Differences in conformational properties of the second intracellular loop (IL2) in 5HT2C receptors modified by RNA editing can account for G protein coupling efficiency

Irache Visiers, Sergio A. Hassan and Harel Weinstein,1

Department of Physiology and Biophysics, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adenosine-to-inosine RNA editing events that have been demonstrated for 5HT 2C receptors resulted in alterations of the amino acid sequence at positions 156, 158 and 160 in the intracellular loop 2 (IL2) region. The edited receptor isoforms were shown to have reduced basal activity, but similar maximum responses to agonist binding. To identify the molecular mechanism of these pharmacological effects of editing we explored the conformational properties of the edited IL2 in comparison with the wild type. The results from conformational studies of the IL2 isoforms, using biased Monte Carlo simulations with an implicit solvent model based on a screened Coulomb potential, show that the compared loops differ in their preferred spatial orientations as a result of differences in the conformational space that is accessible to them by energy criteria. For the IL2 of the unedited (5HT 2C-INI ) receptor, the preference for structures oriented towards the 7TM bundle is larger than for the 5HT 2C-VGV edited receptor. This difference in preferred orientation can affect the association of IL2 with other intracellular loop domains involved in G protein coupling and hence the coupling efficiency. The results illustrate the high sensitivity of the system to small changes in the interaction surface presented to other intracellular loops, and/or the G protein.

Keywords: coupling efficiency/G protein coupling surface/implicit solvent model/loop conformation/Monte Carlo simulation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The 5HT 2C subtype of serotonin receptors is a member of the G protein coupled receptor (GPCRs) superfamily. In numerous structure–function studies, a large variety of experimental approaches have been used to identify the functional role of the individual transmembrane segments of the GPCRs and the loop region connecting them, in both ligand recognition and signal transduction (for some reviews see Sealfon, 1995; Wess, 1997, 1999; Gether, 2000). For the second intracellular loop (IL2) that connects transmembrane helices (TMH) 3 and 4 in GPCRs such studies have demonstrated an important role in G protein coupling ( Kuhn and Hargrave, 1981Go ; Konig et al.1989Go ; Franke et al.1990Go , 1992Go ; Farahbakhsh et al.1993Go , 1995Go ; Moro et al.1993Go ; Arora et al.1995Go ; Ballesteros et al.1998Go ). However, full G protein activation has been shown to require both the second (IL2) and the third (IL3) intracellular loops, suggesting that they act in synergy (Wong et al.1988Go ; Konig et al.1989Go ; Cypess et al.1999Go ). A naturally occurring variation in IL2 of the 5HT 2C receptor was demonstrated recently as a result of adenosine-to-inosine RNA editing (Burns et al.1997Go ; Herrick-Davis et al.1999Go ; Niswender et al.1999Go ). Such editing was shown to produce an alteration of the amino acid sequence at positions 156, 158 and 160 in the second intracellular loop (IL2) (Figure 1 Go ). Pharmacological characterization of the edited constructs identified functional impairments compared with the unedited receptor. For the fully edited 5HT 2C-VGV receptor in which the sequence containing I156, N158, I160 (the INI sequence) at the edited positions was replaced by VGV, these included a decrease in agonist potency as well as a 5-fold reduction in the levels of basal [ 3 H]inositol monophosphate generation (Niswender et al.1999Go ). Competition binding experiments revealed that only the unedited receptor (5HT 2C-INI ) exhibited the highest affinity for serotonin. Taken together, these results indicate that the edited receptor couples less efficiently to G proteins, leading to the suggestion that the editing of 5HT 2C receptors may represent a regulatory mechanism of receptor signal transduction at serotonergic synapses (Burns et al.1997Go ).



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Fig. 1. Helical net representing the amino acid sequence of the unedited 5HT 2C receptor. Residues in IL2 are highlighted by a thick dark circle.

 
To identify the mechanistic basis for the altered properties of the edited 5HT 2C receptor, we investigated the effect of the mutations on the conformational properties of IL2 using biased Monte Carlo simulations to explore the conformational preferences of the various IL2 constructs. The resulting conformations were examined in the structural context of a receptor model in order to identify the potential role of the changes produced by the editing in the function of IL2. We find that the differences in the population of conformations between the edited and unedited forms relate to the orientation of the loop relative to the 7 TMH bundle and IL3. Given the likely role of the association of IL2 with other intracellular loop domains in producing optimal coupling to the G protein, these differences in the population of IL2 conformations can relate the conformational effect of loop editing to the coupling efficiency of the edited receptors.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exploration of the conformational space

