atrial natriuretic peptide (anp) is a high-affinity substrate for rat insulin-degrading enzyme

Eur. J. Biochem. 202, 285-292 (1991) c) FEBS 1991

Atrial natriuretic peptide (ANP) is a high-affinity substrate for rat insulin-degrading enzyme Dieter MULLER, Hans BAUMEISTER, Friedrich BUCK and Dietmar RICHTER Institut fur Zellbiochemie und klinische Neurobiologie, UKE, UniversitHt Hamburg, Hamburg, Federal Republic of Germany

(Received June 14, 1991) - EJB 91 0784

A cytosolic protein specifically binding to and degrading atrial natriuretic peptide (ANP) was purified from rat brain homogenate. Based on partial amino acid sequences and enzymatic properties, this protein with an apparent molecular mass of 112 kDa has been identified as the rat insulin-degrading enzyme (IDE). In addition to the known substrates, insulin and transforming-growth-factor c1 IDE binds also with high affinity (apparent Kd 60 nM) to ANP. Competition studies with structural variants of ANP demonstrate that both the C terminus and the disulfide loop of the molecule are essential for high-affinity binding. The data suggest that IDE might be involved in the cellular processing and/or metabolic clearance of ANP.

Atrial natriuretic peptide (ANP) is a hormone primarily synthesized and secreted by the heart. It is mainly involved in the regulation of blood pressure by stimulating natriuresis, diuresis and vasodilation. As known for many other peptide hormones originally isolated from peripheral tissues, ANP is also present and active in the central nervous system [I -41.

Three plasma-membrane receptors for ANP have been described. Two of them (the ANP-A and ANP-B receptors) contain, apart from the extracellular binding domain, a single putative membrane-spanning region and a cytosolic domain with guanylate cyclase activity [5 - 71. Activation of this guanylate cyclase by bound hormone is believed to mediate many, but not all, of the physiological effects of ANP [8, 91. The third receptor (the ANP-C receptor) has only a short cytoplasmic tail and is suggested to be involved in metabolic clearance of ANP from the circulation [lo] but functions in signal transduction are also discussed [9, 111. Receptor-mediated endocytosis of ANP [12- 151 followed by cleavage of the peptide [14, 151 has been repeatedly reported. The specific contributions of the different ANP receptors to this process have not yet been determined. However, the ANP-C receptor seems to be mainly responsible for the metabolic clearance of ANP at least in kidney and vascular tissues [16, 171.

Insulin-degrading enzyme (IDE), also called insulin protease or insulinase, is a neutral cytosolic protease with a remarkable substrate and cleavage specificity. Data derived from molecular cloning, and immuno-labeling and affinity-

Correspundence to D. Richter, Institut fur Zellbiochemie und klinische Neurobiologie, UKE, Universitlt Hamburg, Martinistrasse 52, W-2000 Hamburg 20, Federal Republic of Germany

Ahhreviutions. ANP, atrial natriuretic peptide. In this study, ANP designates the rat ANP (99 - 126). The amino acids are numbered according to the ANP precursor (126 amino acids), from which the circulating hormone is released by cleavage between residues 98 and 99. BNP, brain natriuretic peptide; IDE, insulin-degrading enzyme (insulinase); EGF, epidermal growth factor; TGF,, transforming growth factor CI.

Enzymes. Insulin-degrading enzyme (EC 3.4.22.1 1); enkephalinase (EC 3.4.24.1 1).

labeling studies show that this protein is evolutionary highly conserved [18], developmentally regulated [I 91 and occurs in all tissues tested until now [20]. The enzyme, a thiol metalloproteinase [21], appears to be unique with respect to known protease classes. This is infered from amino acid sequence comparisons [22] and from a cleavage specificity, which is not yet fully understood but is obviously different from the properties of other characterized proteases [23, 241. The identified cleavage sites in insulin point to some preference for a-helical regions around leucine - tyrosine bonds [23].

Although IDE has been known for more than 40 years to be involved in insulin metabolism, its biological function and subcellular localization are still unclear. Several lines of evidence indicate that IDE is a cytosolic [25] nonlysosomal [26] enzyme. Microinjected monoclonal antibodies to IDE inhibit cellular insulin degradation [27] verifying the significance of this proteolytic activity in vivo. Moreover, the insulin cleavage sites in vivo have been found to be identical with those gener- ated by the purified enzyme 1241. It has been shown that insulin degradation by IDE occurs subsequent to the receptor- mediated internalization process [24]. But the fundamental question how the internalized peptide comes into contact with the cytoplasmic protein is not yet settled. Furthermore, the biological significance of insulin degradation by IDE is still a matter of debate. The IDE-initiated peptide degradation may be mainly important for switching off responses to the hormone by metabolic clearance [28]. Alternatively or additionally, the specific cleavage to peptide intermediates may serve in generating new signal transmitters [29, 301.

