©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand-induced Cleavage of the V Vasopressin Receptor by a Plasma Membrane Metalloproteinase (*)

(Received for publication, May 18, 1994; and in revised form, January 16, 1995)

Elzbieta Kojro Falk Fahrenholz (§)

From the Max-Planck-Institute of Biophysics, Kennedyallee 70, 60596 Frankfurt am Main, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The proteolytic cleavage of a G protein-coupled peptide hormone receptor, the renal V(2) vasopressin receptor, by a plasma membrane proteinase was investigated. In the absence of protease inhibitors during incubation of bovine kidney membranes with a photoreactive vasopressin agonist, V(2) receptor truncation leads to a labeled receptor fragment with M(r) 30,000. The V(2) receptor-degrading enzyme could be completely inhibited by zinc ions yielding the native V(2) receptor glycoprotein with M(r) 58,000. Studies with inhibitors of metalloendopeptidases involved in peptide hormone metabolism and with peptide substrates spanning the V(2) receptor cleavage site classify the receptor protease as metalloendoproteinase with specificity for longer substrates. Comparison of the NH(2)-terminal protein sequence of the truncated M(r) 30,000 V(2) receptor with the sequence deduced from the cDNA of the cloned bovine V(2) receptor shows that cleavage occurs between Gln and Val of the second transmembrane helix close to an extracellular agonist binding site. V(2) receptor proteolysis was dependent on the presence of a hormonal ligand. It occurred rapidly after hormone binding and led to a loss of ligand binding properties of the truncated V(2) receptor. The data suggest that the endogenous V(2) receptor-degrading metalloendoproteinase regulates V(2) receptor function. The novel pathway may contribute to the termination of signal transmission.


INTRODUCTION

Receptors for the neurohypophyseal nonapeptide vasopressin belong to the G protein-coupled receptor family that is characterized by seven transmembrane helices. A general property of this signal transduction system is that in spite of continuing presence of a hormonal ligand, signaling becomes attenuated by processes referred to as desensitization(1) . After agonist binding receptor phosphorylation by G protein-coupled receptor kinases has been found to participate in such regulation(2) . Another post-translational receptor modification which might regulate its function is the proteolytic cleavage of the receptor protein. Proteolytic processing of receptor polypeptides by endogenous proteinases has been described in some seven transmembrane receptor systems(3, 4) . However, until now, the regulation and significance of such a receptor cleavage is not known.

Recently, we obtained evidence that the renal V(2) vasopressin receptor is cleaved by a plasma membrane proteinase(5) . The V(2) receptor subtype is located mainly in the distal collecting ducts. It is coupled to the activation of the adenylate cyclase system (6) and mediates the antidiuretic action of vasopressin (7) . After covalent attachment of a radiolabeled photoreactive vasopressin agonist to the membrane-bound bovine V(2) receptor and purification of the labeled M(r) 30,000 protein, NH(2)-terminal sequencing showed that the isolated protein represents a NH(2)-terminal truncated bovine V(2) receptor. From the yield of purified truncated receptor it was concluded that most of the V(2) receptor protein was cleaved during incubation with the photoreactive ligand, although only 3-5% were labeled. By isolation and sequencing of a radioactively labeled receptor fragment peptide, we found that residues of the second extracellular domain are part of the agonist binding site of the renal V(2) receptor(5) . This agonist binding site is in close proximity to the proteolytic cleavage site. Whether a functional connection between ligand binding and truncation of the receptor exists is an open question.

In the present study we examined two major aspects of the V(2) receptor cleavage. By using a variety of protease inhibitors, we classified the V(2) vasopressin degrading enzyme and demonstrated the native V(2) receptor form. Furthermore, we studied whether binding of a hormonal ligand to the V(2) receptor has any influence on its proteolytic cleavage. We report here that the V(2) vasopressin receptor-degrading enzyme is a metalloendoproteinase and that the cleavage of the V(2) receptor on the extracellular side of transmembrane helix two is induced by hormone binding.


