Activation by C-type natriuretic peptide (CNP) of its receptor NPRB results in venodilation and inhibition of cellular proliferation. NPRB-selective antagonists should be useful to understand their physiological implications. We previously observed that Thr 9,Ser 11,Arg 16(N,C-ANP)pBNP (P12) is an antagonist for bNPRB and a potent agonist for bNPRA. The antagonist Ser 11(N-CNP,C-ANP)pBNP(2-26) (P18) displays six-fold selectivity towards hNPRB versus hNPRA. Deletion of the C-terminus in Ser 11(N-CNP,C-ANP)pBNP(2-25) (P19) decreases its affinity for hNPRA but improves its selectivity 35-fold. Peptide libraries based on P19 using phage display methodology yielded two positive clones P20 and P21. P19 behaves as the most potent antagonist, but P20 is the most selective.
The natriuretic peptides ANP, BNP and CNP are involved in the control of natriuresis, diuresis, blood pressure and vasodilation. They are characterized by a disulfide-bridged 17 amino acid loop [13], [28]. Natriuretic peptides interact with three different receptor subtypes. The natriuretic peptide receptor-A (NPRA) and the natriuretic peptide receptor-B (NPRB) consist of a single transmembrane domain, a kinase homology domain, a hinge region, and a guanylyl cyclase domain [17], [25], [31]. Most of the biological activities of the natriuretic peptides are mediated by cGMP [13], [25]. NPRA binds ANP and BNP [13], while CNP selectively binds NPRB [14]. The natriuretic peptide receptor-C (NPRC) is a disulfide-bridged homodimeric membrane protein which contains only a short cytoplasmic domain an which lacks guanylyl cyclase activity. It binds all three peptides with similar affinity and serves as a clearance receptor [2], [27].
Only one structure–activity relationship study has been reported for CNP [8], documenting the role of residues Leu 10, Lys 11, Leu 13 in the specific interaction with NPRB. Physiological studies based on an antagonist have been restricted to the non-selective microbial polysaccharide HS-142-1 [21], [23], [30]. We previously reported that HS-142-1 allosterically interacts with natriuretic peptide receptors to antagonize their activation [24]. Therefore, we considered the need to develop a NPRB-selective peptide with direct competitive antagonist properties. Such a peptide should more specifically oppose CNP-induced venodilation and therefore should be a useful tool to assess the pathophysiological roles of CNP, e.g. in septic shock [9].
In order to develop such an antagonist, we based our approach on the observations of Cunningham et al. [3] who developed by phage display an agonistic analog to ANP, which is specific for human NPRA (hNPRA) versus hNPRC. Three mutations (G9T, R11S, and G16R) appeared to be determinant for the selectivity and specificity of the peptide. When applied to the NPRA superagonist (N,C-ANP) pBNP (P4) [1], [20], these mutations rendered this chimeric peptide a potent NPRB antagonist (P12). In order to improve the selectivity of this antagonist for NPRB and to identify the mutations involved in its antagonistic function, we synthesized a series of analogs based on P4, which were further improved by screening four peptide libraries using phage display methodology. The results document the first development and characterization of peptidic antagonists specific for NPRB.
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The bNPRB construction was reported earlier [7]. The hNPRB and hNPRA was introduced into pBK-CMV (Stratagene) by subcloning from pBS-hNPRB or -hNPRA (Genentech). The strategy for hNPRB deletion mutant (ΔKC) was designed as described earlier [15] using a sense primer 5′-gtcctgaatgagacaatacaggaagg-3′, and two antisense primers 5′-agagcctctttcaccctttcggaaaattaggaagct-3′ (1 pmol), 5′-ttttggtaccttaacctctggtagaagagcctctttcaccctt-3′ (100 pmol). The PCR fragment was subcloned into hNPRB pBK-CMV. The
The chimeric peptide (N,C-ANP)pBNP (P4) was initially obtained in search of a more potent agonist for NPRA [1], [20]. When tested on bNPRB, it also displayed high affinity (p K = 10.4 ± 0.1) and thus limited selectivity for NPRA. Still aiming at a more selective agonist for NPRA, we applied to P4 the three mutations (G9T, R11S, G16R) which had been documented by Cunnigham et al. [3] and which had proved to reduce affinity for other receptor subtypes. Although the resulting peptide P12 was an agonist
This study reports the development of the first selective antagonists for the human NPRB. It also documents the structure activity relationships for receptor NPRB. Insertion of only one residue (Ser 11) in the porcine BNP loop leads to an antagonist. In addition, position 10 should be an Arg, since a Leu, as found in CNP, P6, P13 and P17, restores agonistic activity. The synthetic peptide P19 is the best antagonist developed, with good potency (p A 2 6.8) and selectivity (Δp K 1.57) for NPRB. The
We thank Genentech for providing human NPRA, NPRB and NPRC receptor clones and Myriam Létourneau for technical assistance in peptide synthesis. A.D.L. is recipient of Merck Frosst Canada research Chair in Pharmacology. J.D. is the recipient of F.E.S.-Pharmacology scholarship. This work was supported by grants from the Kidney foundation of Canada and the Canadian Institutes for Health Research.
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