Cloning and properties of a novel natriuretic peptide receptor, NPR-D.
A novel natriuretic peptide receptor, which we have termed natriuretic peptide receptor D (NPR-D), has been cloned and characterized. cDNAs related to the natriuretic peptide receptor (NPR) were amplified by PCR from a template of poly(A)-rich RNA isolated from the eel gill. Sequencing of the PCR products revealed the presence of a new clone that showed about 70% sequence identity to the eel type-C receptor, NPR-C. The PCR fragment was used to determine the tissue distribution of the new NPR-D message by an RNase protection assay, which gave the strongest signal in brain samples, and then used to screen a brain library to obtain a full-length cDNA clone. The cDNA clone predicted a protein of 500 amino acids containing a signal sequence and a hydrophobic transmembrane segment. The predicted sequence also contained the NPR motif which is essential for the binding of natriuretic peptides. The protein NPR-D was expressed in COS cells and shown to have high affinities for eel and rat natriuretic peptides. The newly cloned NPR-D has a short cytoplasmic tail; in this respect, NPR-C and NPR-D are very similar and form a subfamily of the NPR family. Affinity labeling indicated that NPR-D exists as a disulfide-linked tetramer. This is a marked contrast to the homodimeric structure of NPR-C. HS-142-1, a non-peptide natriuretic peptide receptor antagonist of microbial origin previously shown to be selective for the guanylate-cyclase-coupled receptors NPR-A and NPR-B, competitively inhibited the binding of 125I-labeled eel natriuretic peptide to eel NPR-D, whereas it did not affect the binding activity of eel NPR-C, suggesting that HS-142-1 is an antagonist that recognizes the tetrameric structures of NPR since the guanylate-cyclase-coupled receptors have also been demonstrated to exist as tetramers.
Kashiwagi
M
M
Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan.
Katafuchi
T
T
Kato
A
A
Inuyama
H
H
Ito
T
T
Hagiwara
H
H
Takei
Y
Y
Hirose
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004524:Eels / Q000235:genetics* / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000235:genetics; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C