EXPERIMENTAL PROCEDURES

Materials

The radioactive photoreactive vasopressin agonist [^3H] 1-deamino [8-N^6-((4-azido-phenylamidino)lysine)] vasopressin (specific radioactivity 52, 7 Ci/mmol) was prepared by reaction of ^3H-labeled 1-deamino [8-lysine] vasopressin with methyl-4-azidobenzimidate hydrochloride and purified by HPLC (^1)as described previously(8) . [^3H]AVP (specific radioactivity 26 Ci/mmol) was from DuPontNEN.

Synthetic peptides derived from the V(2) receptor sequence were prepared by solid phase synthesis, and their structure was confirmed by mass spectroscopy and amino acid analysis. Bacitracin, antipain, leupeptin, pepstatin, insulin B-chain oxidized 1,10-phenanthroline, phosphoramidon, azocasein, and captopril were from Sigma; Cpp-Ala-Ala-Phe-pAB was from Novabiochem, Switzerland, Pro-Ile was from Bachem, glucagon was from Calbiochem, and AEBSF (Pefabloc SC) from Boehringer Mannheim.

Membrane Preparation

Plasma membranes from bovine kidney medulla were prepared by differential centrifugation followed by Percoll density gradient centrifugation as described previously(9) . Membrane preparations obtained by this procedure had a specific binding capacity of 2-6 pmol of [^3H]AVP/mg of protein. Membranes were stored at -70 °C.

Measurement of V(2) Receptor Cleavage in the Presence of Protease Inhibitors

Bovine kidney plasma membranes (1 mg) were resuspended in 1 ml of binding buffer (50 mM Hepps, pH 8.4, 5 mM MgCl(2)). They were incubated with protease inhibitors and with 20 nM tritium-labeled photoreactive agonist for 30 min at 30 °C. The suspension was cooled in an ice bath for 15 min; then membranes were separated from unbound ligand by centrifugation for 10 min at 10,000 times g. After resuspension of the membrane pellet in 1 ml of ice-cold binding buffer containing 5 mMp-aminophenylalanine as scavenger, the mixture was exposed in a quartz tube to three 1-ms flashes, produced in an apparatus for high energy ultraviolet irradiation(5) . In kinetic experiments, membranes incubated with the photoreactive ligand at 30 °C were diluted after 5, 15, and 30 min with 9 volumes of ice-cold binding buffer and irradiated immediately. After irradiation, plasma membranes were collected by centrifugation for 30 min at 10,000 times g. The pelleted membranes were analyzed by SDS-tube gel (11%) electrophoresis according to Laemmli(10) . After electrophoresis, gels were sliced for liquid scintillation counting and the amount of radioactivity determined in the truncated and native V(2) receptor species. Photoaffinity labeling experiments were performed in the presence of the following reagents in the binding buffer: (A) a mixture of serine, thiol and carboxyl protease inhibitors with 5 µg/ml antipain, 30 µg/ml bacitracin, 6 µg/ml leupeptin, 6 µg/ml pepstatin, 10 µg/ml trypsin inhibitor; (B) 100 µM leupeptin; (C) 100 µM AEBSF; (D) metal chelators: 5 mM EDTA or 1, 5, and 10 mM 1,10-phenanthroline; (E) divalent cations: 0.1 and 1 mM ZnCl(2), 1 mM CuCl(2), 1 mM CdCl(2), 1 mM HgCl(2), 0.1 mM CoCl(2); (F) specific metalloendoproteinase inhibitors: 10 µM phosphoramidon, 200 µM Cpp-Ala-Ala-Phe-pAB, 1 mM Pro-Ile, 10 µM captopril; (E) peptides and proteins H-ADLAVALFQVLPQL-OH (V(2)R 84-97); 1 mM H-FQVLPQL-OH (V(2)R 91-97), 1 mM insulin chain B oxidized, 1 mM glucagon, 5 mg of azocasein.

