QUIRION*t¶ *Douglas Hospital Research Centre and tDepartment of Psychiatry, McGill University, 6875 LaSalle Boulevard, Verdun, PQ, Canada H4H 1R3; tDdpartement de Pharmacologie, Facultd de Mddecine, Universitd de Sherbrooke, Sherbrooke, PQ, Canada JIK 5N4; and §Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, MO 63110

Communicated by Gerald D. Fischbach, July 31, 1989

remain fairly constant, with y-PPT mRNA representing up to 80% of total PPT-I-derived mRNA, p-PPT mRNAs representing '20%, and a-PPT mRNA representing a trace amount (21, 23). It has been shown that all these three PPT-I mRNAs are translatable (24) and can potentially generate multiple biologically active peptides, in addition to the three previously characterized mammalian NKs. For example, it has been demonstrated that a biologically active 21-amino acid peptide, 'y-PPT-(72-92)-peptide amide [y-PPT-(7292)-NH2] (25), is found in rabbit intestine (26) and in the rat CNS (Y.T. and J. E. Krause, unpublished results). However, it is not known if this peptide interacts with known NK receptors or another class of sites to induce its biological effects (25). Similarly, neuropeptide K (NPK), an extended NK polypeptide derived from P-PPT (27, 28), has also been shown to produce various biological effects (27, 29), although its receptor-selective profile is not fully established (30). In this study, 125I-labeled (125I_) y-PPT-(72-92)-NH2 was used to determine the nature of putative 'y-PPT-(72-92)-NH2 binding sites in CNS.

The presence of N-terminally extended forms ABSTRACT of neurokinin A has recently been reported in the mammalian brain. Among them, y-preprotachykinin-(72-92)-peptide amide [y-PPT-(72-92)-NH2], a peptide derived by posttranslational processing of y-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and y-PPT-(72-92)-NH2 are potent competitors of '251-labeled y-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of '2SI-labeled y-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 1251-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and '251-labeled Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that y-PPT-(72-92)-NH2 binds to neurokinin2 receptors and should be considered as a putative endogenous ligand for this receptor class.

The existence of multiple endogenous tachykinins in brain and peripheral tissues is well established (for reviews, see refs. 1-3). Apart from substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have been identified in mammalian brain and peripheral tissues (1-3) and others such as eledoisin (ED), physalaemin, and kassinin have been isolated from amphibians (4). The biological effects of these peptides are mediated by highly specific receptors located on target cells and much evidence has now shown the existence of at least three major classes of neurokinin (NK) receptors, NK-1, NK-2, and NK-3 (for reviews, see refs. 3, 5-10). NK-1 receptors have higher affinity for SP, whereas NKA and NKB preferentially act on NK-2 and NK-3 sites, respectively. However, there are some controversies concerning the existence of NK-2 sites in certain tissues (7, 11-17). The three mammalian NKs, SP, NKA, and NKB, are derived from preprotachykinin I and II genes (PPT-I and -II genes), two different but related genes (1, 3, 18-22). The RNA transcribed from the PPT-I gene is alternatively spliced to yield three different mRNAs encoding the a-, 13-, and 'y-preprotachykin (a-, 13-, and y-PPT, respectively) precursors (1, 3, 19, 21). SP may arise from the posttranslational processing of a-, ,3-, or y-PPT whereas NKA is generated by the processing of 8- or y-PPT (1, 3, 19, 20-22). NKB is exclusively derived from the PPT-II gene (1, 3, 20). The ratio between SP and NKA varies according to the differential splicing of the PPT-I mRNAs in various brain and peripheral tissues (19, 21). In the rat central nervous system (CNS), the relative amounts of a-, ,B-, and y-PPT mRNAs

MATERIALS AND METHODS Synthesis of y-PPT-(72-92)-NH2 and '251-y-PPT-(7292)-NH2. The synthesis and purification of y-PPT-(7292)-NH2 has been reported (25). The purity of this peptide was >90% for these studies and the primary structure was verified by sequence analysis on an Applied Biosystems model 470A protein sequencer. Peptide concentration was determined by amino acid compositional analysis and verified by optical density at 210 nm. The radiolabeling of y-PPT-(72-92)-NH2 with 1251 was performed by the chloramine-T method (31). The purification of 1251-_y-PPT-

