From the Institute of Biological Chemistry, University of Parma,
43100 Parma, Italy, the Institute of Chemistry,
Department of Biochemistry, University of São Paulo, Caixa
Postale 26077, 05599-970, São Paulo, Brazil, and the
¶ Department of Biophysics, Federal University of São
Paulo, Caixa Postale 04044-020, São Paulo Brazil
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ABSTRACT |
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The structural bases that render the third
intracellular loop (i3) of the rat angiotensin II AT1A
receptor one of the cytoplasmic domains responsible for G-protein
coupling are still unknown. The three-dimensional structures of two
overlapping peptides mapping the entire i3 loop and shown to
differently interact with purified G-proteins have been obtained by
simulated annealing calculations, using NMR-derived constraints
collected in 70% water/30% trifluoroethanol solution. While the
NH2-terminal half, Ni3, residues 213-231, adopts a stable
amphipathic The comprehension of the molecular details of G-protein coupled
receptors (GPCRs)1 activation
as well as of G-protein selection and coupling is still speculative.
Similarly, several features of their three-dimensional structure still
need to be defined. In this respect, while a large effort is being
generated to define the orientation and three-dimensional organization
of the transmembrane helices of GPCRs (1-5), not much has been done to
describe the structural properties of the regions exposed to the
extracellular or cytoplasmic media (6).
These considerations, and the results obtained on that subject for
rhodopsin (7-11), for the In a previous work, we focused our attention on the conformational
flexibility of a fragment of the AT1A COOH-terminal tail (17); here we show the existence and propose a model describing the
dynamic features of the structural determinants that characterize the
receptor third intracellular loop (i3) (Fig.
1).
-helix, extending over almost the entire peptide, a more
flexible conformation is found for the COOH-terminal half, Ci3,
residues 227-242. For this peptide, a cis-trans
isomerization around the Lys6
Pro7 peptide
bond generates two exchanging isomers adopting similar conformations,
with an
-helix spanning from Asn9 to Ile15
and a poorly defined NH2 terminus. A quite distinct
structural organization is found for the sequence EIQKN, common to Ni3
and Ci3. The data do suggest that the extension and orientation of the
amphipathic
-helix, present in the proximal part of i3, may be
modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is
considered as a mechanism for G-protein selection and coupling is presented.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
- and
-adrenergic receptors (12-14),
and for the parathormone receptor (15, 16) have prompted us to
undertake a study on some functionally relevant cytoplasmic domains of
the angiotensin II AT1A receptor, mainly seeking to describe the structural and dynamic properties of the receptor surface
that regulate its interaction with the various G-proteins.
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Fig. 1.
Amino acid sequence (32) and schematic
representation of the rat AT1A receptor. The
synthesized peptide sequences studied in this work and named Ni3
(residues 213-231) and Ci3 (residues 227-242) are indicated by
solid lines.
As for various GPCRs such as the -adrenergic, muscarinic, dopamine,
and rhodopsin receptors (18-23), studies involving receptor chimeras
and site-directed mutagenesis (24-28) indicate that i3 is one of the
AT1A functional domains involved in G-protein interaction. In addition, comparison of the i3 of several GPCRs evidences a noticeable heterogeneity in amino acid sequence and size (29-32), suggesting that the secondary structure, rather than the primary sequence and/or the specific length of that domain, plays a key role in
G-protein coupling.
Recent investigations revealed that a synthetic peptide representing
the proximal part of i3 (residues 216-230) is able to activate
purified Gi and G0 proteins, while the peptide
comprising the distal part of that loop (residues 229-242) has no
effect (33). Similar results have been obtained in a study focused on
the activation of Gq, where it has been shown that the
peptide encompassing residues 216-231 is active, while the one
representing the i3 segment 230-241 does not exhibit any activity
(34). Interestingly, the proximal half of i3 has been predicted to have
a very high probability to adopt an amphipathic -helical structure,
whereas the distal half is predicted to form a short helix only at its COOH-terminal end (35, 36).