HPLC Analysis of V(2) Receptor-derived Peptide Cleavage

Peptides V(2)R 84-97 (25 µg, 17 nmol) or V(2)R 91-97 (25 µg, 30 nmol) were incubated with 1 mg of bovine kidney plasma membrane in 1 ml of binding buffer (50 mM Hepps, pH 8, 4, 5 mM MgCl(2)) at 30 °C for 30 min in the presence of various inhibitors. Incubation was terminated by addition of 5 µl of trifluoroacetic acid, the samples were centrifuged at 10,000 times g for 20 min at 4 °C, and the pellets were washed with 100 µl of DMF. The supernatants were applied to Centricon -10 (Amicon) for ultrafiltration to remove proteins, and the filtrates were analyzed by HPLC. Samples were applied onto Lichrosorb RP-18 column (10 µm, 250 times 4.6 mm) at room temperature. Elutions were performed at a flow rate 1.5 ml/min and material absorbing UV light at 220 nm was detected. Peptides were separated with the buffer system 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid, 9.9% water, 90% acetonitril (buffer B) using the following gradient of buffer B: 0% B, 5 min; 0-70% over 45 min. The tetradecapeptide V(2)R 84-97 eluted after 35.2 min, the heptapeptide V(2)R 91-97 after 30.2 min and the COOH-terminal cleavage product, the pentapeptide V(2)R 93-97 (H-VLPQL-OH) after 23.9 min. For the identification of the degradation products derived from peptide V(2)R 84-97, fractions of 1.5 ml were collected and concentrated in a Speed Vac centrifuge (Savant) to dryness. Molecular weights of cleavage products were determined by FAB-mass spectroscopy. The inhibition of peptide V(2)R 84-97 degradation was determined by substracting from the peak area of the uncleaved peptide after incubation with membranes and inhibitors, the peak area of the uncleaved peptide obtained after incubation with membranes in the absence of inhibitors. For calculation of percent inhibition values, the peak area of peptide incubated without membranes and treated identically was used as reference for 100% recovery. Peptide V(2)R 84-97 recovery after incubation with different membranes without inhibitors varied between 20 and 50%.

Determination of V(2) Receptor Cleavage after Preincubation in Binding Buffer

Bovine kidney membranes were preincubated without ligand in binding buffer for 30 min at 30 °C. After preincubation, membranes were collected by centrifugation, resuspended in 1 ml of binding buffer. They were incubated with 0.1 mM Zn(II) to inhibit V(2) receptor cleavage and with 20 nM tritium-labeled photoreactive ligand. Cleavage was determined after photoaffinity labeling as described above.

Determination of Ligand Displacement after Ligand-induced V(2) Receptor Cleavage

Bovine kidney plasma membranes (1 mg) were incubated at 30 °C for 30 min with 20 nM tritium-labeled photoreactive agonist in 1 ml of binding buffer. The suspension was cooled in an ice bath for 15 min, then membranes were separated from unbound ligand by centrifugation for 10 min at 10,000 times g. Membranes were resuspended in 1 ml of binding buffer and incubated with 20 µM AVP (1,000-fold excess) at 30 °C for 30 min. After incubation, membranes were collected by centrifugation, and photoaffinity labeling was performed as described above. The experiments were performed both in the presence and absence of 0.1 mM Zn(II) in the binding buffer.

Deglycosylation of the Photoaffinity-labeled Membranes with N-Glycosidase F

Membranes (1 mg) affinity labeled in the presence of 0.1 mM Zn(II) were prepared for deglycosylation by incubation in 40 µl of buffer (100 mM sodium phosphate, pH 7.2, 50 mM EDTA, 0.5% SDS) for 1 h at room temperature. Enzymatic cleavage was performed by incubation of the membrane suspension containing 1 mg of protein in 100 mM sodium phosphate, pH 7.2, 50 mM EDTA, 0.1% SDS; 1% octyl glucoside, 0.5% 2-mercaptoethanol with 20 or 50 units of enzyme for 24 h at room temperature (total sample volume 200 µl). Membrane proteins were then precipitated using chloroform/methanol method (11) and analyzed by SDS-PAGE.

Receptor Binding Assays

Bovine kidney plasma membranes containing 80-100 µg of protein were incubated with[^3H]AVP for 30 min at 30 °C in binding buffer. The binding assay and data analysis were performed as described previously using a weighted nonlinear least-squares fit to logistic curves(12) .

