Ogoshi M et al. (2021), Adrenomedullin 2 and 5 activate the calcitonin ...

XB-ART-57921

Gen Comp Endocrinol 2021 May 15;306:113752. doi: 10.1016/j.ygcen.2021.113752.

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Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

Ogoshi M , Takahashi M , Aoyagi K , Ukena K , Aizawa S , Takeuchi H , Takahashi S , Takeuchi S .

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The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

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Species referenced: Xenopus tropicalis
Genes referenced: adm adm2 calca calcrl camp dtl ramp1 ramp2 ramp3
GO keywords: adrenomedullin receptor activity [+]

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	Fig. 1. Multiple alignment of amino acid sequences. A: Precursors of adrenomedullins (AMs) in Xenopus tropicalis. Putative mature sequences are boxed. The intramolecular ring structure formed by a disulfide bond is indicated by a square bracket. The arrowhead shows the amidation signal at the C-terminus of putative mature peptide. B-D: Full-length precursors of calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 2 (RAMP2), and RAMP3 in Xenopus, human, and mouse. Predicted transmembrane domains are shadowed. The intramolecular disulfide bonds are indicated by square brackets.
	Fig. 1. Multiple alignment of amino acid sequences. A: Precursors of adrenomedullins (AMs) in Xenopus tropicalis. Putative mature sequences are boxed. The intramolecular ring structure formed by a disulfide bond is indicated by a square bracket. The arrowhead shows the amidation signal at the C-terminus of putative mature peptide. B-D: Full-length precursors of calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 2 (RAMP2), and RAMP3 in Xenopus, human, and mouse. Predicted transmembrane domains are shadowed. The intramolecular disulfide bonds are indicated by square brackets.
	Fig. 1. Multiple alignment of amino acid sequences. A: Precursors of adrenomedullins (AMs) in Xenopus tropicalis. Putative mature sequences are boxed. The intramolecular ring structure formed by a disulfide bond is indicated by a square bracket. The arrowhead shows the amidation signal at the C-terminus of putative mature peptide. B-D: Full-length precursors of calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 2 (RAMP2), and RAMP3 in Xenopus, human, and mouse. Predicted transmembrane domains are shadowed. The intramolecular disulfide bonds are indicated by square brackets.
	Fig. 2. Phylogenetic tree showing relationships of (A) adrenomedullins (AMs), (B) calcitonin receptor-like receptor (CLR) and its subtypes, (C) receptor activity-modifying proteins (RAMPs) between vertebrate lineages depicted by the minimum evolution method. Predicted mature sequences were used in panel A, and full-length precursor sequences were used in panel B and C. Sequences of western clawed frogs (Xenopus tropicalis), The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The scale bar indicates 0.1 (A, B) or 0.2 (C) amino acid replacements per site. The sequences of CLRs and chordate RAMPs were retrieved from databases according to Martins et al., 2014, and Sekiguchi et al., 2016, respectively. GenBank accession numbers are as follow: Xenopus tropicalis (western clawed frog) AM1 (XM_002936741), Xt AM2 (XM_002939371), Xt AM5 (XM_002934624), Homo sapiens (human) AM1 (NM_001124), human AM2 (NM_001369882), Mus musculus (mouse) AM1 (NM_009627), mouse AM2 (NM_182928), Sus scrofa (pig) AM1 (NM_214107), pig AM2 (NM_001206384), pig AM5 (AB287333), Canis lupus familiaris (dog) AM1 (NM_001003183), dog AM2 (XM_038488204), dog AM5 (XM_038656297), Takifugu rubripes (fugu) AM1 (AB120295), fugu AM2 (AB120296), fugu AM5 (AB120299), Oryzias latipes (medaka) AM1 (NM_001104889), medaka AM2 (NM_001104890), medaka AM5 (NM_001104893), Anguilla japonica (Japanese eel) AM1 (AB363985), eel AM2 (AB363986), eel AM5 (AB363988), Eptatretus burgeri (Pacific hagfish) AM (EU807737), Pertomyzon marinus (sea lamprey) AM (EU807738), Xt CLR (LC584178), human CLR (NM_005795), mouse CLR (NM_018782), pig CLR (NP_999260), dog CLR (XP_545560), Takifugu obscurus (obscure pufferfish, mefugu) CLR1 (AB219835), mefugu CLR2 (AB219837), mefugu CLR3 (AB219838), fugu CLR1 (XP_029690338), fugu CLR2 (XP_003966761), fugu CLR3 (XP_011620039), medaka CLR1 (XP_004081714), medaka CLR2 (XP_004066432), medaka CLR3 (XP_023812105), Caenorhabditis elegans CLR (NP_001021171), Xt RAMP2 (LC584179), Xt RAMP3 (LC584180), human RAMP1 (NM_005855), human RAMP2 (NM_005854), human RAMP3 (NM_005856), mouse RAMP1 (NM_016894), mouse RAMP2 (NM_019444), mouse RAMP3 (NM_019511), pig RAMP1 (NM_214199), pig RAMP2 (NM_214082), pig RAMP3 (NM_214089), dog RAMP1 (XM_038435918), dog RAMP2 (XM_038676403), dog RAMP3 (XM_038689253), mefugu RAMP1 (AB219765), mefugu RAMP2a (AB219766), mefugu RAMP2b (AB219767), mefugu RAMP3 (AB219768), mefugu RAMP4 (AB219770), mefugu RAMP5 (AB219771), fugu RAMP1 (XM_003962104), fugu RAMP2a (XM_003964789), fugu RAMP2b (XM_011604154), fugu RAMP3 (XM_029843046), fugu RAMP4 (XM_003966521), fugu RAMP5 (XM_003961068), medaka RAMP1 (XM_004081990), medaka RAMP2 (XM_004071305), medaka RAMP3 (XM_004080950), medaka RAMP5 (XM_004080724), Branchiostoma floridae (Florida lancelet) RAMP1 (AB979722), Bf RAMP2 (AB979723), Bf RAMP3 (AB979724).
	Fig. 3. The conserved synteny around calcrl (clr), ramp2, and ramp3 among the chromosomes of mammals, amphibians, and teleost fish. The chromosome of fugu instead of medaka is shown for ramp3 because ramp3 was not detected in the genome database for medaka. The numbers indicate the location on each chromosome.
	Fig. 4. Effects of Xenopus am1, am2, and am5 on CRE-luciferase reporter activity in HEK293T cells overexpressing clr-ramp2, clr-ramp3, and clr only. Cells were transiently transfected with plasmids for expression of receptor components plasmids in combination with CRE-Luc reporter plasmids. At 24 h post-transfection, the cells were treated with various doses of am1, am2, or am5 for 2 h. The luciferase activities of mock-transfected cells treated with 10−6 M ligands are plotted in each panel. All data shown are means ± SEM (n = 6). P < 0.05, P < 0.01, **P < 0.001.
	Fig. 5. Relative mRNA expression levels of am1, am2, am5, calcrl, ramp2, and ramp3 in various tissues of mature western clawed frogs. Relative mRNA levels are shown as the ratio to that in the liver and described as means ± SEM (n = 5–6). Different lowercase letters on the bars indicate significantly different values (P < 0.05). The mRNA of actb was used as a reference gene to normalize the expression level of each genes. ND: not detected.