Additional questions arise from studies showing that the substrate specificity of the enzyme is not restricted to insulin but extends to some other biologically active peptides. High- affinity binding to the interrelated growth factors, transforming growth factor CI (TGF,) and epidermal growth factor (EGF), and an apparent selective degradation of the former peptide have been described [31]. There are also reports on cleavage of glucagon 129, 321 but the affinity of IDE for this hormone is relatively low [21].

286

Here we report that ANP is specifically recognized and cleaved by rat insulin-degrading enzyme. The affinity of ANP to IDE is in the same range as observed with insulin. Compe- tition studies with structural analogs of ANP refer to a highly specific molecular interaction. Therefore, IDE might be a component of the ANP signaling system.

MATERIALS AND METHODS

Materials

Iz5I-ANP (rat, residues 99- 126) and 'z51-insulin (human, labeled at tyrosine B26), both 2 kCi/mmol, were obtained from Amersham (Braunschweig, FRG). ANP (rat, residues 99 - 126), human ANP (residues 99 - 126), ANP (residues 103 - 123) denoted as atriopeptin I, ANP (residues 103 - 126) denoted as atriopeptin 111 and TGF, (rat) were purchased from Bissendorf (Hannover, FRG). Brain natriuretic peptide (BNP, porcine, 26 amino acids) was from Peninsula (Belmont, CA, USA) and EGF (culture grade, purified from mouse tissue) from Collaborative Research (Waltham, USA). ANP (residues 122- 126) and ANP (residues 104- 126, linear) denoted as des Cyslo5, des Cys,,,-ANP(104- 126), synthesized by the solid phase method, were kindly provided by W. Kullmann. All other peptides were from Sigma (Munich, FRG).

All protease inhibitors were purchased from Sigma, except for bacitracin (Calbiochem, La Jolla, USA) and 1,10- phenanthroline (Serva, Darmstadt, FRG). The cross-linking reagent disuccinimidyl suberate was from Pierce (Rockford, IL, USA).

Standard affinity-labeling assay

Samples of the 112-kDa protein were incubated for 10 rnin at 25°C with 20 fmol 1251-ANP (2 kCi/mmol) in a total volume of 20 p120 mM Hepes buffer, pH 7.5, 1 mM dithiothreitol, 2 mM EDTA, 2 mM 1,lO-phenanthroline and 150 mM NaCl. 2 p1 disuccinimidyl suberate (0.01 M in dimethyl sulfoxide) were added and the incubation was continued for 10 min. The reaction was stopped by the addition of 11 pl 375 mM Tris/HCI, pH 6.8, including 30 g/1 SDS, 20% (by vol.) glycerol, 200 mM dithiothreitol and 0.6 g/l bromophenol blue. Samples were heated for 1 rnin at 100°C and analyzed by SDSjPAGE according to Laemmli 1331 on 6% separation gels. Gels were stained with Coomassie brilliant blue R 250, dried and exposed to X-ray film at -70°C between inten- sifying screens. Molecular masses were determined by using the following markers from Sigma : carbonic anhydrase (29 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase B (97 kDa), P-galactosidase (116 kDa) and myosin (205 kDa).

Purification of the 112-kDa protein

7 g total brain from Wistar rats, stored in liquid nitrogen, were pulverized in a mortar and suspended in 70 ml 50 mM TrisjHCl buffer, pH 7.5, containing 1 mM EDTA, 1 mM dithiothreitol and 0.5 mM phenylmethylsulphonyl fluoride. The suspension was homogenized in a Potter-Elvehjem homogenizer (3 strokes) and subsequently cell debris and nuclei were removed by a centrifugation at 3000 x g for 8 min. The supernatant fractions of three such preparations (i.e. from 21 g brain) were collected on ice and then centrifuged for 60 min at 100000 x g at 4°C. The cytosolic supernatant

obtained (560 mg protein) was fractionated by ammonium sulfate precipitation. The 40- 65% precipitate (92 mg protein) was suspended in 8 ml20 mM Tris/HCI, pH 7.5, including 2mM EDTA and subjected to gel filtration through a Sephacryl S 200-column (94 x 2.5 cm), equilibrated and developed with the same buffer at 4°C. Fractions with ANP- binding activity were pooled (22 mg protein) and concentrated fivefold to 5 ml by means of a Centriprep-30 ultrafiltration device (Amicon).