Molecular Cloning of the Bovine V(2) Receptor

mRNA was isolated from freshly prepared inner medulla of bovine kidney by the standard guanidinium thiocyanate method and oligo(dT)-cellulose chromatography. A gt10 cDNA library was constructed by methods described recently(13) . Screening of the cDNA library with a P-labeled fragment of the pig V(2) receptor cDNA (13) yielded a full-length clone which was sequenced. Details and the complete sequence will be published elsewhere. (^2)


RESULTS

Classification of V(2) Receptor-degrading Enzyme

To classify the V(2) receptor-degrading enzyme, incubation of the membrane-bound receptor was performed in the presence of various protease inhibitors. The activity of the enzyme was estimated from the relative amount of truncated and native V(2) receptor determined by photoaffinity labeling and SDS-gel analysis. Incorporation of a tritium-labeled photoreactive agonist into the V(2) receptor species allowed a quantitative estimation of their relative amounts. The photoreactive vasopressin analogue used as ligand in this study shows prior to photoactivation properties very similar to the natural hormone: a high rat antidiuretic activity in vivo(14) , and a binding affinity for the renal bovine V(2) receptor in the nmolar range that is identical to that of AVP(15) . In a typical experiment, the membrane-bound V(2) receptor was incubated with the photoreactive agonist for 30 min at 30 °C in the absence or presence of proteinase inhibitors. After incubation most of the unbound ligand was removed by centrifugation and membranes were resuspended at 4 °C. Covalent incorporation of the V(2) receptor-bound ligand was achieved by flash photolysis. In the absence of inhibitors during incubation, the V(2) receptor was almost completely cleaved yielding the truncated form with M(r) of 30,000 (Fig. 1A). In some experiments a protein with M(r) 58,000 was labeled with much less efficiency. Its relative amount was maximally 5% of the truncated V(2) receptor. When incubation with the photoreactive ligand was performed at 4 °C overnight, specific and exclusive labeling of the receptor protein with M(r) 30,000 was also obtained. A mixture of serine, thiol, and carboxyl protease inhibitors (see ``Experimental Procedures'') usually applied in receptor biochemistry was added during incubation and had no influence on V(2) receptor cleavage. Further affinity labeling experiments with 100 µM of either leupeptin which inhibits serine and cysteine proteases or AEBSF which inhibits a broad range of serine proteases also resulted in the exclusive labeling of the truncated V(2) receptor.


Figure 1: Inhibition of metalloproteinase mediated cleavage of the native V(2) receptor by zinc ions. Bovine kidney membranes (1 mg) containing 6 pmol of V(2) receptor/mg of protein were incubated for 30 min at 30 °C with 20 nM photoreactive vasopressin agonist. After removing most of the free ligand by centrifugation, the membranes were resuspended and irradiated. Membrane proteins were subjected to electrophoresis on SDS-PAGE, and gels were sliced for counting. Experiment A was performed without inhibitors. In experiment B 0.1 mM ZnCl(2) was present during incubation. To prove the specificity of the native V(2) receptor labeling, incubation with Zn(II) was performed in the absence of the specific V(2) agonist DDAVP (bullet) and in the presence of 2 µM DDAVP (circle).



To examine the role of metalloproteinases, either metal chelators or transition metal ions (16) which are known to inhibit metallopeptidases were included. Metal chelators like EDTA and phenanthroline reduced binding of vasopressin or the photoreactive agonist to the V(2) receptor. Therefore, higher concentrations (5-10 mM) included in the binding buffer prevented labeling of the V(2) receptor protein. Metal ions such as Cu(II), Hg(II), and Cd(II) also reduced binding of the photoreactive agonist to the V(2) receptor.The presence of zinc ions (0.1 mM) had no influence on V(2) receptor binding properties. Apparent dissociation constants for binding of [^3H]vasopressin of 8.3 ± 1.2 and 7.4 ± 0.8 nM were determined on binding experiments in the presence and absence of 0.1 mM Zn(II), respectively. A higher concentration (1 mM) of Zn(II) slightly decreased the affinity of vasopressin for the V(2) receptor (K(d)=19 nM). To exclude the influence of Zn(II) on V(2) receptor binding properties, a concentration of 0.1 mM was used in photoaffinity labeling experiments.

