Abstract
A recombinant vaccinia virus has been used to direct the expression of the atrial natriuretic peptide clearance receptor (ANP C-receptor) in mammalian cell lines normally deficient in this protein. The recombinant receptor binds 125I-ANP-(102-126) in a specific and saturable manner and carboxyl-terminal truncated and internal-deleted ANP analogs bind to this site with high affinity. Following the covalent attachment of 125I-ANP-(102-126) to the recombinant ANP C-receptor, the protein exhibits an electrophoretic mobility identical to that of the native ANP C-receptor of cultured vascular cells. Expression of the ANP C-receptor in heterologous cells does not affect ANP-stimulated cyclic GMP accumulation, confirming previous reports that this novel ANP receptor subpopulation is not coupled to cyclic GMP metabolism. Furthermore, specific antisera, generated by inoculating rabbits with living recombinant virus, block 125I-ANP binding to the ANP C-receptor but do not inhibit ANP stimulation of cyclic GMP, supporting the existence of two receptor subpopulations that are functionally and immunologically distinct.
Highlights
$ T o whom all correspondence should be addressed California Biotechnology, Inc., 2450 Bayshore Parkway, Mountain View,CA 94043
Recent reports have identified a second class of ANP receptors in cultured vascular cells that do not appear to be linked directly to cyclic GMP metabolism (Scarborough et al, 1986; Leitman et al, 1986).While the high affinity binding of ANP to the guanylate cyclase-coupled receptor form requires full length disulfide-bridgedANP ligands, the cyclic GMP-uncoupled receptor exhibits relaxed structural binding requirements
Peptide analogs containing internaldeletions of amino acids bind to this site with high affinity (Scarborough et al, 1987; Maack et al, 1987)
Summary
$ T o whom all correspondence should be addressed California Biotechnology, Inc., 2450 Bayshore Parkway, Mountain View,CA 94043. To determine the ligand specificity of this cloned ANP receptor, the ability of several different ANP analogs to compete for 1251-ANPbinding to recombinant vaccinia-infected MDCK cells was investigated (Fig. 3A).
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