The conformational space of the loops was explored with a modified version of the biased Monte Carlo method described earlier, the Conformational Memories (CM) method ( Guarnieri and Wilson, 1995Go ; Guarnieri and Weinstein, 1996Go ). The method was modified with the inclusion of a new implicit solvent model described below. Briefly, the application of the CM technique involves a two-stage process consisting of an exploratory phase and a biased sampling phase. In the exploratory phase, several Monte Carlo-simulated annealing (MC/SA) runs are performed to a final temperature of 300 K. For the peptides studied here, the starting temperature was sufficiently high (3000 K) to guarantee randomization of the starting structures. The cooling schedule was T n + 1 = 0.9 T n and the interval of temperatures was divided into 18 steps; 10 4 trial moves per temperature were generated. The trial conformations were obtained by randomly picking two rotatable dihedral bonds and assigning random values between ±180° (peptide bonds were considered fixed at 180° throughout the simulation). Acceptance of a trial conformation followed the standard Metropolis algorithm with a Boltzmann distribution. After several runs have been performed, a probability distribution of each torsional angle in the mean field of all other rotatable bonds can be obtained at the lower temperature. The number of MC runs is determined by a convergence criterion of the probability distributions ( Guarnieri and Weinstein, 1996Go ). These distributions are used in the second phase of the simulation, in which the Monte Carlo sampling only explores the populated regions of the conformational space weighted by the probability distribution generated in the first stage ( Guarnieri and Weinstein, 1996Go ). In the second stage, the ensemble of structures at T = 300 K is obtained from a second simulated annealing process.

The solvent environment model

The aqueous environment of IL2 in the cytoplasmic compartment was modeled with a recently developed implicit solvent model that is based on a screened Coulomb potential formulation (the SCP-ISM) (Hassan et al.2000aGo , bGo ). In this model, the Coulomb interaction among particles is screened by a distance-dependent dielectric function of sigmoidal form. The SCP-ISM introduces the self-energy of the atoms as derived from the integral form of the Born equation and makes use of a novel approach to describe the Born radii of atoms in the protein environment. An algorithm for hydrogen bonding interaction is incorporated, which is based on the degree of exposure of the polar hydrogens to the proton acceptor environment. The model was implemented in the CHARMM package (Brooks et al.1983Go ) and parameterized in the context of the all-atom PAR22 force field of CHARMM. As implemented in the biased sampling Monte Carlo, this SCP-ISM was shown to predict with accuracy the most favorable conformation of peptides in water (Hassan et al.2000aGo , bGo ).

Loop conformation in the context of the molecular model of the receptor

The simulations were performed with the IL2 loop anchored to the cytoplasmic ends of TM3 and TM4 in a model of the transmembrane bundle of 5HT 2C . The spatial relative orientation of the helices is taken from a 3D model of the 5HT 2C receptor constructed as described elsewhere ( Ballesteros and Weinstein, 1995Go ), using a template described by Baldwin (Baldwin et al.1997Go ) according to the density map from cryo-electronmicroscopy for rhodopsin (Unger et al.1997Go ). This model is characterized by a number of structural motifs consisting of spatially adjacent residues that adopt specific interaction patterns. These structural motifs have been shown to constitute functional microdomains (Ballesteros et al.1998Go ) and the network of interactions that stabilizes the structural pattern of these motifs (Sealfon et al.1995Go ; Ballesteros et al.1998Go ; Konvicka et al.1998Go ) have been confirmed recently by the breakthrough X-ray structure of rhodopsin (Palczewski et al.2000Go ). These key features of the receptor model that were confirmed by the crystal structure include (1) the hydrogen bonding network between the conserved N1.50 (asparagine 55 in rhodopsin) and the H-bonded region of interaction between TM2 and TM7 (compare Figures 7 and 8 in Konvicka et al.1998 with Figure 5C Go in Palczewski et al.2000)—a spatial adjacency that has been verified experimentally in many GPCRs (Sealfon et al.1995; Flanagan et al.1999; and references therein); (2) the `arginine cage' motif at the cytoplasmic end of TM3 that involves the highly conserved DRY sequence (Ballesteros et al.1998Go ) which brings together the ends of TM3 and TM6 in the `inactive' state of the receptor through an H-bonding interaction between D3.49 (corresponding to E134 in rhodopsin), R3.50 (R135 in rhodopsin) and E6.30 (E247 in rhodopsin), as seen from the comparison of Figure 2 Go in Ballesteros et al. (1998) and Figure 5D Go in Palczewski et al. (2000).



