Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II
Stéphane A. Laporte,
Antony A. Boucard,
Guy Servant,
Gaétan Guillemette,
Richard Leduc and
Emanuel Escher
Department of Pharmacology Medical School Université
de Sherbrooke Sherbrooke, Québec J1H 5N4 Canada
 |
ABSTRACT
|
---|
To identify ligand-binding domains of Angiotensin
II (AngII) type 1 receptor (AT1), two different
radiolabeled photoreactive AngII analogs were prepared by replacing
either the first or the last amino acid of the octapeptide by
p-benzoyl-L-phenylalanine (Bpa).
High yield, specific labeling of the AT1
receptor was obtained with the
125I-[Sar1,Bpa8]AngII
analog. Digestion of the covalent
125I-[Sar1,Bpa8]AngII-AT1
complex with V8 protease generated two major fragments of 15.8 kDa and
17.8 kDa, as determined by SDS-PAGE. Treatment of the
[Sar1,Bpa8]AngII-AT1
complex with cyanogen bromide produced a major fragment of 7.5 kDa
which, upon further digestion with endoproteinase Lys-C, generated a
fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to
hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we
circumscribed the labeling site of
125I-[Sar1,Bpa8]AngII
within amino acids 285 and 295 of the AT1
receptor. When the AT1 receptor was
photolabeled with
125I-[Bpa1]AngII, a
poor incorporation yield was obtained. Cleavage of the labeled receptor
with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon
deglycosylation showed an apparent molecular mass of 7.5 kDa,
delimiting the labeling site of
125I-[Bpa1]AngII
within amino acids 147 and 199 of the AT1
receptor. CNBr digestion of the hAT1 I165M
mutant receptor narrowed down the labeling site to the fragment
166199. Taken together, these results indicate that the seventh
transmembrane domain of the AT1 receptor
interacts strongly with the C-terminal amino acid of
[Sar1, Bpa8]AngII,
whereas the N-terminal amino acid of
[Bpa1]AngII interacts with the second
extracellular loop of the AT1 receptor.
 |
INTRODUCTION
|
---|
The octapeptide hormone angiotensin II (AngII) exerts its numerous
physiological effects on cardiovascular, endocrine, and neuronal
systems by interacting with specific receptors (1). Two
pharmacologically distinct types of receptors have been identified for
this vasoactive peptide: AngII type 1 (AT1) and AngII type
2 (AT2) (2). The AT1 receptor mediates all the
known physiological actions of AngII, including regulation of blood
pressure and of water and electrolyte balance (3). The functional roles
of the AT2 receptor are not well defined, but recent
studies suggest that it could act as a physiological antagonist of
AT1-mediated pressor effect and that it could regulate
central nervous system functions related to locomotion and exploratory
behavior (4, 5). The AT1 and AT2 receptors have
been cloned from several species and are members of the G
protein-coupled receptor family (GPCRs) characterized by their
topography consisting of seven
-helical transmembrane domains. The
two receptors exhibit high affinities for AngII even though they
display a low degree of sequence identity at the amino acid level
(34%).
The elucidation of primary structures of numerous GPCRs has prompted
investigators to identify domains in receptors directly involved in
ligand binding. One approach to the identification of the sites
interacting between a ligand and its receptor is to covalently link a
selective radioactive ligand to its cognate receptor and, by chemical
or enzymatic fragmentation of the complex, to identify
photoligand-binding domains of the receptor. This technique has been
invaluable in mapping the ligand-binding pocket of the ß-adrenergic
receptors (6, 7), the GnRH receptor (8), adenosine receptor (9), NK-1
receptor (10), and more recently, by our group, the AT2
receptor (11). This approach complements some of the limitations
of site-directed mutagenesis, deletion analysis, or
chimeric receptors in that it is a more direct attempt to identify
ligand-binding domains. We previously reported the use of photoreactive
AngII analogs for covalent labeling of AT receptors (12). In the
present study, two photoreactive analogs were prepared by replacing the
position 1 aspartyl or the position 8 phenylalanine of AngII with
p-benzoyl-L-phenylalanine (Bpa) (13). These two
photoreactive AngII analogs were used to covalently label the human
AT1 (hAT1) receptor expressed in COS-7 cells.
Targeted enzymatic and chemical fragmentation of the photoaffinity
labeled receptor identified two distinct receptor domains that are
specifically labeled by the two photoreactive AngII analogs.
 |
RESULTS
|
---|
Specificity of Photoreactive Analogs in Binding Experiments and
Photoaffinity Labeling
Figure 1
shows the primary
structures of AngII and photoreactive AngII analogs used in this study.
Asp1 and Phe8 were respectively replaced by Bpa
to give [Bpa1]AngII and
[Sar1,Bpa8]AngII. By competitive binding
assays, we first determined that
[Sar1,Bpa8]AngII and
[Bpa1]AngII exhibit respective IC50 values of
0.8 ± 0.2 nM (n = 3) and 17.7 ± 1.3
nM (n = 3) for the hAT1 receptor expressed
in COS-7 cells. The functional properties of the two analogs were then
analyzed by measurement of the ligand-induced
inositol-1,4,5-trisphosphate production in COS-7 cells expressing the
hAT1 receptor. Incubation of the cells with a high
concentration of [Bpa1]AngII (1 µM) caused
an increase of inositol 1,4,5-trisphosphate comparable to that obtained
with AngII, whereas the same treatment with [Bpa8]AngII
had no effect on inositol phosphate production (results not shown),
thus suggesting the agonist and the antagonist nature of
[Bpa1]AngII and
[Sar1,Bpa8]AngII, respectively. It had been
previously shown that [Sar1,Bpa8]AngII is a
competitive antagonist on the rabbit aorta preparation (13).