Inclusion of 0.1 mM Zn(II) during incubation resulted in a complete inhibition of the V(2) receptor-degrading enzyme. Instead of the 30 000 M(r) form, a protein with M(r) of 58 000 was specifically labeled with the same yield as the truncated form (Fig. 1B). The labeling of this protein was almost completely suppressed by a 200-fold excess of either the specific V(2) receptor agonist DDAVP (Fig. 1B) or vasopressin. The presence of 0.1 mM Co(II) in the reaction mixture had no influence for V(2) receptor cleavage, only the truncated form of the receptor with M(r) 30,000 was labeled. Inclusion of zinc ions only during photoactivation of ligand yielded exclusively the truncated V(2) receptor. These results demonstrate that the renal bovine V(2) receptor with M(r) 58,000 is cleaved during incubation with the photoreactive agonist at 30 °C by a membrane-bound metalloendoproteinase which can be inhibited by Zn(II).

Determination of Cleavage and Glycosylation Site

By NH(2)-terminal sequencing of the purified truncated bovine V(2) receptor, the sequence -X-X-Pro-Gln-Leu-Ala-Trp-Asp has been obtained(5) . To determine the complete sequence of the cleavage site, the cDNA of the bovine V(2) receptor was cloned and sequenced. Fig. 2shows the amino acid sequence of the second transmembrane region and the first extracellular loop obtained from protein sequencing (5) and cDNA cloning. The hexapeptide found by NH(2)-terminal sequencing is located at the interface of plasma membrane and extracellular region. Comparison with the amino acid sequence deduced from cDNA cloning shows that cleavage occurs between Gln and Val which are located at the extracellular side of the second transmembrane helix. The region spanning the cleavage site is highly conserved in receptors for the neurohypophyseal hormones vasopressin and oxytocin: the sequence from His to Trp is identical in all four cloned V(2) receptors (13, 17, 18) and the heptapeptide Phe to Leu is found also in V(1)(19) and oxytocin (20) receptors.


Figure 2: Localization of the proteolytic cleavage site of the V(2) vasopressin receptor in a two-dimensional model of the V(2) receptor. The amino acid sequence of the second transmembrane helix and the first extracellular loop is shown; it was deduced from the nucleotide sequence of the cloned bovine V(2) receptor. The position of the transmembrane helix was predicted from hydrophobicity analysis(32) . By NH(2)-terminal protein sequencing of the purified truncated V(2) receptor, the sequence XXPQLAWD was obtained (5) . Cleavage by the metalloendoproteinase occurs between Gln and Val (). The photoreactive vasopressin agonist binds covalently to residues of the first extracellular loop (Thr and Arg)(5) . In the extracellular NH(2)-terminal part, a N-glycosylation site was found (), to which carbohydrates are linked.



A molecular weight of 30,517 was calculated from the primary structure of the truncated V(2) receptor. This is in good agreement with the value of 31,000-32,000 obtained from SDS-PAGE that includes the molecular weight of the covalently bound vasopressin nonapeptide. The molecular weight calculated for the protein core of the cloned V(2) receptor (40,236) is significantly lower than that found by SDS-PAGE after affinity labeling in the presence of Zn(II). All cloned V(2) receptors including the bovine V(2) receptor contain a conserved N-glycosylation motif (Asn-Xaa-Ser) at its NH(2) terminus (Asn). To prove that the native V(2) receptor protein with M(r) 58, 000 is glycosylated, membranes affinity-labeled in the presence of Zn(II) were treated with N-glycosidase F which cleaves asparagine bound N-glycans. SDS-PAGE analysis after such treatment yielded a new radiolabeled protein with M(r) 49,000 (Fig. 3). Under our experimental conditions (20 or 50 units of N-glycosidase F), roughly 50% of the labeled V(2) receptor were converted to the protein with M(r) 49,000. Comparison with the molecular weight calculated for the protein core suggests that the protein obtained after N-glycosidase F treatment is not a final digestion product.


Figure 3: Deglycosylation of the photaffinity labeled V(2) vasopressin receptor with N-glycosidase F. Membranes (1 mg) photoaffinity labeled in the presence of 0.1 mM Zn(II) were resuspended in 200 µl of cleavage buffer and incubated with 20 units of N-glycosidase F for 24 h at room temperature. Membrane proteins were then precipitated with chloroform/methanol and analyzed by SDS-PAGE.



Effect of Metalloendopeptidase Inhibitors and Peptide Substrates

There is recent evidence that several metalloendopeptidases in kidney membranes are involved in the metabolism of biologically active peptide hormones. To test whether one of these enzymes is responsible for the V(2) receptor cleavage, inhibitors known to be specific for these enzymes were included during incubation experiments. The results of these experiments are summarized in Table 1.



