Molecular and functional characterization of adrenomedullin receptors in pufferfish.
The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AM1 (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AM1), CLR2-RAMP2 (AM1), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells).
Nag
Kakon
K
Department of Biological Sciences, Tokyo Institute of Technology,Yokohama 226-8501, Japan.
Kato
Akira
A
Nakada
Tsutomu
T
Hoshijima
Kazuyuki
K
Mistry
Abinash Chandra
AC
Takei
Yoshio
Y
Hirose
Shigehisa
S
D053607:Adrenomedullin; D000595:Amino Acid Sequence; D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002460:Cell Line; D005880:Gills / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010802:Phylogeny; D058265:Receptors, Adrenomedullin; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D023721:Tetraodontiformes / Q000378:metabolism*
D010455:Peptides; D058265:Receptors, Adrenomedullin; D018000:Receptors, Peptide; D053607:Adrenomedullin; D015740:Calcitonin Gene-Related Peptide