In photoaffinity labeling experiments,
125I-[Sar1,Bpa8]AngII (Fig. 2A
) and
125I-[Bpa1]AngII (Fig. 2B
) specifically
labeled the hAT1 receptor that migrated as a glycoprotein
of 110 kDa, as previously described (14). The labeling of the
hAT1 receptor by the two photoreactive analogs was
completely abolished by L158,809 (1 µM), an
AT1 receptor-selective ligand and by AngII (1
µM) (Fig. 2
), thereby confirming the specificity and the
selectivity of the labeling. Although both photoreactive analogs
successfully labeled the hAT1 receptor, determination of
covalent incorporation yields (calculated from the ratio of total
radioactivity found in isolated bands to total specific binding
observed before photolysis) revealed that
125I-[Sar1,Bpa8]AngII, with an
approximately 30% yield of covalent incorporation, was far more
effective than 125I-[Bpa1]AngII (
1% yield
of covalent incorporation). This difference is well illustrated in Fig. 2
where the intensity of the band labeled with
125I-[Sar1,Bpa8]AngII is clearly
higher (2 h autoradiography) than the one labeled with
125I-[Bpa1]AngII (20 h autoradiography).

View larger version (14K):
[in this window]
[in a new window]
|
Figure 1. Amino Acid Sequence of the Photoreactive AngII
Analogs
Residues represented in bold characters correspond to
amino acid modifications. The absorption of a photon at approximately
350 nm by the Bpa moiety results in the formation of a diradicaloid
state in which the electrophilic electron-deficient oxygen interacts
with C-H bonds to produce a benzpinacol-type compound.
|
|

View larger version (62K):
[in this window]
[in a new window]
|
Figure 2. Specific Photoaffinity Labeling of the Human
AT1 Receptor Expressed in COS-7 Cells
Cells expressing hAT1 receptor were photolabeled with
125I-[Bpa8]AngII (A) or
125I-[Bpa1]AngII (B) in the absence or
presence of 1 µM L-158,809 or 1 µM AngII.
Samples (40 µg of protein/lane) were run on 8% acrylamide separating
gel followed by autoradiography for 2 h (A) or 20 h (B).
14C-labeled protein standards of the indicated molecular
masses (kDa) were run in parallel.
|
|
Mapping the
125I-[Sar1,Bpa8]AngII
Contact Point
125I-[Sar1,Bpa8]AngII-photolabeled
hAT1 receptor was partially purified, as indicated in
Materials and Methods, and digested with glycopeptidase-F
(PNGase-F). Treatment of the photolabeled complex with PNGase-F shifted
the mobility of
125I-[Sar1,Bpa8]AngII-hAT1
receptor complex to an apparent molecular mass (Mr) of 34
kDa, corroborating the glycosylated nature of hAT1 receptor
(Fig. 3
). Digestion of the photolabeled
receptor with Endoproteinase Glu-C (Staphylococcus aureus V8
protease) in ammonium carbonate buffer [a condition under which it
specifically cleaves peptide bonds on the C-terminal of glutamic acid
(15)] produced two fragments of 15.8 kDa and 17.8 kDa (Fig. 3
).
Treatment of the 15.8-kDa and 17.8-kDa fragments with PNGase-F had no
effect on the mobility of the two V8 protease digestion products,
indicating that they were not glycosylated (results not shown). The
best candidate for the 15.8-kDa fragment (estimated Mr of
the peptide + photolabel) is the peptide located between glutamate 227
and glutamate 357 (Fig. 4
). The 17.8-kDa
fragment would correspond to the incomplete digestion of peptide
186357.

View larger version (92K):
[in this window]
[in a new window]
|
Figure 3. Enzymatic Digestions of
125I-[Sar1,Bpa8]AngII-Labeled
AT1 Receptor
Partially purified photolabeled hAT1 receptor
(50,000100,000 cpm) was incubated in the absence (control) or in the
presence of PNGase-F (80 U/ml) for 24 h at room temperature.
Photolabeled wild-type receptor was incubated with 800 µg/ml of V8
protease for 4 days at room temperature. Photolabeled A221E mutant
hAT1 receptor was incubated with 800 µg/ml of V8 protease
for 24 h at room temperature. Samples were then run on a 16.5%
acrylamide Tris-Tricine separating gel followed by autoradiography.
14C-labeled protein standards of the indicated molecular
masses (kDa) were run in parallel.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Figure 4. Two-Dimensional Representation of the Primary
Structure of the hAT1 Receptor and Its Potential Sites of
Enzymatic Cleavage and Chemical Hydrolysis
Arrows indicate recognition sites for V8 protease;
glutamic acid residues 173, 185, 227, and 357, as well as the A221E
mutant bold circles indicate recognition sites for endo
Lys-C; solid circles indicate sites of hydrolysis by
CNBr; C indicates cysteine residues. Putative N-glycosylation sites on
asparagines 4, 176, and 188 are also shown.
|
|
To confirm the location of the binding domain for
125I-[Sar1,Bpa8]AngII, we used a
previously described mutant of the hAT1 receptor in which a
glutamate residue was introduced at position 221 (16). We suspected
that by introducing another glutamate residue near the
Glu227, we could eliminate the partial digestion product of
17.8 kDa, since the mutant would become more susceptible to cleavage by
V8 protease at position 221. The hAT1A221E mutant receptor
displays activation and affinity profiles highly similar to those of
the wild-type hAT1 receptor (16). Photolabeling of
hAT1A221E with
125I-[Sar1,Bpa8]AngII resulted in
incorporation yields identical to those obtained with the
hAT1 receptor. Figure 3
shows that V8 protease treatment of
the partially purified receptor mutant released a major digestion
product of 15.8 kDa comparable to fragment 228357 generated by the
same proteolytic cleavage of the wild-type receptor. This experiment
clearly identifies the 222357 fragment as the label-bearing segment
of the receptor protein (see Fig. 4
). Interestingly, the enzymatic
cleavage of the hAT1A221E mutant receptor with V8 protease
was almost complete after a 24-h incubation, whereas the digestion of
the wild-type receptor was still incomplete even after a 4-day
incubation.
