Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase

Abstract

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.

The Biochemical, Biological, and Pathological Kaleidoscope of Cell Surface Substrates Processed by Matrix Metalloproteinases

“…Interestingly, the human hippocampus of Alzheimer's disease patients shows increased levels of pro-MMP-9 near the amyloid plaques (Backstrom et al, 1996) and the same increase was reported in amyloidpositive beagle brains (Lim et al, 1997). In addition, levels of circulating MMP-9 are significantly elevated in the plasma of Alzheimer's disease patients compared (Parvathy et al, 1997) Batimastat (BB-94) Broad spectrum MMP and TACE TAPI-2 Zn 2+ -dependent MP TAPI (-1) Collagenase, gelatinase, ADAM-10, TACE (Schwager et al, 1998;Schwager et al, 1999) ANF-R / EDTA MP (Abe and Misono, 1992) AR / Batimastat (BB-94) Broad spectrum MMP and TACE (Vecchi et al, 1998;Brown et al, 1998 MMP-1, MMP-8 > MMP-9 > MMP-3 Not inhibited by TAPI(-1) EGF / EDTA, EGTA MP (Dempsey et al, 1997) Batimastat (BB-94) Broad spectrum MMP and TACE FcyRIII CD16 GI5402 MMP-1,-3,-9,-13 and TACE (Dekkers et al, 2000) O-phen MP (Bazil and Strominger, 1994) (Galon et al, 1998) RU36156 MMP-8, MMP-9 and TACE (Galon et al, 1998;Mota et al, 2004) 168 B. Cauwe et al …”

Aortic smooth muscle contains guanylate‐cyclase‐coupled 130‐kDa atrial natriuretic factor receptor as predominant receptor form

“…Similarly, it is possible that Creceptor-like low-molecular-mass receptors previously observed in the adrenal cortex (Meloche at al., 1986a;Shinjo et al, 1986;Takayanagi et al, 1987) and the kidney membranes (Koseki et al, 1986;Hamada et al, 1987) may also have been derived through artificial proteolysis of high-molecular-mass ANF receptors which have been reported by others (Yip et al, 1985;Misono et al, 1985;Meloche et al, 1986b) to be predominant in these organs. Indeed, it was found that the 130-kDa ANF receptor in bovine adrenal cortex membranes was readily cleaved by an endogenous proteasc (Misono, 1988;Abe and Misono, 1992), or by exogenously added trypsin (Liu et al, 1989) to generate an ANFbinding fragment with a molecular mass around 60 kDa. Moreover, the adrenal receptor cleaved by the endogenous protease was found to exhibit C-receptor-like properties, such as relaxed ligand specificity and lack of guanylate cyclase activity (Abe and Misono, 1992).…”

“…Indeed, it was found that the 130-kDa ANF receptor in bovine adrenal cortex membranes was readily cleaved by an endogenous proteasc (Misono, 1988;Abe and Misono, 1992), or by exogenously added trypsin (Liu et al, 1989) to generate an ANFbinding fragment with a molecular mass around 60 kDa. Moreover, the adrenal receptor cleaved by the endogenous protease was found to exhibit C-receptor-like properties, such as relaxed ligand specificity and lack of guanylate cyclase activity (Abe and Misono, 1992).…”

Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase

“…The ECD contains a highly conserved chloride‐binding site near the ECD dimerization interface [17,18]. The ECD also contains in its juxtamembrane region a highly conserved structural motif, referred to as the receptor‐GC signaling motif [19]. Single‐residue mutations in this motif either render the receptor unresponsive to ligand binding or cause constitutive activation of GC activity [20], suggesting that this conserved structure plays a critical role in transmembrane signal transduction.…”