Aliquots of 1 ml were subjected to chromatography on a Mono Q HPLC column (Pharmacia HR 5/5, 5 x 0.5 cm), equilibrated with 20 mM Tris/HCl, pH 7.5. This solution (buffer A) was used together with buffer B (buffer A plus 1.5 M NaCI) for elution of bound proteins at a flow rate of 1 nd/min. The linear gradient steps were (a) 30 min from 0- 33% buffer B and (b) 5 min from 33 - 100% buffer B. The 112- kDa protein eluted in three fractions (3 ml) at 270-300 mM NaC1. The pooled material from five separations (15 ml, 1.3 mg protein) was desalted and concentrated to 800 pl by means of Centriprep-30.

Aliquots of 160 ~1 (160 pg protein) plus 240 p1 buffer C (2 M ammonium sulfate, 250 mM ammonium acetate) were applied to a hydrophobic interaction chromatography (HIC) column (Aquapore HIC 300, 30 x 4.6 mm, Applied Biosys- terns) and proteins were eluted at a flow rate of 0.4 ml/min with buffers C and D (buffer D: 0.2 M ammonium sulfate, 25 mM ammonium acetate) during the following steps: (a) 4min 30% buffer D, (b) 31 rnin 30-100% gradient buffer D, (c) 1 rnin 100% buffer D. The 112-kDa protein eluted maximally at 0.38 M ammonium sulfate and was collected from two fractions (0.8 ml). The material from five runs was pooled. After desalting and concentrating about tenfold by using Centricon-10 ultrafiltration columns (Amicon) with 20 mM Hepes buffer, pH 7.5, this solution was the partially purified enzyme preparation used in most studies. Analysis by SDSjPAGE and silver staining (Gelcode, Pierce) revealed the presence of several other protein bands in addition to the prominent 112-kDa band. To estimate the concentration of the 112-kDa protein in our preparation, we compared the intensity of the silver stained 112-kDa protein with reference proteins (phosphorylase b and P-galactosidase from Sigma) of known concentrations running on the same gel. From this, the amount of 112-kDa protein, obtained from 21 g rat brain, was roughly estimated to be 500 ng. In the final solution, the 112-kDa protein (1 - 2 ng/ml) represents about 20% total protein.

The purification of the 112-kDa protein was monitored by the standard affinity-labeling assay. If required, samples were desalted prior to the cross-linking assay, Protein concentration was determined by using the Bio-Rad protein assay with bovine serum albumin (Sigma, fraction V) as standard.

Analytically, a further HPLC on a C8 reverse-phase column [Aquapore RP 300,2.1 x 30 mm, Applied Biosystems, buffer E: 0.1% (by vol.) trifluoro acetic acid in H 2 0 ; buffer F: 70% (by vol.) acetonitrile/0.08% trifluoro acetic acid in H20] was used to prove the identity of the silver stained 112- kDa protein with the ANP-binding activity. Because of the denaturing conditions in this separation system, a small amount of the protein was affinity-labeled prior to the run. The fractions were then analyzed by SDSjPAGE followed by autoradiography or silver staining. The silver-stained 112- kDa protein and the radiolabeled protein nearly coelute. The observed small difference (1 min) in retention times is assumed to result from the influence of the covalently linked ANP.

287

Microsequencing

For microsequencing, eight preparations as described above were pooled, concentrated by lyophilization and fractionated by SDS/PAGE. The proteins were then electroblotted at 35 V (chamber model TE 50, Hoefer Scientific Instruments) onto a polyvinyldifluoride membrane (Immobilon-P, Milli- pore) in 5-1 transfer buffer [25 mM Tris/HCl, pH 8.3,192 mM glycine, 15% (by vol.) methanol] at 4°C for 11 h. The blot was stained with 0.2% (massjvol.) Coomassie brilliant blue R-250 in 40% (by vol.) methanol, 10% acetic acid and des- tained in a solution of 40% (by vol.) methanol, 10% (by vol.) acetic acid for 40 min. After extensive washing in H20, the stained 112-kDa protein band was excised and dried. Minced membrane strips were treated in an Eppendorf tube for 90 s with 200 ~ 1 0 . 2 % (by vol.) polyvinylpyrrolidone in methanol. After washing (3 x 500 pl H 2 0 ) the strips were incubated for 13 h with 2 pg trypsin (sequencing grade, Boehringer, Mannheim) in 200 pl 0.1 M Tris/HCl, pH 8.5, at 37°C. The sample was then vigorously vortexed, shortly centrifuged and the solution was submitted to HPLC on a C8 reverse-phase column (as described above). Peaks were collected manually and re-run on a CIS reverse-phase column (Vydac C18,30 nm, 2.1 x 100 mm). Sequencing of isolated peptide peaks was performed in an automated gas-phase sequencer (Applied Bio- systems, model 470A, equipped with an on-line 120A PTH analyzer).