To further delineate the binding domain for
125I-[Sar1,Bpa8]AngII,
photolabeled and undigested hAT1 receptor was submitted to
hydrolysis with CNBr, which cleaves specifically at the
carboxy-terminal side of methionine residue (17). Figure 5A
shows that, under these conditions, a
major digestion product of 7.5 kDa was obtained. Considering the
previous results obtained with V8 protease digestion and the location
of the methionine residues in this sequence (243, 284, and 334), this
apparent molecular mass identifies the fragment as the 285334
polypeptide of the seventh transmembrane domain/cytoplasmic tail of the
hAT1 receptor. To support this assumption and to narrow
down the size of the label-bearing receptor segment, the partially
purified 7.5-kDa CNBr fragment was submitted to Endo Lys-C proteolysis,
which specifically cleaves on the carboxylic side of lysine residue
(18). Figure 5B
shows that digestion of the 7.5-kDa CNBr fragment with
Endo Lys-C produced a 3.6 kDa fragment that could correspond to either
of the three peptides located between proline 285 and lysines 307, 308,
or 310 of the hAT1 receptor (Fig. 4
). The 7.5-kDa CNBr
fragment was also submitted to chemical hydrolysis with
2-nitro-5-thiocyanobenzoic acid (TNB-CN), which cleaves at the
amino-terminal side of cysteine residue (19). Figure 5B
indicates
that the 7.5-kDa CNBr fragment contained at least one cysteine residue
since it was converted to a 3.1-kDa fragment, upon cleavage with
TNB-CN. Indeed, the
125I-[Sar1,Bpa8]AngII binding
domain contains two cysteine residues at positions 289 and 296 (Fig. 4
). Complete cleavage at these residues should produce either the
labeled peptides 285288 or 289295. Both fragments are expected to
migrate with an estimated Mr of 1.7 and 2.2 kDa,
respectively. However, we could only detect a 3.1-kDa fragment probably
corresponding to the partial hydrolysis of the peptide at position 296,
which would generate the labeled 285295 fragment. The possibility
that the labeled fragment corresponds to peptide 296334 must be
excluded because it would be expected to migrate with an approximative
Mr of 6.4 kDa. Taken together, these results indicate that
125I-[Sar1,Bpa8]AngII labels the
fragment encompassing residues 285295 within the seventh
transmembrane domain of hAT1 receptor.

View larger version (78K):
[in this window]
[in a new window]
|
Figure 5. Chemical and Enzymatic Fragmentation of
125I-[Sar1,Bpa8]AngII-Labeled
AT1 Receptor
A, Partially purified photolabeled hAT1 receptor (65,000
cpm) was incubated in the absence (control) or the presence of CNBr
(100 mg/ml) for 24 h at room temperature in the dark. Samples were
then run on a 16.5% acrylamide Tris-Tricine separating gel followed by
autoradiography. The 7.5-kDa fragment was recovered by passive elution
from gel slices and in panel B was incubated in the absence (control)
or the presence of endo Lys-C (40 µg/ml) or 25 mM TNB-CN
as described in Materials and Methods. Samples were run
on a 16.5% acrylamide Tris-Tricine separating gel followed by
autoradiography. 14C-labeled protein standards of the
indicated molecular masses (kDa) were run in parallel.
|
|
Mapping the
125I-[Bpa1]AngII
Contact Point
125I-[Bpa1]AngII-photolabeled
hAT1 receptor was partially purified, as indicated in
Materials and Methods, and digested with Endo Lys-C, which
produced a broad band of approximately 31 kDa (Fig. 6A
). The high Mr and the
broadness of the band suggested a polypeptide that was glycosylated.
Treatment of the endo Lys-C digestion product with PNGase-F generated a
7.5-kDa fragment that migrated as a sharp band, indicating that the
polypeptide was completely deglycosylated (Fig. 6
). Similar results
were obtained with a simplified protocol in which the photolabeled
hAT1 receptor was simultaneously digested with Endo Lys-C
and PNGase-F (results not shown). Since potential sites for the
N-glycosylation of the hAT1 receptor are located in the
amino-terminal ectodomain and in the second extracellular loop, the
cleavage by Endo Lys-C must have occurred at the two lysine residues
located at positions 146 and 199 (Fig. 4
). Cleavage at these residues
would yield a fragment of 7.5 kDa (including the photolabeled analog of
1.3 kDa). Further narrowing of the contact point in this fragment was
not achieved on the native hAT1 receptor due to the very
low labeling yield obtained with 125I-[Bpa1]
AngII. To narrow down the contact point of
125I-[Bpa1]AngII, a mutant
hAT1I165M receptor was constructed and expressed in COS-7
cells. This mutant receptor showed a high affinity (0.9 nM)
for AngII and was expressed at a density (1.5 pmol/mg) comparable to
that of native AT1 receptor. Digestion of the photolabeled
mutant receptor with CNBr yielded a glycosylated fragment, which after
deglycosylation migrated with a Mr of 10.1 kDa,
corresponding to the only possible fragment 166243 (see Fig. 4
).