On the basis of these evidences, we have studied the solution conformation of two synthetic fragments mapping the entire i3 loop, the NH2-terminal 19-mer TSYTLIWKALKKAYEIQKN-NH2, Ni3 (residues 213-231), and the C COOH-terminal 16-mer EIQKNKPRNDDIFRII-NH2, Ci3 (residues 227-242). The underlined residues indicate the overlapping region between the two peptides.
The structures of the two peptides in 70% H2O/30% TFE
were obtained by means of restrained molecular dynamics calculations using, as restraints, the NMR-derived proton distances and dihedral angles.
The results show that Ni3 is characterized by a well defined
amphipathic -helix extending over almost the entire peptide sequence.
For Ci3, the data indicate the existence of cis-trans
isomerization about the Lys6Pro7 peptide bond
giving rise to two slowly exchanging conformational states. The
resulting isomers adopt very similar secondary structures characterized
by a poorly defined NH2 terminus and by a flexible amphipathic
-helix in the COOH-terminal stretch
(Asn9-Ile15). Interestingly, the sequence EIQKN
common to Ni3 and Ci3, and corresponding to the central part of i3,
adopts a quite distinct structural organization in the two peptides
suggesting, for Pro233, a functional role as structure
breaker or modulator (37, 38).
The results of this work, together with our previous study (17),
provide the basis for disclosing some conformational features of the
native receptor cytosolic face. In particular, the data support the
hypothesis that the capability of specific domains of the receptor to
form amphipathic -helices is essential for receptor activation and
G-protein selection and coupling.
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MATERIALS AND METHODS |
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Peptide Synthesis and Purification-- The two peptides corresponding to residues 213-231 (Ni3) and 227-242 (Ci3) of the i3 loop of the rat angiotensin II AT1A receptor (Fig. 1) have been synthesized by the solid phase method using t-butoxycarbonyl chemistry. Optimized coupling conditions were introduced according to the resin solvation theory (39). The peptides were purified by high pressure liquid chromatography, and their purity and molecular weight were confirmed by mass spectrometry. Fig. 5 shows the sequences with both the numbering for the residues of the peptides and the one from the primary sequence of rat AT1A receptor (32).
Circular Dichroism Spectroscopy--
Far UV CD spectra were
recorded on a Jasco J-715 spectropolarimeter using a Peltier system
PTC-348 WVI for cell temperature control. Ellipticity is reported as
the mean residue molar ellipticity, [] (deg cm2
dmol
1). The instrument has been calibrated with
recrystallized d-10-camphorsulfonic acid. The
H2O/TFE cross-titration experiments were carried out mixing
the appropriate aliquots of two 0.15 mM stock solutions, one in water at pH
4 and the other in TFE. A 1 mm cell was used.
NMR Spectroscopy--
NMR samples were prepared in 70%
H2O/30% TFE-d3 (v/v), pH 4, to yield a peptide concentration of about 2 mM for both
peptides. All two-dimensional 1H-NMR experiments were
recorded and processed by the procedures elsewhere described (17).
Computational Procedures--
All calculations were carried out
on a Silicon Graphics ONYX computer as described previously (17). The
structures of the two peptides were computed using the final set of
NOE-derived distance constraints listed in Table III together with the
dihedral angles. The quality of the final structures was verified
on the basis of the minimum number of NOE distance and
dihedral
angle violations as well as of the minimum root mean square deviation values of the backbone atoms in the region of interest. For NOE distance and
dihedral angle violations, an upper limit of 0.3 Å and 5 degrees has been used, respectively.
