Molecular cloning of an anuran V 2 type [Arg 8] vasotocin receptor and mesotocin receptor: functional characterization and tissue expression in the Japanese tree frog ( Hyla japonica)

Abstract

In most amphibians, [Arg 8] vasotocin (VT) has an antidiuretic effect that is coupled to the activation of adenylate cyclase. In contrast, mesotocin (MT) has a diuretic effect and acts via the inositol phosphate/calcium signaling pathway in amphibians. To further clarify the mechanisms of VT and MT activation, we report the molecular cloning of a VT receptor (VTR) and a MT receptor (MTR) from the Japanese tree frog, Hyla japonica. Tree frog VTR or MTR cDNA encoded 363 or 389 amino acids, and their amino acid sequences revealed close similarity to the mammalian vasopressin V 2 (51–52% identity) or toad MT (94% identity) receptors, respectively. Using CHO-K1 cells transfected with tree frog VTR, we observed elevated concentrations of intracellular cAMP following exposure of the cells to VT or other neurohypophysial hormones, whereas the cells transfected with MTR did not exhibit altered cAMP concentrations. The cells transfected with VTR exhibited the following efficiency for cAMP accumulation: VT= hydrin 1 ≧ vasopressin ≧ hydrin 2 > MT= oxytocin> isotocin . VTR or MTR mRNA exhibits a single 2.2- or 5.5-kb transcription band, respectively, and both are expressed in various tissues. VTR mRNA is clearly expressed in brain, heart, kidney, pelvic patch of skin, and urinary bladder, whereas brain, fat body, heart, kidney, and urinary bladder express MTR mRNA. Specifically, VTR mRNA in the pelvic patch or MTR mRNA in the dorsal skin is present at elevated levels in the skin. Characteristic distribution of VTR and MTR on osmoregulating organs indicates the ligands for these receptors would mediate a variety of functions. Further, the distribution of VTR in the skin would make the regional difference on cutaneous water absorption in response to VT in the Japanese tree frog.