A N P degradation assay

To test the effect of protease inhibitors upon ANP degradation the following assay was used: ANP at a concentration of 10 ph4 (containing 7.5 fmol lZ5I-ANP) in a total volume of 15 p120 mM Hepes buffer, pH 7.5, including 5 mM MnCl and 0.5 mM dithiothreitol, was incubated with 2 pl(3 ng 112- kDa protein) of the purified IDE preparation at 25°C. At different times, 2.5-11 aliquots were withdrawn and the reaction was stopped by addition of 1 ~ 1 5 mM N-ethylmaleimidel 25 mM EDTA/2 mM 1 ,lo-phenanthroline. Samples were then analyzed by thin-layer chromatography and the ' 251-ANP spot was quantified in a Berthold BF 5300 gamma-counter. In the absence of inhibitors, 12'I-ANP is cleaved into a radiolabeled product of higher mobility within 60 rnin ( t l i z = 8 min). When using inhibitors, these were incubated with the enzyme for 15 min at 4°C.

To test whether different peptides might interfere with the degradation of ANP by IDE, assays were performed as described above, but using 0.1 pM ANP and less enzyme (1 ng 112-kDa protein). The competing peptides were present at a 50-fold molar excess (5 pM).

Thin-layer chromatography

Samples (up to 3.5 pl) were applied to silica gel 60 plastic sheets (layer thickness 0.2 mm, Merck 5748) and the chromatograms were developed in the following solvent system: n-butanollpyridinelacetic acid/H20 (20: 10: 3 : 10, by vol.). After a running time of 4 - 5 h, the sheets were dried and exposed to X-ray film. The migration of unlabeled reference compounds was visualized by ninhydrin treatment. The following RF values were measured: ANP 0.35, '251-ANP 0.36, tyrosine 0.55, iodine-125 0.70. This chromatography system is also suitable for monitoring degradation of '251-insulin (RF 0.23). After incubation of the B-27 labeled peptide with the purified rat enzyme preparation, three radiolabeled com-

M C ' - + I ' - + ' ANP,IO-~ M

kDa

-205

c-- 148

- 116 c-- 112

- 97

- 66

Fig. 1. Cross-linking of l2'1-AMP toproteins of the membrane ( M ) or the cytosolic fraction ( C ) of rat oCfactory bulb homogenate. 75 fmol lZ5I-ANP together with 11.5 pl (50 pg protein) of the membrane suspension (prepared as described in [41]) were incubated in a total volume of 50 p120 mM Hepes buffer, pH 7.5, including 1 mM EDTA, 150 mM NaC1, 5 mM MnC1, 1 mg/ml bacitracin, I pg/ml each pepstatin A and amastatin, 8 pg/ml leupeptin and 4 pg/ml phosphor- amidoa, for 15 rnin at 25 "C. 140 fmol'ZSI-ANP were used in reactions with the cytosolic fraction (30 pl of the I00000 x g supernatant fraction, containing 40 pg protein). Different from the membrane assay, the total volume was 80 pl and the solution additionally contained 1 mM dithiothreitol and 10 mM EDTA. Cross-linking was initiated in both reactions by addition of disuccinimidyl suberate (0.01 M in dimethyl sulfoxide) to a final concentration of 0.5 mM and allowed to proceed for 10min at the same temperature. Reactions were stopped by the addition of 0.5 vol. 375 mM Tris/HCl, pH 6.8, containing 150 g/l SDS, 20% (by vol.) glycerol, 200 mM dithiothreitol and 0.6 g/l bromophenol blue). After heating for 2 rnin at lOO"C, the samples were analyzed by SDSjPAGE and autoradiography. Reac- tions in the presence of unlabeled ANP and the positions of marker proteins are indicated

pounds of greater mobility appear, one of which comigrates with the labeled product of ANP cleavage.

