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 PC R from a template of poly(A)-rich RNA isolated from the eel gill. Sequ encing of the PCR products revealed the presence of a new clone that s howed about 70% sequence identity to the eel type-C receptor, NPR-C. T he PCR fragment was used to determine the tissue distribution of the n ew NPR-D message by an RNase protection assay, which gave the stronges t 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 transm embrane segment. The predicted sequence also contained the NPR motif w hich is essential for the binding of natriuretic peptides. The protein NPR-D was expressed in COS cells and shown to have high affinities fo r 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 a nd form a subfamily of the NPR family. Affinity labeling indicated tha t NPR-D exists a disulfide-linked tetramer. This is a marked contrast to the homodimeric structure of NPR-C. HS-142-1, a non-peptide natriur etic 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 I-125-labeled eel natriu retic peptide to eel NPR-D, whereas it did not affect the binding acti vity of eel NPR-C, suggesting that HS-142-1 is an antagonist that reco gnizes the tetrameric structures of NPR since the guanylate-cyclase-co upled receptors have also been demonstrated to exist as tetramers.