title: Molecular identification and characterization of three isoforms of tachykinin NK(1)-like receptors in the cane toad Bufo marinus.
abstract: The tachykinin peptide bufokinin, isolated from the cane toad intestine, is important in intestinal and cardiovascular regulation in the toad. In this study, three tachykinin NK(1)-like receptor isoforms, bNK(1)-A, bNK(1)-B, and bNK(1)-C, encoding proteins of 309, 390, and 371 amino acids, respectively, were cloned from the toad brain and intestine. These isoforms differ only at the intracellular COOH terminus. The bNK(1)-A and bNK(1)-B isoforms are similar to the truncated and full-length forms of the mammalian NK(1) receptor, whereas bNK(1)-C is unique and does not correspond to any previously described receptor. RT-PCR studies demonstrated that three isoform transcripts are widely distributed in the toad with high expression in gut, spinal cord, brain, lung, and skeletal muscle. When expressed in COS-7 cells, bufokinin showed similar high affinity (IC(50) 0.6-0.8 nM) in competing for (125)I-labeled Bolton-Hunter bufokinin binding at all receptors, but the binding affinities of substance P (SP) and neurokinin A (NKA) were very different at each isoform. When expressed in Xenopus oocytes, the truncated isoform, bNK(1)-A, was inactive, whereas bNK(1)-B and bNK(1)-C produced changes in chloride current when stimulated by tachykinins (minimum concentrations: bufokinin, 0.1 nM; SP, 1 nM; and NKA, 10 nM). A marked desensitization of the response was seen to subsequent applications of tachykinins, as experienced by the mammalian NK(1) receptor. In summary, our study describing three isoforms of NK(1)-like receptor from the toad suggests that the alternative splicing of NK(1) receptor is a physiologically conserved mechanism and raises a fundamental question as to the physiological role of each isoform.
authors.affiliation: School of Medical Sciences, Univ. of New South Wales, Sydney 2052, Australia. lu.liu@unsw.edu.au
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.forename: Irit
authors.initials: I
authors.lastname: Markus
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Vandenberg
authors.forename: Brett A
authors.initials: BA
authors.lastname: Neilan
authors.forename: Michael
authors.initials: M
authors.lastname: Murray
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Burcher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002024:Bufo marinus; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005260:Female; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D008024:Ligands; D020033:Protein Isoforms; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1