RESULTS

identification and pur ifical ion of a cytosolic ANP-binding protein

In the course of studying ANP receptors in rat brain, affinity-labeling experiments were performed to identify ANP- binding proteins in the olfactory bulb. This brain region has been shown to contain a high density of ANP binding sites [34]. Fig. 1 demonstrates the cross-linking of 12'I-ANP to a membrane protein of rat olfactory bulb. The protein has an apparent relative molecular mass of 120 kDa presumably rep- resenting the ANP-A membrane receptor [5, 81. When monitoring the cytosolic fraction (100000 x g supernatant fraction) of olfactory bulb homogenate for ANP-binding activity, two other proteins were detected. Analysis by reducing SDS/ PAGE reveals proteins of apparently 112 kDa and 148 kDa, which are specifically labeled by '251-ANP (Fig. 1).

In order to characterize one of these soluble proteins, the more strongly labeled 112-kDa protein was purified. Since other brain regions examined also contain this soluble ANP- binding activity (data not shown), the following experiments

288

1 EHPFQEEHLK 4 NEFIPTNFEILALE 2 NPGHYLG (0 A

5 FIIOSEKPPHYLES 3 AEGPQEFVFQE

(W

L Z . 3 4 5 - - 1 I

500 1000

Fig. 2. Amino acid sequences of tryptic peptides f rom the rat 112-kDa protein. The localization of these peptides within the human IDE sequence (1019 amino acids) is indicated. Two amino acid residues which are different in the human IDE sequence are shown in paren- theses

were performed with the 100000 x g supernatant fraction of total rat brain homogenate. The purification steps were ammonium sulfate fractionation, gel filtration and HPLC on Mono Q anion-exchange and hydrophobic-interaction columns (for details see Materials and Methods). Analysis of the ANP-binding activity after the final chromatography step by SDSjPAGE and silver staining revealed a prominent protein band of apparently 112 kDa. To confirm that the silver stained 112-kDa protein is identical to the ANP-binding protein a sample of the pooled material, together with trace amounts of previously affinity-labeled 112-kDa protein was rechromatographed on a different separation system (C8 reverse-phase HPLC). Fractions were analyzed by PAGE followed by autoradiography or silver staining (not shown). The observed coelution of the '"I - ANP-labeled and the stained protein points to the identity of both activities.

In order to isolate the 112-kDa protein, the partially purified protein preparation was separated by SDSjPAGE and electroblotted onto a membrane filter. After excision of the 112-kDa protein band and digestion with trypsin, five tryptic peptides could be isolated and sequenced by automated Edman degradation (Fig. 2). Comparison with proteins present in the NBRF-PIR database revealed that all peptide sequences match the cDNA-derived amino acid sequence of human IDE [22, 351. This protein has been characterized as a cytosolic enzyme responsible for the cellular processing of insulin 127, 361. Fig. 2 illustrates that the five peptides match different regions of the human IDE sequence. Except for two residues (one each in peptides 3 and 4) the amino acid similarity is perfect. Together with the fact that human IDE migrates in SDS/polyacrylamide gels [21, 351 like the purified ANP-binding protein, these findings strongly suggest that the 112-kDa protein represents the rat IDE homolog.

The 112-kDa protein is an ANP-degrading enzyme

To investigate whether the 112-kDa protein has not only ANP binding but also degrading activity, eluted fractions of the hydrophobic interaction chromatographic separation were analyzed for their ability to degrade ANP. For IDE, divalent metal ions have been reported to be essential for proteolytic activity [32]. Therefore, in contrast to binding assays, in which chelating of divalent ions by EDTA or 1,10- phenanthroline has been found to strongly enhance the yield of affinity-labeling, degradation assays were performed in the presence of free metal ions ( 5 mM MnC12). Cleavage of 1251- ANP, which is labeled at the C-terminal tyrosine, was examined by thin-layer chromatography followed by autoradiography. As shown in Fig. 3, the fractions containing ANP- binding activity also possess proteolytic activity indicating

B

Fig. 3. ANP-degrading ( A ) and ANP-binding activity ( B ) in fractions o f the hydrophobic interaction chromatography. (A) 1.5 pl each of the desalted fractions were incubated in a total volume of 3 p1 20 mM Hepes buffer, pH 7.5, including 1 mM dithiothreitol and 5 mM MnCl with 5 fmol 1Z51-ANP for 15 min at 37°C. The samples were then directly applied to a thin-layer sheet. After developing (see Materials and Methods) the sheet was dried and exposed to X-ray film. R, reference lZ5I-ANP. The migration distances of unlabeled ANP and tyrosine are marked. (B) 2.5 pl of the same fractions used in (A) were incubated in a total volume of 10 p1 40 mM Hepes buffer, pH 7.5, including 2 mM dithiothreitol, 4 mM EDTA, 4 mM 1,lO-phenanthroline and 150 mM NaCl with 10 fmol Iz51-ANP for 10 min at 25°C. Cross-linking was performed by addition of 1 pl 0.01 M disuccinimidyl suberate and further incubation for I5 min. The samples were analyzed by PAGE and autoradiography