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RESULTS |
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CD Experiments--
The CD spectra of both peptides did not
indicate the existence of any preferential secondary structure in
aqueous solution (Fig. 2), and they were
not affected by changes in concentration within the 0.15-1.5
mM range.2
However, upon addition of small aliquots of TFE, it was found that Ni3
exhibited a transition to an -helical conformation the extension of
which increased up to 30% TFE (Fig. 2A), where it stabilized at an helical content of ~55%, as calculated according to
the method of Chen et al. (40). For Ci3, instead, only a partial folding into an ill defined helical structure could be observed
at high TFE concentration (Fig. 2B). In this case, however, due to the complexity of the spectrum suggesting the existence of a
multiple conformation equilibrium, the use of the same method to
evaluate the peptide
-helical content (Fig. 2B) appears
not to be ideal. A detailed analysis of the CD data will be the subject of a work in preparation.2
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1H-NMR Experiments--
According to the CD results,
all 1H-NMR experiments were recorded in 70%
H2O/30% TFE-d3 (v/v). The complete
assignment of the proton resonances for the two peptides was obtained
using standard two-dimensional methods (41). The spectra of Ci3
displayed two distinct sets of resonances for the stretch
Lys6-Arg14 (Table
I), suggesting the existence of a
cis-trans isomerization about the
Lys6Pro7 peptide bond. The correct
assignments of the protons corresponding to the two conformers was
based on the observation, in the NOESY and rotating frame nuclear
Overhauser effect spectra, of the characteristic connectivities between
Lys6
H and Pro7
H for the cis
isomers and between Lys6
H and Pro7
H for
the trans isomers. The relative intensity of the peaks indicates that the cis-trans molar ratio turns out to be
~1:1.5, suggesting that the two isomers are energetically quite
similar.
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The chemical shifts for Ci3 and Ni3 are listed in Tables I and II, respectively. The proton resonances of the common sequence EIQKN present significant differences in the two peptides, indicating that the amino acids flanking that domain play an important role in defining its secondary structure.
Evidence of Secondary Structure--
The conformational features
of the two peptides have been derived by the analysis of complementary
NMR parameters such as the -proton chemical shift perturbation, the
3JHN-
coupling constants and the
NOEs pattern.
The -proton residual chemical shifts (42) are reported in Fig.
3. In the case of Ni3 (Fig.
3A), it is evident that, except for Ser2 and
Leu5, all the
-proton resonances move upfield with
respect to the random coil values, thus suggesting the existence of a
helical structure extending from Ile6 to
Asn19.
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The presence of sequential dNN (i,
i + 1) NOEs, spanning almost the entire Ni3 sequence in the
150-ms NOESY spectrum (Fig. 4A), indeed supports the
existence of an -helical conformation whose stability is confirmed
by the characteristic d
N (i, i + 3) connectivities encompassing residues
Tyr3-Asn19 (Fig. 4B). The summary
of the interresidue NOEs is reported in Fig.
5A, showing a good correlation
with the
CH secondary shifts (Fig. 3A). Finally, except
for Asn19, the majority of the NH-
CH coupling constants
(3JHN-
) are in the range of
5.60-6.5 Hz (Table II), as expected for
a peptide in
-helical conformation.
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On the other hand, knowing that a minimum of 4 adjacent residues with a
negative deviation of the CH chemical shifts is necessary to assess
the presence of a stable helical organization, and that its stability
is proportional to the intensity of such deviation (42), the secondary
shifts plot for Ci3 (Fig. 3B) indicates that, in this case, a helical
folding is possible only in the peptide COOH-terminal portion. In
addition, except for Arg8 and Asp10, all the
residues do not exhibit any significant difference between the
trans and the cis isomers (Fig. 3B) suggesting
that Ci3, in the two states, adopt very similar conformations.
Indeed, the analysis of the NOESY and rotating frame nuclear Overhauser
effect spectra carried out for both the trans and cis isomers evidenced nearly no difference. Thus, Fig. 5B
shows the summary of the interresidue NOEs only for the relatively more populated trans isomers. As for Ni3, a good correlation with
the CH secondary shifts (Fig. 3B) can be observed. In fact, adjacent NH-NH correlations, together with medium and weak intensities d
N (i, i+3) and
d
(i, i+3) NOEs were found from Asp10 to Ile16, indicating the presence of a
helical conformation only in the COOH-terminal portion of the peptide.
Moreover, the coexistence of medium and weak intensity (i,
i+3) NOEs together with the intense d
N
(i, i+1) correlations, suggests the presence of
conformational fluctuations in that stretch.