The enzyme responsible for V(2) vasopressin receptor cleavage could be distinguished from metalloendopeptidases EC 24.11(21) , EC 24.15(22) , EC 24.16(23) , and angiotensin converting enzyme (24) since it was not effected by micromolar concentrations of their specific inhibitors.

The substrate specificity of the V(2) receptor-degrading enzyme was examined by competition experiments of V(2) receptor cleavage in the presence of several peptides (Table 1). The tetradecapeptide H-ADLAVALFQVLPQL-OH corresponding to residues 84-97 in the V(2) receptor sequence that spans the cleavage site partly inhibited V(2) receptor proteolysis: 68% of the labeled V(2) receptor corresponded to the truncated form, 32% to the native species. The shorter heptapeptide corresponding to residues 91-97 which also contains the cleavage site did not inhibit V(2) receptor cleavage. To examine whether the V(2) receptor-derived tetradecapeptide is a substrate of the V(2) receptor-degrading enzyme, its enzymatic cleavage by kidney membranes was analyzed by HPLC in the presence of various inhibitors (Table 2). Degradation of the tetradecapeptide could be inhibited by Zn(II) and to a lesser extent by Cu(II). To examine the degradation after inhibition of other peptidases present in kidney membranes, incubation of the peptide was performed in the presence of inhibitors for metalloendopeptidases listed in Table 1and inhibitors of serine, thiol, and carboxyl proteases. Addition of either Zn(II) or 1,10-phenanthroline to this mixture of inhibitors further increased the recovery of the tetradecapeptide. To show that the synthetic peptide is cleaved in the absence of specific inhibitors at the same site as the intact V(2) receptor, fractions from HPLC which correspond to the elution position of the expected cleavage products were analyzed by FAB-mass spectroscopy. At the elution position of the COOH-terminal pentapeptide VLPQL, the corresponding product (m/z = 569; MH) was identified. When the tetradecapeptide was incubated in the presence of the serine, thiol, and carboxypeptidase inhibitor mixture, the cleavage between Gln and Val yielding the COOH-terminal pentapeptide was not inhibited. These results suggest that the same enzyme may cleave the V(2) receptor and the V(2) receptor-derived tetradecapeptide.



Concerning the length of substrates, the enzyme degrading the V(2) receptor and the V(2) receptor tetradecapeptide resembles metalloendoproteinases as meprin which have a preference for substrates longer than seven amino acids and which are insensitive to phosphoramidon(25) . We therefore examined whether substrates of meprin such as azocasein, insulin B-chain, and glucagon (25, 26, 27) have an inhibitory effect on V(2) receptor cleavage. No effect was observed for azocasein and glucagon (Table 1). HPLC analysis showed that glucagon was not cleaved by kidney membranes. Insulin B-chain yielded a 20% inhibition of V(2) receptor cleavage (Table 1).

Influence of Ligand Binding on V(2) Receptor Cleavage

By isolation and sequencing of a radioactive labeled fragment, we had recently shown that residues of the first extracellular loop (Arg and Thr) are part of the agonist binding site(5) . This agonist binding site is in close proximity to the proteolytic cleavage site (Fig. 2). We therefore examined whether ligand binding influenced V(2) receptor cleavage and compared cleavage in the presence and absence of ligand. Membrane-bound V(2) receptor was incubated with the photoreactive agonist for 5, 15, and 30 min at 30 °C in the absence of zinc ions before activation of the ligand. Cleavage was terminated by 10-fold dilution with ice-cold buffer before activating of the ligand. V(2) receptor labeling after these incubation times yielded roughly 80 (Fig. 4), 90, and 100% of the truncated V(2) receptor, demonstrating that receptor cleavage in the presence of the vasopressin agonist occurs rapidly.


Figure 4: Influence of ligand binding on V(2) receptor cleavage. Membranes (1 mg) with 3 pmol of V(2) receptor/mg of protein were preincubated without ligand for 30 min at 30 °C in binding buffer; after that time zinc ions (0.1 mM) and photoreactive ligand (20 nM) were added, and after 30 min of incubation at 30 °C photoaffinity labeling was performed. Exclusive labeling of the native M(r) 58,000 V(2) receptor was found (bullet). For comparison, membranes were incubated with photoreactive agonist for 5 min at 30 °C without zinc ions. After 10-fold dilution with ice-cold buffer, they were irradiated; 80% of the V(2) receptor was truncated, yielding the M(r) 30,000 form (circle).