Taken together, these results suggest that
125I-[Bpa1]-AngII labels the fragment
encompassing residues 166199, which is essentially the second
extracellular loop of AT1 receptor.

View larger version (72K):
[in this window]
[in a new window]
|
Figure 6. Endo Lys-C Digestion of
125I-[Bpa1]AngII-Labeled AT1
Receptor
Partially purified photolabeled hAT1 receptor (150,000 cpm)
was initially incubated with Endo Lys-C (80 µg/µl) for 18 h at
37 C. An aliquot was then subjected to deglycosylation by
incubating with PNGase-F (80 U/ml) for 24 h at room temperature.
Samples were run on a 10% acrylamide separating gel followed by
autoradiography. 14C-labeled protein standards of the
indicated molecular masses (kDa) were run in parallel.
|
|
 |
DISCUSSION
|
---|
Numerous structure-activity studies performed on AngII have
demonstrated that the aromatic group of Phe8 at the C
terminus of the peptide carries the informationneeded for the
biological response (20). In the present work, we have used a
photoaffinity labeling approach with the aim to identify contact points
between the AT1 receptor and its peptide ligands. Our
results indicate that the C-terminal end of
[Sar1,Bpa8]AngII interacts with the inner
portion of the seventh transmembrane domain. Using a similar approach,
Desarnaud et al. (21) had suggested that the C-terminal
portion of Ang II interacts with the third transmembrane domain of
AT1 receptor. This apparent discrepancy is probably due to
the photochemical properties and pathways that are fundamentally
different for the two labeling moieties. Desarnauds study used the
4'-azidophenyl moiety, whereas in the present study we used the much
more efficient 4'-benzoyl-phenyl moiety. Although structurally the
reactive part of both molecules is at the same position corresponding
to the para-hydrogen of the phenyl nucleus in the endogenous ligand
AngII, they follow very distinct pathways. It was previously believed
that the azido-phenyl moiety produces an irreversible aryl nitrene upon
light activation and that this nitrene inserts into a carbon-hydrogen
bond in the close vicinity. Recent studies, however, have shown that
this type of arylnitrene is extremely short-lived and immediately
rearranges into a ketenimine (22). This latter moiety has a much longer
lifetime and is not able to undergo a C-H insertion but is susceptible
to nucleophile attack from solvent or from a polar protein sidechain.
This ketenimine is therefore either hydrolyzed (thus not incorporated)
or in the absence of water, it reacts with a Lys, Cys, Ser, or Asn
sidechain in the immediate vicinity or, if none of these side chains is
available, it reacts further away. The Bpa residue, however, produces a
reversible benzophenone radical that either reacts in its vicinity in a
nondiscriminative manner or hydrolyzes or rearranges. In the latter two
cases the benzophenone is reconstituted and can be reactivated again.
The C-H insertion reaction of the benzophenone radical is therefore
only possible in its immediate vicinity, and the incorporation yield is
inversely related to the distance. If high incorporation yields are
observed, then a close and tight interaction between the photosensitive
moiety and the receptor can be assumed. Low incorporation yields, on
the other hand, may be due to longer distances, the presence of water,
or a combination of both.
These different labeling mechanisms could explain the different results
obtained between the present study and that reported by Desarnaud
et al. Interestingly, this raises the possibility that the
third transmembrane domain is in close proximity to the seventh
transmembrane domain of AT1 receptor, since the secondary
photoproduct of the arylnitrene, the cyclic ketenimine, has to search
for a nucleophilic group. This suggestion is strongly supported by a
site-directed mutagenesis study that proposed that the spatial
proximity of Asn111 in the third transmembrane domain and
Asn295 in the seventh transmembrane domain of the
AT1 receptor could be responsible, in part, for maintaining
the receptor in a conformational constraint that would be relaxed upon
agonist binding (23). The proximity of the third and the seventh
transmembrane helices has also been suggested in three-dimensional
models of other GPCRs (24, 25, 26).
Binding domains on the AT1 receptor have also been
extensively studied using mutagenesis approaches coupled with molecular
modeling (for review, see Refs. 27, 28). Activation of the
AT1 receptor appears to require interaction of the
Phe8 side chain of AngII with His256 in the
sixth transmembrane domain, an interaction that is stabilized by
docking of the
-COOH group of Phe8 to Lys199
in the fifth transmembrane domain of AT1 receptor (29, 30).
It was also suggested that the aromatic Trp253 in the sixth
transmembrane domain stabilizes the ionic bridge formed between
Lys199 in the fifth transmembrane of AT1
receptor and the carboxylate anion of Phe8 of AngII (31).
The C-terminal residue of AngII would therefore be confined within a
binding pocket circumscribed by transmembrane domains III, V, VI, and
VII. Again, this is in agreement with the crucial role played by the
Phe8 side chain of AngII in activating the AT1
receptor (20) and the involvement of intrahelical amino acids in GPCR
activation upon agonist binding (32). In a related study, we have
previously shown that [Sar1,Bpa8]AngII
incorporates in the third transmembrane domain of AT2
receptor (11) (Fig. 7
). On the basis of
these results, it is tempting to speculate that the binding pockets of
AT1 receptor and AT2 receptor are different.
However, as mentioned for AT1 receptor, the third and the
seventh transmembrane domains of AT2 receptor may be in
close proximity, and hence local conformation brought about by the
different primary structure of AT2 receptor would
contribute to the formation of a distinct binding pocket.