that the 112-kDa protein is an ANP-degrading enzyme. The radiolabeled cleavage product of 251-ANP migrates similar to (unlabeled) tyrosine. We have not yet established the identity of this molecule, but a cleavage site at or near the C- terminal tyrosine might be implicated.

Effect of various inhibitors on A N P degradation

To prove further the identity of the 112-kDa protein as the rat counterpart of IDE the ability of selected agents to inhibit ANP degradation was investigated (Table 1). The SH- modifying agents N-ethylmaleimide and p-hydroxymercuribenzoate as well as the chelating agents EDTA and 1,lO- phenanthroline strongly reduce the protease activity. Sensi- tivity to these agents is characteristic for IDE, which is regard- ed as a thiol metalloproteinase [37]. The similarity of the inhibitor profile is further documented by the inhibitory effect of bacitracin and the insensitivity to phenylmethylsulphonyl fluoride, phosphoramidon, pepstatin, leupeptin, bestatin and amastatin [21]. Moreover, the reported pH optimum or IDE (7-7.5) is in the same range as that determined for ANP degradation by the rat 112-kDa enzyme (data not shown).

289

I -peptide Insulin ANP I I I

kDa -205

-116 - 97

66

Fig. 4. Competition of variouspeptides with lZ5I-ANP binding to IDE. 2 pl (3 ng 112-kDa protein) of the purified enzyme preparation were incubated in 20 mM Hepes buffer, pH 7.5, including 0.5 mM dithiothreitol, 5 mM EDTA, 2 mM 1, 10-phenanthroline, 100 mM NaCl with 75 fmol ‘251-insulin (I5000 cpm) or 10 fmol lZ51-ANP (40000 cpm) for 10 min at 25°C in a total volume of 10 pl. After addition of 2 ~10.01 M disuccinimidyl suberate the reaction was continued for 10 min and then analyzed by PAGE and autoradiography. Reactions performed in the presence of unlabeled peptides are indicated. The unspecifically labeled protein at 66 kDa is bovine serum albumin, which is present in the 1z51-labeled peptide preparations

High-ufinity binding of A N P to IDE

To get more insight into the binding specificity of the cytosolic rat protein, various peptides were tested as competitors for affinity-labeling by 12’I-ANP. As expected for IDE, the protein can also be labeled by ‘251-insulin (Fig. 4; the less intense band is due to a 1251-insulin preparation of lower specific activity in this experiment). In both cases labeling is blocked by an excess of ANP or insulin, indicating that these unrelated pepetides share a common binding site on the enzyme. In competition assays with other biologically active peptides only the growth factors TGF, and EGF were potent in competing with 12’I-ANP binding. While the former peptide totally prevented the protein labeling, EGF reduced the extent of labeling to about 40% indicating a weaker affinity for the protein compared to TGF,, ANP and insulin. The peptides glucagon, somatostatin, bradykinin, angiotensin I1 and vasopressin are incapable of inhibiting 12’I-ANP cross- linking. These binding data correlate with results obtained by degradation studies (Table 1). Only those peptides which compete in the binding assay are able to interfere with 1251- ANP degradation. This apparent high substrate specificity is a remarkable feature of IDE. The observed high affinity to TGF, and the lower affinity to EGF are also reported for this enzyme [31]. Although the degradation of glucagon by IDE has been claimed by others [29, 321, we failed to recognize any effect of glucagon in our competition assays. This is in agreement with reports on a relatively low (& > 1 pM) affinity of glucagon for IDE [21].