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Fig. 5. Values of {theta} for the conformations of the IL2 loop in ( A ) the unedited 5HT 2C-INI receptor and ( B ) the edited 5HT 2C-VGV receptor. Both angle values (left) and corresponding energies (right) are shown for each structure.

 


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Fig. 2. Definition of the conformational propensities of IL2. ( A ) set of geometrical parameters. P 1 and P 2 are the coordinates of two fixed points corresponding to the C {alpha} atoms of R168 and A155; P 3 corresponds to the position of the C {alpha} of residue 164; point M is the middle point of the segment P 1 P 2 . ( B ) Definition of the angle {theta}: vector a is the axis of TM4; point M is the middle point of the segment connecting points P 1 and P 2 defined in (A); the ellipse defines the plane of the membrane; vector R o = P 1 x a; V i x P 3 x a; {theta} is the angle between V i and R o for each conformation of the loop.

 
The ends of all the TMH in the model were determined as described in detail ( Ballesteros and Weinstein, 1995Go ; Ballesteros et al.1998Go ) based on a variety of results from sequence alignment analysis of conservation combined with biophysical criteria and direct data from experiments that offer specific constrains ( Ballesteros and Weinstein, 1995Go ). For example, R157 that follows immediately the highly conserved DRYXXI motif in the serotonin receptor was identified as the last residue of the TM3 helix (Ballesteros et al.1998Go ), thus starting IL2. Similarly, spin-labeling studies in rhodopsin (Farahbakhsh et al.1995Go ) and also secondary structure prediction and results from scanning with the substituted cysteine accessibility method (Javitch et al.2000Go ) had identified S167 as the first residue of TM4, thus ending IL2. These features of the model are verified in the structure of rhodopsin (Palczewski et al.2000Go ); the calculated C {alpha} r.m.s.d. for TM3 and TM4 relative to the crystal structure of rhodopsin (PDB 1F88) is 2.7 Å.

Geometrical description of the conformational space

In order to define quantitatively the conformational space accessible to the loops in the context of the receptor model, we defined a set of geometrical parameters described in Figure 2A and B Go . The coordinates of two fixed points corresponding to the C {alpha} atoms of R168 and A155 are defined as points P 1 and P 2 , respectively (Figure 2A Go ). The axis of TM4, characterized by vector a , defines an orientation perpendicular to the plane of the membrane. The middle point of the segment P 1 P 2 defines the point M. Figure 2B Go shows a schematic representation of the geometrical parameters, with the plane of the membrane (perpendicular to TM4) represented by an ellipse. For the sake of clarity the vector a defining the TM4 axis is translated to point M that is considered the origin of the coordinates system. The cross product P 1 x a yields vector R o that is perpendicular to both and thus contained in the ellipse that represents the plane of the membrane. To describe the conformational variations, a variable vector P 3 is defined, where point P 3 is chosen to correspond to the position of the C {alpha} of residue 164 (see Figure 2 Go ) whose position changes for each conformation. The cross product P 3 x a yields vector V i perpendicular to both and in the same plane as R o . The angle {theta} between the different V i and R o provides a measure of the space swept by the energetically accessible conformations of the loop, termed the `swing' of IL2 (Figure 4 Go ).



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Fig. 4. Representative structures of the most populated conformations of IL2 of the unedited (blue) and edited forms of the 5HT2CR receptor (green) in the context of the receptor model viewed parallel to the membrane. The red arrow indicates the broad area of the conformational space swept by the loop.

 

    Results
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 Materials and methods
 Results
 Discussion
 References
 
Conformational families of the second intracellular loop (IL2)

The conformational studies were carried out for two 14-residue peptides that represent the loops of the fully edited and the unedited receptors (nine residues), flanked by three residues from TM3 and two from TM4. The peptide representing the unedited sequence (INI) is AIRNPIEHSRFNSR and for the edited sequence (VGV) the peptide is AVRGPVEHSRFNSR (see Figure 1 Go ). Throughout the simulations, the orientation of fragments 155–157 and 167–168 are constrained with harmonic potentials to fit the orientations of TM3 and TM4 at their cytoplasmic ends that must remain helical. In addition, the inter-residue distances between the two helical fragments were restrained to the corresponding distance in the receptor model.