View larger version (36K):
[in this window]
[in a new window]
|
Figure 7. Two-Dimensional Representation of AngII-Binding
Domains of the Human AT1 and Rat AT2 Receptors
The ligand-binding domains are represented by solid
circles and numbered 1 for those domains interacting with the
Bpa moiety of [Bpa1]AngII and 8 for those domains
interacting the Bpa moiety of
[Sar1,Bpa8]AngII. The amino-terminal end of
AngII would make contact with the hAT1 receptor within
residues 147199, whereas it would make contact with the extracellular
amino-terminal tail of the rAT2 receptor (11 ). The
carboxy-terminal end of AngII would make contact with the seventh
transmembrane domain of the hAT1 receptor (residues
285295), whereas it would make contact with the third transmembrane
domain of the rAT2 receptor (residues 129138) (11 ).
|
|
The results of the present study also indicate that the N-terminal
portion of [Bpa1]AngII interacts with a segment of the
second extracellular loop of AT1 receptor. These results
are in agreement with a previous study demonstrating how charged
residues of the second extracellular loop are major docking points for
the N-terminal amino acid of AngII. Using a site-directed mutagenesis
approach, Feng et al. (30) suggested that the N-terminal Asp
of AngII interacts with His183 found in the second
extracellular loop of AT1 receptor. Further studies on
receptor mutants with additional cleavage sites will be necessary to
narrow down the site of contact between AT1 receptor and
the N terminus of AngII.
The AngII molecule has an estimated length of 30 Å (probably less in
solution), which is about the size of a transmembrane domain (
40
Å). To account for the interaction of our AngII analogs with the
contact sites identified in this study, the second extracellular loop
must be in proximity to the seventh transmembrane domain of
AT1 receptor. In a previous study on the AT2
receptor, we also showed that the N terminus of the photoligand
interacts with an extracellular domain (the N-terminal ectodomain),
whereas the C terminus of the photoligand interacts with the third
transmembrane domain of AT2 receptor (Fig. 7
) (11). A
similar pattern of peptide binding was also found when the binding
domains of NK-1 receptor were examined with its cognate ligand
Substance P. Using a photoaffinity-labeling approach, it was proposed
that the C terminus of Substance P inserts into a binding pocket formed
between transmembranes II and VI, whereas the N-terminus of the peptide
interacts with an extracellular loop of the receptor (10).
It has been determined that the binding domain of heptahelical
receptors for several small physiological ligands, like bioamines, are
formed by residues located within the transmembrane helices (32). On
the other hand, glycoprotein hormone receptors provide only
extracellular regions as docking sites for their much larger ligands.
Indeed, although extracellular domains of receptors have been
implicated for the binding of glycoprotein hormones like TSH and FSH,
recent studies have shown that small ligands, such as glutamate and
calcium ion, can also interact with these regions. These ligands appear
to bind to extracellular sites located in an elongated N terminus of
their respective receptors (33). As suggested in the present study, the
principle that governs small peptide hormone binding probably
encompasses a mixture of both these above mentioned situations. The
size of peptides conceivably allows them to find their way to the lower
portion of the transmembrane domains. The fact that [Sar1,
Bpa8]Ang was characterized as an antagonist in functional
studies should not invalidate our findings with respect to the
agonistic behavior of AngII. It has been suggested by site-directed
mutagenesis that nonpeptide antagonists do not dock to the same parts
of the AT1 receptor. Contrary to nonpeptide ligands,
structure-activity relationship studies by us and others (20, 35),
using a variety of AngII peptide analogs, indicate that AngII peptide
antagonists and agonists have highly similar interaction profiles on
the AT1 receptor.
Furthermore, in favor of a similar positioning of
[Sar1]AngII (Kd of 0.9 mM) and of
[Sar1, Bpa8]AngII within the AT1
receptor are studies on the agonist-to-antagonist transition observed
with analogs having aromatic substitutions in position 8. Increasing
the bulk of the aromatic nucleus gradually decreases efficacy until, on
rabbit aorta strips and on IP3 production, agonistic
behavior can no longer be observed but strong binding is evident
(37). It is therefore highly unlikely that the
Phe8
Bpa8 transition substantially alters or
disturbs the peptide orientation in the AT1 binding
pocket.
In conclusion, we have identified two peptide-binding regions in the
AT1 receptor with the use of specific AngII photoreactive
analogs. This approach also allowed the determination of the ligand
molecules orientation in its binding pocket. Our results would
support a model in which the C terminus of AngII interacts with the
seventh transmembrane domain of AT1 receptor and the
N-terminus of AngII interacts with the second extracellular loop of
AT1 receptor. In future experiments, it will be important
to identify which amino acid, within the identified fragments,
interacts with the photoreactive analog. In addition, systematic
mapping of the contact points between the AT1 receptor and
AngII analogs containing the Bpa moiety at all possible positions could
be revealing. At present, we believe that models of the
three-dimensional structure of the AT1 receptor-binding
pocket should accommodate the results of the present study.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Infection/Transfections
COS-7 cells were grown in DMEM containing 2 mM
L-glutamine and 10% (vol/vol) FBS. Cells were seeded in
10-cm2 plates and infected at a density of 1.52 x
106 cells with a Vaccinia virus recombinant
expressing the T7 RNA polymerase (VV:TF73) as previously described
(38). Briefly, cells were infected for 1 h at 37 C with VV:TF73
(multiplicity of infection = 2), washed once with serum-free DMEM,
and transfected with 4 µg of pcDNA3 into which was inserted the DNA
encoding human AT1 (kindly provided by Dr. S. Meloche,
Université de Montréal) and 30 µl of Lipofectamine
(GIBCO/BRL, Gaithersburg, MD) in 8 ml of serum-free DMEM, according to
the manufacturers instructions. The cells were incubated for 5 h
at 37 C, and the media were replaced with complete DMEM containing 100
IU/ml penicillin and 100 µg/ml streptomycin. Infected/transfected
cells were grown for 1824 h before binding assays or photoaffinity
labeling. The hAT1 I165M mutant was constructed as already reported
elsewhere (16) and transfected as described above.
