The atrial natriuretic peptide (ANP) receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. We observed that the receptor, when bound with ANP, was rapidly cleaved by endogenous or exogenously added protease to yield a 65-kDa ANP-binding fragment. No cleavage occurred without bound ANP. This ligand-induced cleavage abolished GCase activation by ANP. Cleavage occurred in an extracellular, juxtamembrane region containing six closely spaced Pro residues and a disulfide bond. Such structural features are shared among the A-type and B-type ANP receptors but not by ANP clearance receptors. The potential role of the hinge structure was examined by mutagenesis experiments. Mutation of Pro(417), but not other Pro residues, to Ala abolished GCase activation by ANP. Elimination of the disulfide bond by Cys to Ser mutations yielded a constitutively active receptor. Pro(417), and Cys(423) and Cys(432) forming the disulfide bond are strictly conserved among GCase-coupled receptors, while other residues are largely variable. The conserved Pro(417) and the disulfide bond may represent a consensus signaling motif in the juxtamembrane hinge structure that undergoes a marked conformational change upon ligand binding and apparently mediates transmembrane signal transduction.

The beta-form of S-adenosylmethionine synthetase among three isozymes has been purified from rat liver, and proven to be homogeneous. The molecular weight of this enzyme was estimated to be 100,000 by gel filtration on Sephacryl S-200, and the enzyme was shown to be composed of two subunits of 48,000 daltons. A rabbit antiserum against the normal rat liver beta-form of S-adenosylmethionine synthetase was used for immunochemical characterization. The alpha- and beta-forms of isozyme are immunochemically identical, but the antiserum did not react with the gamma-form from rat kidney.

Photoaffinity labeling of atrial natriuretic factor (ANF) receptor in the plasma membranes from bovine aortic smooth muscle tissue using Na5-(4-azidobenzoy1)-ANF-(5-28)-peptide labeled with yielded a 130-kDa band. However, when smooth muscle cells from the same bovine aorta were placed in culture, the 130-kDa receptor quickly disappeared and a 60-kDa band began to appear at high density. After three passages, essentially no 130-kDa band was found and only the 60-kDa band was strongly labeled. The primary structures of the two receptor forms were compared by radiochemical peptide mapping after endoproteinase Glu-C digestion of photoaffinity-labeled and detergent-solubilized 130-kDa receptor from the aorta or the 60-kDa receptor from the cultured cells. The peptide mapping showed courses of digestion that were significantly different from each other, suggesting difference in their primary structures. The basal guanylate cyclase activity in the aortic membranes was 1.0 pmol cGMP produced . min-' . mg protein ' at 37 "C using Mn2+-GTP as substrate. The corresponding activity in the membranes from the cultured cells was 20fmol cGMP . min-' . mg protein-'. Binding studies gave a density of binding sites (BmJ of 82 fmol/mg protcin for the aortic membranes and 850 fmol/mg protein for the cultured cell membranes. These data suggest that the major form of ANF receptor in the cultured cells, namely the 60-kDa receptor, lacked guanylate cyclase activity. Northern blot analysis of poly(A)-RNA extracted form bovine thoracic aorta or adrenal cortex gave a single 3.6-kb band when 32P-labeled human A-type ANF receptor cDNA was used as a hybridization probe. However, no band was detected when C-receptor cDNA was used as a probe. In addition to the major 130-kDa band, extended SDS/PAGE revealed two additional faint bands with estimalcd molecular masses of 126 kDa and 135 kDa. Treatment with endoglycosidase H resulted in disappearance of the 126-kDa band and appearance of a 100-kDa band. The 130-kDa and 135-kDa bands were unchanged. Treatment by endoglycosidase F or glycopeptidase F reduced all three bands to a single 100-kDa band. These results suggest that the slight difference in mobility is due to different states of glycosylation. Competitive protection experiments showed binding specificity in the order of ANF > BNP S-CNP, AP-I for all three bands, indicating that the state of glycosylation had no effect on the ligand specificity. Photoaffinity labeling of bovine adrenal cortex membranes gave a single 130-kDa band. The same experiment with lung membranes yielded a 130-kDa band and a 60-kDa band at about 1 : 1 ratio. The data presented in this report indicate that the vascular smooth muscle contains predominantly the 130-kDa guanylate-cyclase-coupled ANF receptor in vivo, and that C-receptors found in culturcd vascular smooth muscle cells have been induced artificially from cell culturing.Atrial natriuretic factor (ANF) is a potent natriuretic (deBold et a]., 1981) and vasorelaxant peptide (Currie et al., 1983;Grammer et al., ...

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH (5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 1251-labeled W4-azidobenzoyl-ANF(4 -28) followed by SDSjPAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDSjPAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANFbinding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4 -28), but not by atriopeptin I [ANF(5 -25)] or C-ANF [des-(18 -22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecularmass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.

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