To evaluate the binding affinity of ANP, affinity-labeling experiments were performed in the presence of different concentrations of unlabeled ANP. As shown in Fig. 5, the protein labeling is already strongly reduced at 0.1 pM. From a more detailed study, quantified by counting the radiolabeled protein

Table 1 . Eflect of various inhibitors and peptides on ‘251-hNP degradation by the purified 112-kDa protein

ANP degradation (after 60 min) was measured as described in Materials and Methods

Inhibitor/peptide Inhibition

N-Ethylmaleimide (2 mM) p-Hydroxymercuribenzoate (200 pg/ml) EDTA (10 mM) 1,lO-Phenanthroline (2 mM) Bacitracin (1 mg/ml) Phenylmethylsulfonyl fluoride (1 mM) Phosphoramidon (0.1 mM) Pepstatin A (20 pg/ml) Leupeptin (20 pg/ml) Bestatin (20 pg/ml) Amastatin (20 pg/ml) ANP (5 pM) Insulin (5 pM) Glucagon (5 pM) Bradykinin ( 5 pM)

% 61

100 94 88 63 2 0 0 0 0 0

86 79

0 0

bands, a competitive binding curve was derived (data not shown), from which an apparent dissociation constant (Kd) of 60 nM was calculated. Thus the affinity of ANP for the cytosolic protein is very similar to the reported affinity of insulin for IDE (& = 0.1 pM) [31].

Competition studies with ANP-related peptides

After establishing that ANP has to be added to the list of peptides with high affinity for IDE, we looked for structural requirements of this molecular interaction. The structural properties determining whether a peptide is an IDE substrate or not are essentially unknown. Therefore we used structural variants of ANP and examined their potency as competitors for 12’I-ANP binding. As demonstrated in Fig. 5, most of these peptides (at 1 pM) have lost the ability to displace the labeled ANP. The exceptions are human ANP and ANP(103-126). Human ANP differs from rat ANP by a single amino acid conversion (Ile to Met) at position 110 (Fig. 5 ) . This amino acid exchange does not lead to any re- cognizable effect with respect to binding affinity. The trunc- ated ANP(103 - 126) lacking the four N-terminal amino acids also retains significant competing potency. The C terminus of ANP, however, appears to be essential for high-affinity binding to IDE. Loss of the three C-terminal amino-acids as in ANP(103 - 123) correlates with the loss of competing activity at the concentration used. However, the C terminus alone [ANP(122 - 126)] is not sufficient for 12’I-ANP displacement. Also ANP(104-126, linear) devoid of the two cysteine residues essential for the formation of the molecule’s ring structure is unable to displace 1251-ANP. This suggests that both the ring structure and the C terminus of ANP are prerequisites for high affinity-binding to IDE.

Furthermore, we looked for the ability of the related BNP to compete with ANP binding. The disulfide-linked loop of 17 amino acids is highly conserved between BNP and ANP, whereas the sequences of the C-terminal and N-terminal ex- tensions are different [38]. In the presence of 1 pM porcine BNP, only a slight reduction of I2’I-ANP cross-linking is detectable. Hence the affinity of IDE for porcine BNP is more

290

-112 kDa

99 103 110 123 126 ANP ~ L R R ~ S ~ F G G R i [ ) R I G A ~ S G L G ~ ~ S ~ R ~ . . Fig. 5. Effect of ANP-related peptides on affinity-labeling of IDE by l Z 5 f - A N P . Each 0.6 pI of the purified IDE preparation (0.9 ng, 112- kDa protein) were incubated in a total volume of 10 p120 mM Hepes buffer, pH 7.5, including 1 mM dithiothreitol, 2 mM EDTA, 2 mM 1,lO-phenanthroline and 100 mM NaCI, with 5 fmol "'1-ANP for 1 5 min at 25°C. Unlabeled peptides were present as indicated. 1 yl 0.01 M disuccinimidyl suberate was then added and the reaction continued for 15 min. The samples were analyzed by PAGE and autoradiography. For clarity, the amino acid sequence of ANP is depicted and partially numbered. The disulfide bridge is indicated

than one order of magnitude lower than for ANP. This is remarkable, because porcine BNP and ANP have similar af- finities for the ANP-C receptor [39] and porcine BNP is also an efficient activator of guanylate cyclase-linked ANP receptors [6, 401.

DISCUSSION

Two cytosolic proteins specifically binding '251-ANP have been detected in rat brain homogenate. While the identity of the 148-kDa protein remains to be elucidated, the 112-kDa protein was purified and characterized as the rat IDE. This identification is based on the particular substrate specificity and the characteristic inhibitory profile of the enzyme and is corroborated by structural information concerning amino acid sequences and the apparent molecular mass. Though initially detected in and purified from brain, the 11ZkDa protein was also observed in all peripheral tissues examined (data not shown), in accordance with the reported ubiquitous occurrence of IDE [20]. The amino acid sequence data of the isolated rat protein are still very limited. However, the comparison with the cDNA-deduced human sequence already allows the prediction of a very high degree of similarity. This is consistant with studies from Duckworth et al. [42], who compared antigenic and catalytic properties of purified rat

and human IDE and could show, that both contain identical antigenic sites and generate the same products from insulin. The high conservation even between human and Drosophila IDE became evident recently by cDNA analysis [18].