Binding Assays and Photoaffinity Labeling
[Bpa1]AngII and
[Sar1,Bpa8]AngII were synthesized in our
laboratories as previously described (13). 125I-AngII,
125I-[Bpa1]AngII, and
125I-[Sar1,Bpa8]AngII (specific
radioactivity,
1000 Ci/mmol) were prepared with iodogen as described
by Fraker and Speck (39) but in an acetic acid buffer (pH 5.4). The
labeled peptides were purified by HPLC on a C-18 column (10 µm)
(Allteck Associates Inc., Deerfield, IL; No 29004) with a 2040%
acetonitrile gradient. The specific radioactivity of the labeled
hormones was determined by self-displacement and saturation binding
analysis.
Infected-transfected COS-7 cells were gently scraped and
centrifuged at 200 x g for 10 min at 4 C. The pellet
was washed once with ice-cold binding medium without BSA (25
mM Tris-HCl, pH 7.4, 100 mM NaCl, 5
mM MgCl2, 0.1 mg/ml soybean trypsin inhibitor,
0.1 mg/ml bacitracin) and resuspended in binding medium containing 2
mg/ml BSA. In binding experiments, COS-7 cells were incubated in
binding medium containing 0.2 nM of
[125I]AngII and selected concentrations of
[Sar1,Bpa8]AngII or
[Bpa1]AngII. Incubations were performed at room
temperature for 60 min. Nonspecific binding was measured in the
presence of 1 µM of unlabeled AngII. Bound radioactivity
was separated from free ligand by filtration through GF/C filters
presoaked in binding buffer. In photoaffinity-labeling experiments,
cells were incubated with 5 nM of
125I-[Sar1,Bpa8]AngII or 45
nM of 125I-[Bpa1]AngII in the
presence or absence of 1 µM L158,809 (an
AT1-selective, nonpeptide analog, Merck & Co., Inc.,
Wilmington, DE) or 1 µM AngII. After a 60-min
incubation period at room temperature, cells were washed once with
ice-cold binding medium (without BSA) and irradiated for 60 min at 0 C
under filtered (Raymaster black light filters number 5873, George W.
Gates & Co. Inc., Franklin Square, NY) UV light (365 nm) (2
x 100 W mercury vapor lamp JC-Par-38, Westinghouse Electric Corp.,
Pittsburgh, PA). Cells were centrifuged at 200 x g for 10 min at
4 C. The pellet was solubilized in a buffer containing 100
mM Na2HPO4, pH 8.5, 25
mM EDTA, 0.1 mg/ml soybean trypsin inhibitor, and 1%
(vol/vol) Nonidet P-40 and then submitted to one cycle of freezing and
thawing. Cell lysates were centrifuged at 13,000 x g
for 10 min at 4 C to remove insoluble material, and supernatants were
kept at -80 C until further analysis.
Partial Purification of the Labeled Complex
The solubilized photolabeled receptors were diluted with
an equal volume of 2x loading buffer [120 mM Tris-HCl, pH
6.8, 20% (vol/vol) glycerol, 4% (wt/vol) SDS, 200 mM
dithiothreitol, and 0.05% (wt/vol) bromophenol blue] and incubated
for 1 h at 37 C. SDS-PAGE was performed as described by Laemmli
(40) using 8% gels. The gel was then dried and exposed to x-ray film
(Kodak BIOMAX MS; Eastman Kodak, Rochester, NY) with an intensifying
screen. After autoradiography, radioactive bands were excised from
dried gels and rehydrated with appropriate digestion buffer. The gel
slices were macerated and eluted with 2 ml of buffer for 34 days at 4
C under gentle agitation. The eluted proteins were filtered (Acrodisc
0.45 µm; Gelman Sciences, Inc., Ann Arbor, MI), and the gel slices
were washed with 10 ml of digestion buffer. The whole eluate (1215
ml) was concentrated approximately 150 times using Centriprep-10 and
Centricon-10 cartridges (Amicon, Inc., Beverly, MA), and the partially
purified proteins were aliquoted and kept at -80 C. This passive
elution protocol is similar to that described by Blanton and Cohen
(41).
Proteolytic, Chemical, and Endoglycosidase Digestions
For endoglycosidase digestions, partially purified photolabeled
receptors were resuspended in digestion buffers containing 0.2%
(vol/vol) Nonidet P-40. PNGase-F (33100 U/ml) was added and samples
were incubated overnight at room temperature.
For proteolytic digestions, partially purified photolabeled
receptors (10,000300,000 cpm) were resuspended in 25 µl of
digestion buffer containing 100 mM
NH4HCO3, pH 8.0, and 0.1% (wt/vol) SDS.
Samples were incubated for 34 days at room temperature with indicated
amounts of V8 protease. Partially purified photolabeled receptors were
also digested for 1824 h at 37 C with indicated amounts of endo Lys-C
in 25 µl of digestion buffer containing 25 mM Tris-HCl,
pH 8.5, 1 mM EDTA, and 0.1% (wt/vol) SDS. All digestions
were terminated by adding an equal volume of 2x loading buffer and
boiling the samples for 3 min. When subsequent digestions were needed,
products of the first digestion were identified by SDS-PAGE followed by
autoradiography and recovered from nonfixed gels by passive elution for
1218 h at 37 C with 500 µl of digestion buffer. Extracted proteins
were lyophilized and submitted to chemical or enzymatic digestion as
described.