The N-terminal half of Ci3, including the first five amino acids that
belong to a well defined -helix in the COOH-terminal half of Ni3,
clearly does not adopt any stable secondary structure. Except for the
Asn5 NH-Lys6 NH connectivity, the other medium
range NOEs associated to the presence of helical regions are
unambiguously absent from the spectra. Finally, in the Pro-containing
stretch, NOE connectivities reminiscent of the presence of a
-turn
were not found.
Three-dimensional Structure Calculations of Ni3 and Ci3-- Following the restrained molecular dynamics protocol described under "Materials and Methods," 80 possible three-dimensional structures were calculated for both peptides. After minimization, 20 structures for each peptide were selected to represent their solution conformation. The structural statistics are reported in Table III.
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Fig. 6A displays the Ni3
structures superimposed, for the minimum backbone deviation, between
residues Tyr3 and Lys18. The existence of a
stable and well defined -helical folding, involving most of the
peptide, is also supported by the analysis of the main chain hydrogen
bonds (Table III) and of the
and
dihedral angles (data not
shown), carried out using the program DSSP (43).
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In the case of Ci3, Fig. 6B refers to the
trans-Pro conformers, and the superimposition of the
backbone atoms has been made for the
Asn9-Ile15 stretch. As previously mentioned,
discussing the NMR data, the structures of the cis and
trans isomers are quite similar, with an -helical
conformation extending over the COOH-terminal peptide stretch, only.
Because of the limited number of NMR constraints available, the
NH2-terminal region preceding Asn9 is poorly
defined and no elements of stable ordered secondary structure have been detected.
Fig. 6 (bottom) shows a schematic representation of the
least energy structure for the two peptides evidencing the amphipathic nature of the -helical regions.
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DISCUSSION |
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Peptide Structural Features-- There is a general consensus (6, 44-46) on the hypothesis that the specific conformational changes of GPCRs cytosolic domains, induced by agonist binding, are critical for their activation. Since experimental observations with substitution or deletion in receptor mutants cannot rule out the induction of indirect conformational effects, the use of synthetic peptides representing defined receptor regions allows the performance of experiments in a more conformationally controlled manner. The validity of this approach is based on the recognition that if the isolated fragments retain the biological function they have in the receptor (e.g. G-protein activation), it is likely that their folding and conformational dynamics are similar to the ones they undergo in the native receptor (33, 44).
Due to the recognized relevance of the i3 loop as a critical region for GPCRs to express their activity, many attempts are being carried out to identify the domains involved in the complex molecular process of G-protein coupling (6, 44, 45, 47-53). In the specific case of the rat AT1A receptor, i3 has been found to play a pivotal role, with distinct biological functions exerted by its proximal and distal portions (24, 25, 54).
In this study, to verify the existence of structural determinants that may help to describe the conformation and dynamics of the i3 domains involved in G-protein coupling, we have characterized the solution structure of two overlapping fragments of the AT1A i3 loop, corresponding to its proximal and distal halves (residues 213-231 and 227-242, respectively) and encompassing the regions that have been suggested to exhibit distinct biological activity (33, 34).
Not surprisingly, in aqueous solution both peptides turned out to be unstructured, as clearly indicated by their CD spectra (Fig. 2). The tendency of small peptides to adopt spontaneously a preferential conformation is often very weak; in fact, they rapidly interchange between various conformations, each of them adopted only by a small fraction of the molecules at any time. Thus, a useful tool to explore their intrinsic capability to fold into some sort of helical conformation (helicity) is the use of TFE as a cosolvent (55-58).
Secondary structure prediction indicates that the proximal part of i3
has a high propensity to adopt a helical structure in the receptor (35,
36). Indeed, the data presented in this study (Figs. 2A and
6A) clearly show that upon addition of small amounts of TFE,
Ni3 folds into an amphipathic -helix extending over almost the
entire peptide. Since most of the helical peptides do not present
medium range NOE connectivities to their termini, at room temperature,
either in aqueous solution (59, 60) or in the presence of the
helix-stabilizing solvent TFE (55, 61), the observation that for Ni3
the helical structure in those regions is not extensively frayed
strongly suggests that, in this case, when formed the helix is highly
stable. In fact, only the first two amino acids, residues 213 and 214 in the receptor, appear to be flexible (Fig. 6A).