(72-92)-NH2 by HPLC, the determination of the sites of radioiodination (histidyl residues at positions 4, 9, and 12) and the ligand-specific radioactivity was determined as described (Y.T. and J.E.K., unpublished results). Receptor Binding Assays in Membrane Preparations. For the membrane binding assays, the method used was essentially as described (32). Male Sprague-Dawley rats (250300 g; Canadian Breeding Farm, Saint Constant, PQ) were decapitated and their brains minus cortex and cerebellum [for 125I-y-PPT-(72-92)-NH2 and 125I-NKA] or the cortex only [for 125I-Bolton-Hunter reagent-labeled (125I-BH) ED (125I-BHED)/NK-3] were used for assays (32). After the final cenAbbreviations: SP, substance P; NK, neurokinin; ED, eledoisin; PPT, preprotachykinin; NPK, neuropeptide K; CNS, central nervous system; BH, Bolton-Hunter; 251-, 1251-labeled; y-PPT(72-92)-NH2, y-PPT-(72-92)-peptide amide; Sar, sarcosine. 11To whom reprint requests should be addressed at: Douglas Hospital Research Centre, 6875 LaSalle Boulevard, Verdun, PQ, Canada H4H 1R3.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 87 (1990)

trifugation, pellets were washed three times with 50 mM Tris HCl (pH 7.4) at 40C (buffer A) before final resuspension in 60 vol of this buffer. Aliquots were taken for protein determination (33). Membrane suspension (0.2 ml) was incubated for 90 min at 250C in 0.4 ml of a buffer containing 50 mM Tris-HCI (pH 7.4), 3 mM MnCI2, 0.02% bovine serum albumin (ICN), bacitracin (40,ug/ml; Sigma), chymostatin (2 ,ug/ml; Sigma), and leupeptin (4 Ag/ml; Sigma) (buffer B) plus 25 pM 125I-y-PPT-(72-92)-NH2 (2175 Ci/mmol; 1 Ci = 37 GBq), 25 pM 125I-NKA (2200 Ci/mmol; Amersham), 25 pM 1251-BH-ED (2200 Ci/mmol; New England Nuclear) or various concentrations of 125I-y-PPT-(72-92)-NH2 for saturation experiments. Nonspecific binding was determined in the presence of 1 ,uM y-PPT-(72-92)-NH2, 1 ,uM NKA, or 1 ,uM ED, respectively. Incubations were terminated as described (32). Binding data were analyzed using a computerized program (BioSoft, Elsevier). Receptor Autoradiography. For receptor autoradiography, rat brain sections were prepared as described (11, 32). Tissue sections were incubated for 90 min at 25°C in buffer B containing =25 pM 125I-y-PPT-(72-92)-NH2, 50 pM 125I[ BH-SP, 50 pM 1251-NKA, or 50 pM 125I-BH-ED. Specific binding was determined as the difference in binding observed in the presence of 1 ,uM ry-PPT-(72-92)-NH2, 1 aM SP, 1 ,uM NKA, or 1 ,M ED, respectively. Incubations were terminated as described (32). Incubated slides were juxtaposed tightly against tritium-sensitive film and stored at room temperature as follows: 10 days for 125I-y-PPT-(72-92)-NH2, 7 days for 125I-BH-SP, 30 days for 125I-NKA, and 20 days for 1251-BH-ED. Films were developed and analyzed as described (32).

RESULTS Characterization of '251-y-PPT-(72-92)-NH2 Binding Sites in Rat CNS. 125I-y-PPT-(72-92)-NH2 bound with high affinity (pM range) to a low density of specific sites in rat brain membranes prepared from subcortical regions (Kd = 0.46 nM; Bmax = 4.17 fmol/mg of protein). Table 1 reveals the relative potency of various tachykinins in competing for 125I-y-PPT-(72-92)-NH2 binding in rat brain membrane preparations. The ligand selectivity pattern shows that y-PPT(72-92)-NH2 > NKA > ED > SP. Thus y-PPT-(72-92)-NH2 was the most potent peptide to compete for 1251-y-PPT(72-92)-NH2 binding with an IC50 of 5.1 nM. NKA and ED had 2-3 times lower potencies whereas SP was much weaker. Furthermore, the selective NK-1 {[Sar9, Met(02)11]SP, where Sar is sarcosine} and NK-3 ([MePhe7]NKB) ligands (34) were virtually inactive (IC50 > 1.0 ,uM) in competing for these sites. y-PPT-(72-92)-NH2 was also very potent against 1251_ NKA binding as were unlabeled NKA and ED, whereas SP was very weak (Table 1). Finally, we also observed that Table 1. Relative potency of various tachykinins in competing for brain '251-y-PPT-(72-92)-NH2 and 1251-NKA binding in rat brain membrane preparations

'251-y-PPT-(72-92)-NH2 Tachykinin y-PPT-(72-92)-NH2 NKA ED SP

IC5o, nM

1251-NKA

RP, % IC50, nM 100 2.4 ± 1.7 85 16.5 ± 4.8 7.4 + 4.7 69 14.5 ± 6.1 128 ±63 4 >1000 [Sar9,Met(02)11SP >1000 1000

gamma-Preprotachykinin-(72-92)-peptide amide: an endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors.

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-...
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