In comparison, cleavage was studied in the absence of ligand: membrane-bound V(2) receptor was preincubated at 30 °C for 30 min without hormonal ligand. After this time, Zn(II) chloride was added to inhibit the V(2) receptor-degrading enzyme, and the extent of cleavage during preincubation without ligand was determined by photoaffinity labeling. Under these conditions exclusive labeling of the native V(2) receptor protein with M(r) 58,000 was found, but the truncated form was not labeled (Fig. 4). The amount corresponded to more than 70% of the value which was found in identical experiments performed without preincubation. The reduction in total V(2) receptor could be due to denaturation and the formation of aggregates during longer incubation. This result suggests that proteolytic cleavage of the V(2) receptor in the absence of a hormonal ligand does not occur or only with a low rate.

To examine the V(2) receptor function after ligand-induced cleavage, the membrane-bound V(2) receptor was first incubated with 20 nM tritium-labeled photoreactive agonist for 30 min at 30 °C. After that time membranes were incubated with a 1000-fold excess of AVP to displace the receptor-bound photoreactive ligand. The extent of displacement was determined by photoaffinity labeling. This experiment was performed both in the presence and absence of Zn(II) in binding buffer (Fig. 5). In the presence of Zn(II), no V(2) receptor labeling was detected indicating complete exchange of photoreactive ligand by vasopressin. On the other hand, the labeling of the truncated V(2) receptor in the absence of Zn(II) shows that after ligand-induced cleavage a substantial part of the photoreactive ligand could not be displaced on the truncated V(2) receptor by a large excess of vasopressin, which apparently was unable to bind to the cleaved V(2) receptor.


Figure 5: Control of the receptor function after ligand induced cleavage. Membranes with 2.6 pmol of V(2) receptor/mg of protein were preincubated with 20 nM tritium-labeled photoractive ligand for 30 min at 30 °C in binding buffer, then membranes were cooled to 4 °C, collected by centrifugation, resuspended in binding buffer with 20 µM AVP, and after 30 min of incubation at 30 °C photoaffinity labeling was performed. Experiments were performed in the absence (bullet) and presence (circle) of 0.1 mM Zn(II) in binding buffer.




DISCUSSION

In this report we provide evidence, that the renal V(2) vasopressin receptor is cleaved by a plasma membrane metalloendoproteinase. The receptor truncation could be completely inhibited by zinc ions yielding a specific labeling of the native V(2) receptor protein with M(r) 58,000. Deglycosylation of the labeled V(2) receptor shows that the native V(2) receptor is N-glycosylated. This is in accordance with the existence of a N-glycosylation site, which is at the extracellular NH(2) terminus and is conserved in all cloned V(2) receptors.

Several zinc proteinases are known to be inhibited by an excess of Zn(II) (e.g. carboxyl-peptidase-a, thermolysin, and other neutral endopeptidases)(16) . The metalloenzyme responsible for truncation of the renal vasopressin receptor is neither sensitive to phosphoramidon, an inhibitor of endopeptidase-24.11(21) , well characterized in kidney of several species, nor to specific inhibitors of other kidney membrane metalloendopeptidases that metabolize peptide hormones.

Cleavage of the V(2) receptor was partly inhibited by the tetradecapeptide corresponding to residues 84-97 in the V(2) receptor. This sequence is part of the 20 residue long peptide conserved in all cloned V(2) receptors which spans the cleavage site. The results of these studies suggest that the V(2) receptor-derived synthetic tetradecapeptide is a substrate of the V(2) receptor-degrading enzyme. Its degradation by this enzyme can be inhibited by divalent cations and by 1,10-phenanthroline. The shorter heptapeptide corresponding to residues 91-97 which contains the cleavage site did not inhibit the V(2) receptor-degrading enzyme and was a poor substrate. These results suggest that the V(2) receptor-degrading enzyme belongs to a class of metalloendoprotease with specificity for longer substrates. As kidney membranes contain several endo- and exopeptidases, the detailed substrate specificity and characterization of the V(2) receptor-degrading enzyme can only be determined after its purification.