For CNBr hydrolysis, partially purified photolabeled receptors
(60,000450,000 cpm) were resuspended in 50 µl of 70% (vol/vol)
trifluoroacetic acid, and CNBr (50 µl) was added to a final
concentration of 100 mg/ml. Samples were incubated at room temperature,
in the dark, for 2436 h. Reactions were terminated by adding 500 µl
of water. Samples were lyophilized, resuspended in denaturing buffer,
and analyzed by SDS-PAGE. TNB-CN hydrolysis was performed using a
modified procedure (19). Briefly, the isolated CNBr fragment was
resuspended in digestion buffer containing 100 mM Tris-HCl,
pH 8.5, 5 mM DTT, and 0.1% (wt/vol) SDS and incubated at
37 C for 30 min. A 5-fold excess of TNB-CN (25 mM) over
thiol present was added, and the pH was readjusted, if necessary, to
8.5 with NaOH. The mixture was then allowed to stand at room
temperature for 60 min. An equal volume of 200 mM sodium
borate buffer, pH 10.5, was added and the reaction was incubated at 70
C for 2 h. Hydrolysis was terminated by adding an equal volume of
2x loading buffer.
Analysis of Products of Proteolysis and Chemical Cleavage
The products of proteolysis and deglycosylation were analyzed by
SDS-PAGE using 16.5% acrylamide Tris-Tricine gels (Bio-Rad, Richmond,
CA) followed by autoradiography on x-ray films (Kodak BIOMAX MS).
14C-labeled low molecular protein standards (GIBCO/BRL)
were used to determine apparent molecular masses. Running conditions
and fixation and coloration of gels were performed according to the
manufacturers instructions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Emanuel Escher, Ph.D., Département de pharmacologie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, CANADA. E-mail: e.escher{at}courrier.usherb.ca
This work was supported by grants from the Heart and Stroke Foundation
of Canada (HSFC) and the Medical Research Council of Canada (MRCC).
S.A.L. and J.P. are recipients of studentships from HSFC. G.S. is
the recipient of a studentship from MRCC-Ciba-Geigy. E.E. is the
recipient of the J.C. Edwards Chair in Cardiovascular Research. G.G. is
the recipient of an MRCC Scientist Award. R.L. is a scholar from the
Fonds de la Recherche en Santé du Québec.
This work is part of the Ph.D. thesis of S.A.L.
Received for publication May 18, 1998.
Revision received December 17, 1998.
Accepted for publication January 14, 1999.
 |
REFERENCES
|
---|
-
Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ,
Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, PBM Timmermans WM 1989 Identification of angiotensin II receptor subtypes. Biochem
Biophys Res Commun 165:196203[Medline]
-
Bumpus FM, Catt KJ, Chiu AT, De Gasparo M,
Goodfriend T, Husain A, Peach MJ, Taylor DG, PBM Timmermans WM 1991 Nomenclature for angiotensin receptors. A report of the Nomenclature
Committee of the Council for High Blood Pressure Research. Hypertension 17:720721[Medline]
-
Peach MJ 1981 Molecular actions of angiotensin.
Biochem Pharmacol 80:27452751[CrossRef]
-
Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK 1995 Behavioural and cardiovascular effects of disrupting the
angiotensin II type-2 receptor in mice. Nature 377:744747[CrossRef][Medline]
-
Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa
Y, Fogo A, Niimura F, Ichikawa I, Hogan BLM, Inagami T 1995 Effects on
blood pressure and exploratory behaviour of mice lacking angiotensin II
type-2 receptor. Nature 377:748750[CrossRef][Medline]
-
Dohlman HG, Caron MG, Strader CD, Amlaiky N,
Lefkowitz RJ 1988 Identification and sequence of a binding site peptide
of the beta-adrenergic receptor. Biochemistry 27:18131817[Medline]
-
Wong SK-F, Slaughter C, Ruoho AE, Ross EM 1988 The
catecholamine binding site of the ß-adrenergic receptor is formed by
juxtaposed membrane-spanning domains. J Biol Chem 263:79257928[Abstract/Free Full Text]
-
Janovick JA, Haviv F, Fitzpatrick TD, Conn PM 1993 Differential orientation of a GnRH agonist and antagonist in the
pituitary GnRH receptor. Endocrinology 133:942945[Abstract]
-
Kennedy AP, Mangum KC, Linden J, Wells JN 1996 Covalent modification of transmembrane span III of the AT1
adenosine receptor with an antagonist photoaffinity probe. Mol
Pharmacol 50:789798[Abstract]
-
Li Y-M, Marnerakis M, Stimson ER, Maggio JE 1995 mapping peptide-binding domains of the substance P (NK-1) receptor from
P388D1 cells with photolabile agonists. J Biol Chem 270:12131220[Abstract/Free Full Text]
-
Servant G, Laporte SA, Leduc R, Escher E,
Guillemette G 1997 Identification of angiotensin II-binding domains in
the rat AT2 receptor with photolabile angiotensin analogs.