The substrate and cleavage specificity of IDE is an in- triguing issue. The question as to which specific properties are shared by IDE substrates is not yet answered. Cleavage data available so far can not be explained by simple peptide-bond recognition. By investigating the different cleavage sites in '251-insulin, Duckworth et a]. [24] showed, that most of them are in close physical proximity when viewing the three-dimen- sional structure of insulin. Similar results were reported from Stentz et al. [23], who used unlabeled insulin. In addition, these authors noted a preference for cleavage in the proximity to leucin and tyrosine adjacent to a-helical turns. Our results upon the loss of competition efficiency in the linear ANP analog are consistant with a model in which the three-dimen- sional structure is a main recognition element. The elucidation of the cleavage site(s) in ANP will provide more precise information and will be helpful for a better understanding of the substrate recognition and cleavage site specificity of IDE.

Receptor-mediated internalization and subsequent degradation by IDE appeares to be the main clearance mechanism for insulin [37]. In the case of ANP, at least two mechanisms for the inactivation of the peptide exist, one of which is receptor independent. It has been shown that enkephalinase, an ectoenzyme occurring mainly in renal brush-border mem- branes, contributes to ANP degradation in vivo [43, 441. The other degradation pathway is linked to receptor-mediated endocytosis of the peptide [14, 151. Some recent reports de- scribe a soluble proteolytic activity which may be involved in the intracellular ANP degradation by cutting off the C-terminal tripeptide [14, 45, 461. With respect to the effect of inhibitors and the pH optimum, this activity shows similar enzymatic properties as IDE. Unfortunately, structural information which would allow a more detailed comparison with IDE are presently not available.

Apart from the structural implications discussed above it is tempting to speculate on possible functional relationships between IDE substrates. It may be of importance, that all peptides (Insulin, TGF,, EGF and ANP) for which a high affinity to IDE has been documented, possess receptors of the type with a single membrane-spanning region. Those peptides which fail to compete with 1251-ANP binding in our study all are assumed to associate to guanidine-nucleotide-binding regulatory protein linked receptors with a completely different architecture. Ligand-induced endocytosis of receptors of the former type has been extensively reported [12, 47 -501. This internalization, however, may not only serve to clear the hormone, but to deliver additional signal transmitters to the cell. Evidence for insulin accumulation in nuclei [51] and direct effects of intracellular insulin on RNA and protein synthesis [52] as well as on mitochondria1 oxidation [53] have been reported. After entering the cell growth factors like EGF [54] or the basic fibroblast growth factor [55] have been shown to be translocated to the nucleus. However, cleavage products of internalized peptides may also have biological activity [56]. Specific cellular proteases could be involved in these processes by terminating signaling and/or by generating new signal peptides. The involvement of IDE in such a process can be assumed from the results of a study of Kayalar and Wong [57]. The authors show that the morphological and biochemical differentiation of cultured myoblasts requires the activity of IDE. With respect to insulin, the involvement of IDE in terminating [28] or generating [30] signals has been suggested.

29 1

Future studies will have to show whether IDE-initiated cleavage of ANP participates in the signaling of the hormone. It should be noted that some effects of ANP can not be explained by a stimulation of the guanylate cyclase-linked membrane receptors. The inhibition of aldosteron secretion by ANP in the adrenal gland [58] as well as the effect of ANP on thyroglobulin secretion in thyroid cells [l I] appears to be transmitted through a non-guanylate cyclase pathway. More- over, ANP may regulate endothelial permeability by a mechanism independent of cGMP [59]. Modified ANP analogs, which substantially antagonized ANP-induced cGMP accumulation, have been found to be unable to antagonize ANP- induced vasorelaxation [60].

In this report we show that ANP, like insulin, is an in vitro substrate of IDE. Future studies have to reveal the physiological relevance of IDE-mediated ANP degradation and may help to clarify the structural and possibly functional relations between IDE substrates.

We thank Dr Wilhelm Kullmann for synthesizing peptides ANP(l22- 126) and ANP(104- 126, linear), Werner Rust and Marion Daumigen-Kullmann for technical assistance, and Drs Evita Mohr and Hartwig Schmale for critically reading the manuscript. This work was supported by Deutsche Forschungs~emeinschaft, SFB 232 to D. R., and Volkswagen Stiftung 1/65 516.

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