The MC/SA phase of the biased Monte Carlo simulation (see Materials and methods) comprises 50 runs. Each run of the MC/SA consists of a random walk of 180 000 steps. Each rotatable bond block is divided into 18 temperature blocks and the dihedral space is partitioned into 36 10° intervals with normalized populations. The spreadsheet including these data represents the dihedral distribution of a given torsional angle in the mean field of all other rotatable bonds. The data from these runs are sorted and merged into 78 blocks, one for each rotatable bond. The identification of the populated regions in the torsional angle space, the conformational memory ( Guarnieri and Weinstein, 1996Go ), is used in the second phase of the simulation in which biased sampling explores only the populated regions of the conformational space. In this second stage, the ensembles of the populated regions of the conformational structures at 300 K are obtained from a second simulated annealing starting from T = 834.8 K and cooling to 300 K, with sampling performed only from the populated regions of the conformational memories. Population convergence is considered when there is no significant difference in the populations of structures obtained by new independent MC/BS runs (the rate of convergence for the calculated angle {theta} is illustrated in Tables I and II Go Go ).


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Table I. Binning of {theta} angle values (%) for populations of IL2 conformations in the unedited 5HT 2c-INI receptor, calculated from independent Monte Carlo simulations with increasing numbers of runs
 

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Table II. Binning of {theta} angle values (%) for populations of IL2 conformations in the unedited 5HT 2c-VGV receptor, calculated from independent Monte Carlo simulations with increasing numbers of runs
 
The comparative analysis of the IL2 orientations in 5HT 2C-INI and 5HT 2C-VGV receptors, summarized in Figure 3 Go , shows that structures of IL2 in the unedited receptor (in yellow) tend to cluster closer to the center of the bundle, whereas IL2 in the edited receptor (in red) tends to move outward from the center of the bundle. To obtain a quantitative comparison of the ensemble of structures calculated for the unedited and edited receptors, we have defined the geometry of the orientation of the loop with respect of the whole bundle as described in Materials and methods. The ensemble of structures is first positioned in the context of the receptor model so as to fit the orientation of fragments 155–157 and 167–168 and then a quantitative measure of the orientation of the loop with respect the rest of the bundle is determined by the angle {theta} (see Materials and methods for a description; Figure 2 Go ). The values of {theta} for the conformations of the unedited loop and the 5HT 2C-VGV edited IL2 were sorted and are plotted in Figure 5A and B Go , respectively, along with the total energy calculated for each structure. The average value of angle {theta} in the ensemble generated in the Monte Carlo run, for either the INI or VGV receptors, is given by

(1)
where E i is the energy of the system in the loop conformation i in the ensemble and N denotes the number of conformations necessary for convergence; Z is the partition function of the system in the ensemble, k is the Boltzmann constant and the temperature was fixed at T = 300 K.



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Fig. 3. Representative structures of the most populated conformations of IL2 of the unedited form (yellow) and edited form of the 5HT2C receptor (red) in the context of the receptor model viewed from the intracellular side. The hatched area represents the approximate contact surface with G protein ( Fanelli et al.1999aGo , bGo ).

 
Positive values of {theta} correspond to conformations where the C {alpha} of residue 10 is oriented closer to the interior of the bundle. The more negative the {theta}, the more the loop `swings' away from the interior of the bundle (Figures 3 and 4 Go Go ). Note that the loop clusters cover a wide area of the conformational space, as indicated by the red arrow in Figure 4 Go .

The results presented in Figures 3 and 4 Go Go show that the ensemble of structures obtained for IL2 of the unedited and edited receptors cover different areas of the conformational space, but with considerable overlap. To quantify this comparison, the calculated angles {theta} were binned into four regions comprising 25° each. The probability P i ({theta}) of finding a loop conformation in bin i defined by {theta} i <= {theta} <= {theta} i + 25° is given by

(2)
where N i is the number of structures with {theta} in bin i . Convergence of the Monte Carlo simulation was assumed when the probability distribution of Equation 2Go changed less than 1% for every bin i , as illustrated in Tables I and II Go Go .

For the unedited receptor, the average value of the angle <{theta}> is –21.2°, with 41.7% of the structures falling in the narrow region between –25° and 0° (Table I Go ); the population of the same area in the edited receptor is only 31.1% (Table II Go ). Conversely, the average value of the angle in the edited receptor is <{theta}> = –27.5° and the most populated region is between angles –50° and –25°, which contains 41.6% of the structures; the population of the same area in the unedited receptor drops to 30.5%. Note that the area corresponding to {theta} > 0 is also larger in the unedited (18.5%) than in the edited IL2 loop (10.9%); see Tables I and II Go Go .

