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(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
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 In the present study we examined two major
aspects of the V
Synthetic peptides derived from the V
Figure 1:
Inhibition
of metalloproteinase mediated cleavage of the native V
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 Inclusion of 0.1 mM Zn(II) during incubation
resulted in a complete inhibition of the V
Figure 2:
Localization of the proteolytic cleavage
site of the V
A
molecular weight of 30,517 was calculated from the primary structure of
the truncated V
Figure 3:
Deglycosylation of the photaffinity
labeled V
The enzyme responsible
for V The substrate
specificity of the V
Concerning the
length of substrates, the enzyme degrading the V
Figure 4:
Influence of ligand binding on V
In comparison,
cleavage was studied in the absence of ligand: membrane-bound V To
examine the V
Figure 5:
Control of the receptor function after
ligand induced cleavage. Membranes with 2.6 pmol of V
In this report we provide evidence, that the renal V 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 The cleavage site of the V The experiments, where plasma membranes were
preincubated without hormonal ligand and without zinc ions, show that
under these conditions cleavage of the V 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 Metal proteinase-mediated limited proteolysis of the
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6476-6481
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vasopressin Receptor by a Plasma Membrane
Metalloproteinase (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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. 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.
Materials
The radioactive photoreactive
vasopressin agonist [
H] 1-deamino
[8-N
-((4-azido-phenylamidino)lysine)]
vasopressin (specific radioactivity 52, 7 Ci/mmol) was prepared by
reaction of H-labeled 1-deamino [8-lysine]
vasopressin with methyl-4-azidobenzimidate hydrochloride and purified
by HPLC (
)as described previously(8) .
[H]AVP (specific radioactivity 26 Ci/mmol) was
from DuPontNEN. 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
[H]AVP/mg of protein. Membranes were stored at
-70 °C.Measurement of V
Bovine kidney plasma membranes
(1 mg) were resuspended in 1 ml of binding buffer (50 mM Hepps, pH 8.4, 5 mM MgCl Receptor Cleavage in the
Presence of Protease Inhibitors). 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
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
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 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, 1 mM CuCl, 1 mM CdCl, 1 mM HgCl, 0.1 mM CoCl; (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
(VR 84-97); 1 mM H-FQVLPQL-OH
(VR 91-97), 1 mM insulin chain B oxidized, 1
mM glucagon, 5 mg of azocasein.HPLC Analysis of V
Peptides V Receptor-derived Peptide
CleavageR 84-97 (25 µg, 17
nmol) or VR 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) 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 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 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 VR
84-97 eluted after 35.2 min, the heptapeptide VR
91-97 after 30.2 min and the COOH-terminal cleavage product, the
pentapeptide VR 93-97 (H-VLPQL-OH) after 23.9 min.
For the identification of the degradation products derived from peptide
VR 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 VR 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
VR 84-97 recovery after incubation with different
membranes without inhibitors varied between 20 and 50%.Determination of V
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 Receptor Cleavage after
Preincubation in Binding Buffer 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
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 Receptor Cleavage 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[H]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
mRNA was isolated from freshly prepared inner
medulla of bovine kidney by the standard guanidinium thiocyanate method
and oligo(dT)-cellulose chromatography. A Receptor
gt10 cDNA library was
constructed by methods described recently(13) . Screening of
the cDNA library with a 
P-labeled fragment of the pig
V receptor cDNA (13) yielded a full-length clone
which was sequenced. Details and the complete sequence will be
published elsewhere. ()
Classification of V
To classify the V Receptor-degrading
Enzyme 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 receptor determined by photoaffinity labeling and SDS-gel
analysis. Incorporation of a tritium-labeled photoreactive agonist into
the V 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 receptor in the nmolar range that is identical to that of
AVP(15) . In a typical experiment, the membrane-bound V 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 receptor-bound ligand was achieved by flash photolysis.
In the absence of inhibitors during incubation, the V receptor was almost completely cleaved yielding the truncated
form with M of 30,000 (Fig. 1A).
In some experiments a protein with M 58,000 was
labeled with much less efficiency. Its relative amount was maximally 5%
of the truncated V receptor. When incubation with the
photoreactive ligand was performed at 4 °C overnight, specific and
exclusive labeling of the receptor protein with M 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 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 receptor.
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
(
).
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. 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).Determination of Cleavage and Glycosylation
Site
By NH-terminal sequencing of the purified
truncated bovine V 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 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-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 receptors (13, 17, 18) and the
heptapeptide Phe
to Leu
is found also in
V(19) and oxytocin (20) receptors.
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.
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.
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 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.
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. 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.
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).Influence of Ligand Binding on V
By isolation and sequencing of a radioactive labeled
fragment, we had recently shown that residues of the first
extracellular loop (Arg Receptor
Cleavage 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 receptor cleavage and compared cleavage in the presence and
absence of ligand. Membrane-bound V 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 receptor labeling after these incubation
times yielded roughly 80 (Fig. 4), 90, and 100% of the truncated
V receptor, demonstrating that receptor cleavage in the
presence of the vasopressin agonist occurs rapidly.
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
().
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. 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.
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.
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. 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. 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. () 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. 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.
-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.
)))
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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