J Biol Chem 272:86538659[Abstract/Free Full Text]
-
Servant G, Dudley DT, Escher E, Guillemette G 1994 The marked disparity between the sizes of angiotensin type 2
receptors from different tissues is related to differ-ent
degrees of N-glycosylation. Mol Pharmacol 45:11121118[Abstract]
-
Bossé R, Servant G, Zhou L-M, Guillemette G,
Escher E 1993 Sar1-p-benzolyphenylalanine-angiotensin, a new
photoaffinity probe for selective labeling of the type 2 angiotensin
receptor. Regul Pept 44:215223[CrossRef][Medline]
-
Laporte S, Servant G, Escher E, Guillemette G, Leduc
R 1996 The tyrosine within the NPXnY motif of the human angiotensin II
type 1 receptor is involved in mediating signal transduction but is not
essential for internalization. Mol Pharmacol 49:8995[Abstract]
-
Houmard J, Drapeau GR 1972 Staphylococcal protease:
a proteolytic enzyme specific for glutamoyl bonds. Proc Natl Acad Sci
USA 69:35063509[Abstract]
-
Laporte SA, Roy SF, Escher E, Guillemette G, Leduc R 1998 Essential role of leucine 222 in mediating signal
transduction of the human angiotensin II type 1 receptor. Recept
Channels 5:103112[Medline]
-
Nikodem V, Fresco JR 1979 Protein fingerprinting by
SDS-gel electrophoresis after partial fragmentation with CNBr. Anal
Biochem 97:382386[Medline]
-
Jekel PA, Weijer WJ, Beintema JJ 1983 Use of
endoproteinase Lys-C from Lysobacter enzymogenes in protein
sequence analysis. Anal Biochem 134:347354[Medline]
-
Jacobson GR, Schaffer MH, Stark G, Vanaman TC 1973 Specific chemical cleavage in high yield at the amino peptide bonds of
cysteine and cystine residues. J Biol Chem 248:65836591[Abstract/Free Full Text]
-
Regoli D, Park WK, Rioux F 1974 Pharmacology of
angiotensin. Pharmacol Rev 26:69123[Medline]
-
Desarnaud F, Marie J, Lombard C, Larguier R, Seyer
R, Lorca T, Jard S, Bonnafous J-C 1993 Deglycosylation and
fragmentation of purified rat liver angiotensin II receptor:
application to the mapping of hormone-binding domains. Biochem J 289:289297[Medline]
-
Platz MS 1995 Photolysis of di-, tri- and
tetrafluoroinated phenylnitrenes; implications for photoaffinity
labeling. Acc Chem Res 28:487492
-
Balmforth AJ, Lee AJ, Warburton P, Donnelly D, Ball
SG 1997 The conformational change responsible for AT1 receptor
activation is dependent upon two juxtaposed asparagine residues on
transmembrane helices III and VII. J Biol Chem 272:42454251[Abstract/Free Full Text]
-
Baldwin JM 1993 The probable arrangement of the
helices in G protein-coupled receptors. EMBO J 12:16931703[Abstract]
-
Schertler GFX, Villa C, Henderson R 1993 Projection
structure of rhodopsin. Nature 362:770772[CrossRef][Medline]
-
Donnely D, Findlay JBC, Blundell TL 1994 The
evolution and structure of aminergic G protein-coupled receptors.
Receptors Channels 2:6178[Medline]
-
Karnik SS, Husain A, Graham RB 1996 Molecular
determinants of peptide and non-peptide binding to the AT1
receptor. Clin Exp Pharmacol Physiol Suppl 23:S58S66
-
Hunyady L, Balla T, Catt KJ 1996 The ligand binding
site of the angiotensin AT1 receptor. Trends Pharmacol Sci 17:135140[CrossRef][Medline]
-
Noda K, Saad Y, Karnik SS 1995 Interaction of
Phe8 of angiotensin II with Lys199 and
His256 of AT1 receptor in agonist activation.
J Biol Chem 270:2851128514[Abstract/Free Full Text]
-
Feng Y-H, Noda K, Saad Y, Liu X-p, Husain A, Karnik
SS 1995 The docking of Arg2 of angiotensin II with
Asp281 of AT1 receptor is essential for full
agonism. J Biol Chem 270:1284612850[Abstract/Free Full Text]
-
Yamano Y, Ohyama K, Kikyo M, Sano T, Nakagomi Y,
Inoue Y, Nakamura N, Morishima I, Guo D-F, Hamakubo T, Inagami T 1995 Mutagenesis and the molecular modeling of the rat angiotensin II
receptor (AT1). J Biol Chem 270:1402414030[Abstract/Free Full Text]
-
Strader CD, Ming Fong T, Tota MR, Underwood D 1994 Structure and function of G protein-coupled receptors. Annu Rev Biochem 63:101132[CrossRef][Medline]
-
Baldwin JM 1994 Structure and function of receptors
coupled to G proteins. Curr Opin Cell Biol 6:180190[Medline]
-
Deleted in proof
-
Cordopatis P, Manessi-Zoupa E, Theodoropoulos D,
Bosse R, Bouley R, Gagnon S, Escher E 1994 Methylation in positions 1
and 7 of angiotensin II. A structure-activity relationship study. Int J
Pept Protein Res 44:320324[Medline]
-
Deleted in proof
-
Pham ND, Laporte SA, Pérodin J, Bourgeois R,
Escher E 1996 What can angiotensin antagonists do that
converting-enzyme inhibition cant: the case of post-angioplastic
restenosis. Can J Physiol Pharmacol 74:867877[CrossRef][Medline]
-
Leduc R, Molloy S, Thorne BA, Thomas G 1992 activation of Human furin precursor processing occurs by an
intramolecular autoproteolytic cleavage. J Biol Chem 267:1430414308[Abstract/Free Full Text]
-
Fraker PJ, Speck JC 1978 Protein and cell membrane
iodinations with a sparingly soluble chloroamide,
1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res
Commun 80:849857[Medline]
-
Laemmli UK 1970 Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 227:680683[Medline]
-
Blanton MP, Cohen JB 1994 Identifying the
lipid-protein interface of the Torpedo nicotinic acetylcholine
receptor: secondary structure implications. Biochemistry 33:28592872[Medline]