Abstract
The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.
Highlights
From the Departments of $Molecular Biology, §Medicinal and Analytical Chemistry, VBioorganic Chemistry, and IIProtein Chemistry, Genentech, Inc., South SanFrancisco, California 94080
We present here the primary char- bioactivity [9] through the stimulation of cGMP production acterization of these fusion proteins as represented by and is a conserved structural motif in both BNP and CNP
The purified fusion proteins have a variety of pigs, both 22- [3] and 53- [11]amino acid versions of CNP
Summary
The dissociation with 16identical residues inthe region of homology with constant of ‘’‘I-ANP for A-IgG was 1.6 PM and forC- ANP Whereas both ANP and BhNaPve amino acids extend-. The purified fusion proteins have a variety of pigs, both 22- [3] and 53- [11]amino acid versions of CNP have beenisolated andsequenced, each with similar biological properties [3, 6]. This amino-terminal lengthheterogeneity is a reflection of alternate processing sites for prohormone cleavage. Two of these receptors are membrane guanylylcyclases, termed NPR-A or GC-A
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