For the distal part of i3, instead, the prediction (35, 36) indicates the existence of a helical organization only in the COOH-terminal portion. The experimental data show that, in the presence of TFE, not only the NH2-terminal portion of Ci3 is characterized by the absence of any preferential secondary structure (Fig. 6B), but also that the helix present in the COOH-terminal region is quite flexible.
Structure-Function Correlation-- In an attempt to correlate the observed structural determinants with their possible functional role, it is important to accept the hypothesis that conformational adaptability is an essential feature.
The comparison of the secondary structure of Ni3 and Ci3 shows that the
common sequence EIQKN adopts a quite different organization in the two
peptides: it is part of a well defined -helix in Ni3, while it is
highly disordered in Ci3, indicating that the flanking amino acids, in
this case Pro7 (38), play a critical role as secondary
structure modulators.
The cis-trans isomerism observed in the NMR spectra of Ci3
reveals a remarkable feature with respect to the results reported for
the majority of the peptides undergoing that isomerization. While the
cis isomer population is usually significantly smaller than
the trans one, in our case the percentages of the two
isomers as well as their conformation are quite similar, with an
-helix spanning residues 9-15 in both of them (Fig.
7). The dissimilar structural
organization observed for the NH2-terminal domain of the
two isomers is not significant, being solely the result of the limited
number of NOEs available.
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Thus, the conformational and energetic similarities of the trans and cis isomers suggest that the cis-trans isomerization process is likely to occur in the native receptor as well.
In the case of the i3 loop, it is also known that its proximal portion is essential for G-protein coupling (24), while there are evidences that the distal portion of i3 is essential for receptor activation and G-protein selectivity (25). In fact, more generally, there is a tendency to locate the critical motif for GPCRs activation and G-protein selection in that region of the i3 loop (6, 44, 45).
Recognizing that G-protein coupling and activation require the presence
of amphipathic -helices in specific receptor cytosolic regions (17,
18, 25, 62-64), although theoretical predictions indicate that the two
i3 terminal portions must have a helical conformation, we propose that,
in the receptor resting state (65, 66) the cis-trans
isomerization produces a conformational perturbation throughout i3,
leading to a significant reduction of the extent of the helical
structure and to an increase of conformational instability.
Consequently, we believe that the possibility to control the rate of
the cis-trans interconversion can be envisaged as a
modulator of the conformation and/or exposition of the preceding flexible region of i3.
The Angiotensin II AT1 Receptor-- Combining these results with a previous study on the peptide encompassing residues 300-320 (17), we may extend the description of the conformational and dynamical properties of the receptor cytosolic domains involved in G-protein coupling.
Besides accepting that ligand binding to GPCRs causes not yet well
defined conformational changes (6, 44-46), it is now recognized that
reorientation of the transmembrane helices (TM) should play a crucial
role in affecting the conformation of the cytoplasmic surface. In
particular, experimental evidence suggests that, for rhodopsin (67),
-adrenergic receptor (6), and also for angiotensin II
AT1A receptor (5), upon ligand binding the helix-helix
interaction between TM7 and TM3 is removed and a new one between TM7
and TM6 is favored. Furthermore, recently, the TM6 of the
AT1A receptor has been shown to play an essential role in
triggering the response to angiotensin II binding (68).
Previously (17), we proposed that the agonist-induced lateral shift of
the receptor (69) can favor the formation of an amphipathic helix in
the receptor COOH-terminal tail, the hydrophilic side of which is one
of the anchoring points of AT1A with the G-protein
-subunit.
