(Received for publication, May 18, 1994; and in revised form, January 16, 1995)
From the
The proteolytic cleavage of a G protein-coupled peptide hormone
receptor, the renal V 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
receptor truncation
leads to a labeled receptor fragment with M
30,000. The V
receptor-degrading enzyme could be
completely inhibited by zinc ions yielding the native V
receptor glycoprotein with M
58,000. Studies
with inhibitors of metalloendopeptidases involved in peptide hormone
metabolism and with peptide substrates spanning the V
receptor cleavage site classify the receptor protease as
metalloendoproteinase with specificity for longer substrates.
Comparison of the NH
-terminal protein sequence of the
truncated M
30,000 V
receptor with the
sequence deduced from the cDNA of the cloned bovine V
receptor shows that cleavage occurs between Gln
and
Val
of the second transmembrane helix close to an
extracellular agonist binding site. V
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
receptor. The data suggest that the
endogenous V
receptor-degrading metalloendoproteinase
regulates V
receptor function. The novel pathway may
contribute to the termination of signal transmission.
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 vasopressin receptor is cleaved by a plasma membrane
proteinase(5) . The V
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
receptor and purification of the
labeled M
30,000 protein, NH
-terminal
sequencing showed that the isolated protein represents a
NH
-terminal truncated bovine V
receptor. From
the yield of purified truncated receptor it was concluded that most of
the V
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
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 receptor cleavage. By using a variety of
protease inhibitors, we classified the V
vasopressin
degrading enzyme and demonstrated the native V
receptor
form. Furthermore, we studied whether binding of a hormonal ligand to
the V
receptor has any influence on its proteolytic
cleavage. We report here that the V
vasopressin
receptor-degrading enzyme is a metalloendoproteinase and that the
cleavage of the V
receptor on the extracellular side of
transmembrane helix two is induced by hormone binding.
Synthetic peptides derived from the V 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.
Figure 1:
Inhibition
of metalloproteinase mediated cleavage of the native V receptor by zinc ions. Bovine kidney membranes (1 mg) containing
6 pmol of V
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
was present
during incubation. To prove the specificity of the native V
receptor labeling, incubation with Zn(II) was performed in the
absence of the specific V
agonist DDAVP (
) and in the
presence of 2 µM DDAVP
(
).
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 receptor. Therefore, higher concentrations
(5-10 mM) included in the binding buffer prevented
labeling of the V
receptor protein. Metal ions such as
Cu(II), Hg(II), and Cd(II) also reduced binding of the photoreactive
agonist to the V
receptor.The presence of zinc ions (0.1
mM) had no influence on V
receptor binding
properties. Apparent dissociation constants for binding of
[
H]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
receptor (K
=19 nM). To exclude the
influence of Zn(II) on V
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 receptor-degrading enzyme. Instead of the 30 000 M
form, a protein with M
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
receptor agonist DDAVP (Fig. 1B) or vasopressin. The
presence of 0.1 mM Co(II) in the reaction mixture had no
influence for V
receptor cleavage, only the truncated form
of the receptor with M
30,000 was labeled.
Inclusion of zinc ions only during photoactivation of ligand yielded
exclusively the truncated V
receptor. These results
demonstrate that the renal bovine V
receptor with M
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).
Figure 2:
Localization of the proteolytic cleavage
site of the V vasopressin receptor in a two-dimensional
model of the V
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
receptor. The position of the transmembrane helix was
predicted from hydrophobicity analysis(32) . By
NH
-terminal protein sequencing of the purified truncated
V
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
-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 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
receptor (40,236) is significantly lower than that found
by SDS-PAGE after affinity labeling in the presence of Zn(II). All
cloned V
receptors including the bovine V
receptor contain a conserved N-glycosylation motif
(Asn-Xaa-Ser) at its NH
terminus (Asn
). To
prove that the native V
receptor protein with M
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
49,000 (Fig. 3). Under our experimental conditions (20 or 50 units of N-glycosidase F), roughly 50% of the labeled V
receptor were converted to the protein with M
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 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.