When these results are put in the context of the receptor model (Figure 3 Go ), the IL2 of the unedited (5HT 2C-INI ) receptor is seen to have a slightly larger population of structures oriented towards the TM7 bundle (the population with {theta} > 0, which represents 18.5% of the total) than the 5HT 2C-VGV edited receptor (10.9% of the population with {theta} > 0). The schematic illustration in Figure 3 Go shows that the IL2 conformations that are oriented towards the interior of the bundle, where the interaction surface with G proteins is likely to be located according to Fanelli et al . (Fanelli et al.1999aGo , bGo ), also have greater spatial proximity to the region corresponding to the third intracellular loop, IL3.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The exploration of IL2 conformations, with the all-atom CHARMM force field and the new SCP-ISM model for the solvent environment implemented in the Conformational Memories biased MC method, has revealed the differences in the populations of structures accessible to the IL2 loop in the unedited and edited forms of the receptor. We find that the substitution of the INI sequence of residues, i.e. I156, N158 and I160, in the unedited form of the 5HT 2C receptor by a corresponding VGV sequence in IL2 produces a change in the distribution of energetically accessible structures. The unedited loop shows a clear trend towards higher values of {theta} than the edited one. This indicates that the unedited (5HT 2C-INI ) receptor has a slightly larger population of structures oriented towards the 7TMH bundle than the 5HT 2C-VGV edited receptor.

The structures oriented towards the interior of the bundle would also have greater spatial proximity to the third intracellular loop whose role in G protein coupling has been described (Cheung et al.1992Go ). While the distribution of the edited loops overlaps the unedited version in some regions, there is a clear deviation away from the region of potential interactions with IL3. Given the experimental evidence for the role of both intracellular loops in G protein coupling, this difference may be responsible for the observation that the 5HT 2C-VGV edited receptor couples less efficiently to G proteins while retaining V max (Niswender et al.1999Go ). This relation between IL2 and IL3 in determining G protein coupling is suggested by experimental data for various GPCRs. Thus, peptides corresponding in sequence to the IL2 and IL3 of rhodopsin were shown to be involved in interaction with transducin and these peptides had synergistic effects when binding at their respective sites on the G protein transducin (Konig et al.1989Go ). Analogous conclusions were reached for the glucagon receptor (Cypess et al.1999Go ). Similarly, receptor chimera experiments on muscarinic and ß-adrenergic receptors have shown that specific IL2–IL3 combinations are necessary for G protein selectivity (Wong et al.1988Go ). Our present results indicate that the likelihood for a direct interaction between IL2 and IL3 can depend on the conformational properties of IL2, which, in turn, have been shown to depend on the sequence of the loop. From the specific comparison performed here, the interaction between the two loops is predicted to be more favorable for the unedited IL2 loop whose population is higher in regions oriented towards IL3. Although neither the experimental data nor our schematic model demonstrate a direct interaction between these loops, our results suggest that the effects on G protein coupling properties of the compared receptors reflect the likelihood of a structural organization in which the two loops are proximal. In this context, our results provide a molecular basis for the observed synergism between the two loops and a mechanistic basis to explain why the edited 5HT 2C-VGV receptor couples less efficiently to G proteins (Niswender et al.1999Go ).

It is noteworthy that the distribution of {theta} values for the different forms of the IL2 supports the hypothesis that even a small difference in loop orientation is sufficient to account for the observed reduction in G protein coupling. In this model, the reduction is achieved by disrupting the optimal orientation of IL2 relative to IL3 that is involved in their binding at the G protein. This implies a high sensitivity of the system to small changes in the interaction surface presented to the IL3 and/or the G protein. In the context of the whole activation mechanism process, a series of inter- and intramolecular interactions are required for the propagation of the signal from the binding site to the interaction surface with the intracellular components of the signal transduction pathway, such as the G protein (Ballesteros et al.1998Go ). We have described previously some of the structural motifs that are involved in the propagation of the signal (Almaula et al.1996aGo , bGo ; Gether et al.1997Go ; Ballesteros et al.1998Go ; Konvicka et al.1998Go ). These structural motifs constitute functional microdomains that connect directly to the structural determinants presented here for the role of intracellular loop 2.


    Notes
 
1 To whom correspondence should be addressed Back


    Acknowledgments
 
This work was supported by NIH grants DA-12923 and DA-00060 (to H.W.). Computational support was provided by the Cornell Supercomputer Facility and the Advanced Scientific Computing Laboratory at the Frederick Cancer Research Facility of the National Cancer Institute (Laboratory for Mathematical Biology).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 15, 2000; revised January 5, 2001; accepted March 17, 2001.