The cleavage site of the V(2) receptor at the transition between second transmembrane region and first extracellular loop would classify this enzyme as an ecto-enzyme with its active site orientated toward the extracellular space. The concept of specificity may also be sustained by the colocalization of protease and V(2) receptor. As the cleavage site is highly conserved in all V(2) receptors, the endogenous V(2) receptor proteolysis is not limited to the bovine V(2) receptor: a truncated M(r) 30,000 V(2) receptor has been identified in membranes from rat kidney (15) and a pig renal epithelial cell line (28) by affinity labeling. Affinity labeling of the cloned human and bovine V(2) receptor transfected in COS 7 cells derived from monkey kidney also revealed the truncated M(r) 30,000 V(2) receptor form and the existence of the V(2) receptor-degrading enzyme in this cell line. The cleavage of the receptor in COS 7 cells was also completely inhibited by zinc ions. (^3)

The experiments, where plasma membranes were preincubated without hormonal ligand and without zinc ions, show that under these conditions cleavage of the V(2) receptor does not occur or only at a low rate. In contrast, the ligand-occupied V(2) receptor is cleaved with a rate that is comparable to the time course of [^3H]AVP binding(29) . The cleavage of 80% of the V(2) receptor during 5 min of incubation with a photoreactive vasopressin agonist suggests that receptor truncation occurs rapidly after hormone binding. As a hypothesis we propose that binding of the hormonal ligand to a binding domain including the first extracellular loop of the V(2) receptor (5) leads to an exposure of the cleavage site toward the extracellular surface, thereby allowing a more rapid cleavage by the V(2) receptor metalloendoproteinase.

After ligand-induced cleavage, a substantial part of the photoreactive agonist could not be displaced before photoactivation by a large excess of vasopressin. In control experiments with zinc ions which inhibit V(2) receptor cleavage, complete displacement was observed. This result suggests that enzymatic cleavage of the ligand occupied V(2) receptor by the metalloprotease leads to a major distortion of the extracellular hormone-binding site in the truncated receptor with a subsequent change of its hormone binding properties. Recently, it has been reported (30) that the cleavage of another G protein-coupled peptide receptor, the C5a receptor, however, by an exogenous venom metalloendoproteinase occurs at the first extracellular loop between helices 2 and 3. The receptor fragments were unable to bind their natural ligand and to be activated by the C5a glycoprotein.

Metal proteinase-mediated limited proteolysis of the beta-adrenergic receptor on turkey erythrocytes (3) and of the bovine endothelin ET(B) receptor (31) has been described. The cleavage occurs near the NH(2)-terminal end of the first transmembrane domain and did not affect the ligand binding properties. The cleavage of the V(2) receptor described in this report is induced by ligand binding and occurs at the extracellular side of transmembrane helix two, close to a hormone-binding site. This post-translational receptor modification apparently leads to a loss of ligand binding properties. The novel pathway described here may ensure together with other mechanisms the termination of signal transmission. Further experiments on cellular systems should allow a more detailed analysis of V(2) receptor cleavage, its regulation, and function.


FOOTNOTES

*
This work was supported by Grant SFB 169 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Max-PlanckInstitute of Biophysics, Kennedyallee 70, 60596 Frankfurt am Main, Germany. Tel.: 69-6303-267; Fax: 69-6303-244.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; AEBSF, 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride; AVP, [8-arginine] vasopressin; DDAVP, 1- deamino [8-D-arginine] vasopressin; FAB, fast atom bombardment; PAGE, polyacrylamide gel electrophoresis; Hepps, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

(^2)
Ufer, E., Postina, R., Gorbulev, V., and Fahrenholz, F.(1995) FEBS Lett, in press.

(^3)
E. Kojro, E. Ufer, and F. Fahrenholz, unpublished observation.


ACKNOWLEDGEMENTS

We thank Gaby Horter for invaluable technical assistance, Dr. Wolfram Schaefer, Max-Planck-Institute of Biochemistry, Martinsried for mass spectroscopy, Dr. Gerald Gimpl for critically reading, and Solveigh McCormack and Michael Schwalm for typing the manuscript.


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