Molecular identification and characterization of three isoforms of tachykinin NK(1)-like receptors in the cane toad Bufo marinus.

摘要

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.