Indeed, on the basis of the models proposed (5, 6, 67), we may add that
the suggested TMs movements, associated to receptor activation, imply a
decrease of TM6 flexibility that ought to be sensed by the distal part
of the i3 loop. This event should be sufficient to reduce the rate of
the cis-trans isomerization at the
Lys232Pro233 peptide bond. Such a
conformational signal might be converted into a stabilization of the
amphipathic
-helix throughout the i3 loop and/or the exposure of the
correct surface of the proximal portion of the loop, necessary for the
interaction/coupling with the proper G-protein (70, 71). Interestingly,
the receptor residues we suggest to be responsible for that
conformational transition (residues 235-241) are close to the
NH2-terminal side of TM6, and this region has been shown to
play a critical role for G-protein selectivity and activation also in
the muscarinic receptor (25, 29, 44).
Finally, Shirai et al. (33) and Sano et al. (34) showed that only the peptide representing the proximal portion of i3 was able to activate Gi and Gq proteins. We believe that we may justify those results based on the idea of a distinct role played by the two regions of i3, at least in the case of the AT1A receptor, with the distal region being involved in receptor activation and G-protein selectivity, and the proximal one being essential for G-protein coupling.
Receptor activation and G-protein selection should not require interaction with G-protein; instead, they seem to imply conformational modifications and exposition of specific receptor cytoplasmic domains such as the proximal half of i3 that, on the contrary, is expected to selectively interact with the proper G-protein.
Thus, consistent with the hypothesis that peptides representing
fragments of a protein can retain their functional properties, only Ni3
is expected to interact and activate purified Gi and Gq proteins. In fact, Ni3, free in solution, can reach the
G-protein and be induced to assume the conformation necessary for
binding and activation. When inserted in the receptor, due to sterical hindrance, this process cannot be spontaneous; in fact, from the more
general view of receptor activity, it must be controlled. It is this
the step in which the distal portion of the loop comes into play,
regulating the conformational stabilization and proper exposition of
the required loop region without any specific need to interact with the
protein G, thus justifying the lack of activity that characterizes Ci3
in solution.
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CONCLUSIONS |
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G-protein-coupled receptor activation is definitely associated with exposition of a previously buried region to G-protein.
The data obtained thus far on fragments of the i3 loop and of the COOH-terminal tail (17) of the angiotensin II AT1A receptor seem to support that model. In fact, they indicate the existence of structural and dynamic features that we believe relevant to gain further insights on the structure-dynamics-function relationship of the cytoplasmic regions involved in the process of receptor activation and G-protein selection and coupling.
Studies are under way for a complete description of the extramembranous
domains of AT1A and of the dynamics of receptor/G-protein activation.
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ACKNOWLEDGEMENTS |
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The Interfaculty Center for Measurements of the University of Parma (Italy) and the Large-Scale Facility for Biomolecular NMR of Florence (Italy) are gratefully acknowledged for the use of the NMR and computing facility.
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FOOTNOTES |
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* This work was supported in part by Ministero dell'Universitá e della Ricerca Scientifica e Tecnologica (to A. S.), CNR Grant 97.04282.CT04 (to A. S.) and Fundaçao de Amparo à Pesquisa do Estado de São Paulo Grant 93/3457-6 (to. S. S. and C. R. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Conselho Nacional de Desenvolvimento Científico e Tecnológico Ph.D. fellow.
Recipient of a Research Fellowship from the Conselho Nacional
de Desenvolvimento Científico e Tecnológico.
** To whom correspondence should be addressed: Institute of Biological Chemistry, University of Parma, Via Volturno, 39, 43100 Parma, Italy. Tel.: ++39-0521-903807; Fax: ++39-0521-903802; E-mail: aspin{at}ipruniv.cce.unipr.it.
2 Pertinhez, T. A., et al., manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: GPCRs, G-protein coupled receptors; AT1A, type 1A angiotensin II receptor; i3, third intracellular loop of AT1A; Ni3 and Ci3, NH2-terminal fragment 213-231 and COOH-terminal fragment 227-242 of the third intracellular loop; TFE, 2,2,2-trifluoroethanol; NOE, nuclear Overhauser effect; NOESY, two-dimensional NOE spectroscopy; TM, transmembrane helix..
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REFERENCES |
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