The enzyme responsible
for V 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 receptor-degrading enzyme was examined
by competition experiments of V
receptor cleavage in the
presence of several peptides (Table 1). The tetradecapeptide
H-ADLAVALFQVLPQL-OH corresponding to residues 84-97 in the
V
receptor sequence that spans the cleavage site partly
inhibited V
receptor proteolysis: 68% of the labeled
V
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
receptor cleavage. To examine whether the V
receptor-derived tetradecapeptide is a substrate of the V
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
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; M
H) 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
receptor and the
V
receptor-derived tetradecapeptide.
Concerning the
length of substrates, the enzyme degrading the V receptor
and the V
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
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
receptor cleavage (Table 1).
Figure 4:
Influence of ligand binding on V receptor cleavage. Membranes (1 mg) with 3 pmol of V
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
58,000 V
receptor was found (
). 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
receptor was truncated,
yielding the M
30,000 form
(
).
In comparison,
cleavage was studied in the absence of ligand: membrane-bound V receptor was preincubated at 30 °C for 30 min without
hormonal ligand. After this time, Zn(II) chloride was added to inhibit
the V
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
receptor protein with M
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
receptor could be due to denaturation and the
formation of aggregates during longer incubation. This result suggests
that proteolytic cleavage of the V
receptor in the absence
of a hormonal ligand does not occur or only with a low rate.
To
examine the V receptor function after ligand-induced
cleavage, the membrane-bound V
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
receptor labeling was detected indicating complete exchange of
photoreactive ligand by vasopressin. On the other hand, the labeling of
the truncated V
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
receptor by a large excess of vasopressin, which apparently was
unable to bind to the cleaved V
receptor.
Figure 5:
Control of the receptor function after
ligand induced cleavage. Membranes with 2.6 pmol of V 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
(
) and presence (
) of 0.1 mM Zn(II) in binding
buffer.
In this report we provide evidence, that the renal V 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
receptor protein with M
58,000.
Deglycosylation of the labeled V
receptor shows that the
native V
receptor is N-glycosylated. This is in
accordance with the existence of a N-glycosylation site, which
is at the extracellular NH
terminus and is conserved in all
cloned V
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 receptor was partly
inhibited by the tetradecapeptide corresponding to residues 84-97
in the V
receptor. This sequence is part of the 20 residue
long peptide conserved in all cloned V
receptors which
spans the cleavage site. The results of these studies suggest that the
V
receptor-derived synthetic tetradecapeptide is a
substrate of the V
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
receptor-degrading enzyme and was a poor substrate. These
results suggest that the V
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
receptor-degrading enzyme can only be determined
after its purification.
The cleavage site of the V 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
receptor. As the cleavage
site is highly conserved in all V
receptors, the endogenous
V
receptor proteolysis is not limited to the bovine V
receptor: a truncated M
30,000 V
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
receptor transfected in COS 7 cells derived from monkey
kidney also revealed the truncated M
30,000
V
receptor form and the existence of the V
receptor-degrading enzyme in this cell line. The cleavage of the
receptor in COS 7 cells was also completely inhibited by zinc ions. (
)
The experiments, where plasma membranes were
preincubated without hormonal ligand and without zinc ions, show that
under these conditions cleavage of the V receptor does not
occur or only at a low rate. In contrast, the ligand-occupied V
receptor is cleaved with a rate that is comparable to the time
course of [
H]AVP binding(29) . The
cleavage of 80% of the V
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
receptor (5) leads to an exposure of the cleavage site
toward the extracellular surface, thereby allowing a more rapid
cleavage by the V
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 receptor cleavage, complete displacement was observed.
This result suggests that enzymatic cleavage of the ligand occupied
V
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
-adrenergic receptor on turkey erythrocytes (3) and of the
bovine endothelin ET
receptor (31) has been
described. The cleavage occurs near the NH
-terminal end of
the first transmembrane domain and did not affect the ligand binding
properties. The cleavage of the V
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
receptor
cleavage, its regulation, and function.