Molecular identification and characterization of three isoforms of tachykinin NK1-like receptors in the cane toadBufo 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 NK1-like receptor isoforms, bNK1-A, bNK1-B, and bNK1-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 bNK1-A and bNK1-B isoforms are similar to the truncated and full-length forms of the mammalian NK1receptor, whereas bNK1-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 (IC500.6–0.8 nM) in competing for125I-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, bNK1-A, was inactive, whereas bNK1-B and bNK1-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 NK1receptor. In summary, our study describing three isoforms of NK1-like receptor from the toad suggests that the alternative splicing of NK1receptor is a physiologically conserved mechanism and raises a fundamental question as to the physiological role of each isoform.