From the Department of Physiology and Biophysics,
¶ Fishberg Research Center in Neurobiology, and
Department of Neurology, Mount Sinai School
of Medicine, New York, New York 10029 and ** Department of Chemical
Pathology, University of Cape Town, Observatory 7925, Cape Town, South Africa
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ABSTRACT |
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An Arg present in the third transmembrane domain
of all rhodopsin-like G-protein-coupled receptors is required for
efficient signal transduction. Mutation of this Arg in the
gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished
or severely impaired agonist-stimulated inositol phosphate generation,
consistent with Arg having a role in receptor activation. To
investigate the contribution of the surrounding structural domain in
the actions of the conserved Arg, an integrated microdomain modeling
and mutagenesis approach has been utilized. Two conserved residues that
constrain the Arg side chain to a limited number of conformations have
been identified. In the inactive wild-type receptor, the Arg side chain
is proposed to form an ionic interaction with
Asp3.49(138). Experimental results for the
Asp3.49(138) Asn mutant receptor show a modestly
enhanced receptor efficiency, consistent with the hypothesis that
weakening the Asp3.49(138)-Arg3.50(139)
interaction by protonation of the Asp or by the mutation to Asn favors
activation. With activation, the
Asp3.49(138)-Arg3.50(139) ionic bond would
break, and the unrestrained Arg would be prevented from orienting
itself toward the water phase by a steric clash with
Ile3.54(143). The mutation Ile3.54(143)
Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that
solvation of Arg3.50(139) prevents it from functioning in
the activation of the receptor. These data are consistent with residues
Asp3.49(138) and Ile3.54(143) forming a
structural motif, which helps position Arg in its appropriate inactive
and active receptor conformations.
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INTRODUCTION |
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The gonadotropin-releasing hormone (GnRH)1 receptor is a member of the rhodopsin-like G-protein-coupled receptor (GPCR) family (1, 2). These heptahelical proteins include the visual opsins and various receptors for neurotransmitters, peptides, and glycoproteins. Activation of these receptors by their diverse agonists is associated with conformational changes in the receptor that facilitate a signal-propagating interaction with G-proteins (3). These conformational changes can involve relative movement of helices, as reported for rhodopsin (4, 5) and/or rotation of the helices as found in a constitutively active adrenergic receptor (6).
Sequence alignment of GPCRs shows that certain amino acids are highly conserved at corresponding positions within the putative transmembrane domains (TMD) (7). Transitions among receptor conformations may reflect dynamic changes in side chain interactions within the receptor. Two of these conserved residues have been studied by reciprocal mutation in the GnRH and serotonin receptors, and the results suggest that the TMD 2 and 7 side chains have an interdependent role in receptor activation (8, 9). Most likely several other conserved side chains also interact to form the skeleton required for the conformational rearrangements that accompany the transition between inactive and active receptor states.
The elucidation of the intramolecular interactions and conformational changes underlying receptor activation is hindered by the absence of high resolution structural data for any GPCR. The available low resolution projection maps of rhodopsin do not allow inferences about specific side chain interactions (10, 11). A prevalent approach to investigate structure-function relations of GPCRs is to introduce structural perturbations via site-directed mutagenesis and to evaluate their effect on receptor phenotype in binding and signal transduction assays (12). However, determining the phenotype of mutant receptors does not lead to an unequivocal interpretation concerning the structural basis of that phenotype (13).
Molecular modeling has facilitated the integration of experimental observations and biophysical data into a mechanistic scheme for receptor structure and function (12, 14). Structural and functional details of ligand binding (15, 16) and receptor activation by agonist complexing (8, 17) and by constitutively activating mutations (18) have been simulated in such models. The receptor models can thus provide a rationalization of current experimental data within a structural framework in which to explore the mechanisms underlying the functional perturbations induced by activation. However, caveats concerning the computational approaches arise from the complexity of these structures and the relative paucity of pertinent experimental data at atomic detail. Not surprisingly, given a limited number of experimentally determined constraints, the proposed models may exhibit inconsistent interaction patterns. In addition, GPCR models usually are not studied in the appropriate aqueous/membrane interface environment, making it less likely that the key side chain interactions are modeled accurately, especially those involving side chains near or separated by this interface.
To overcome the limitations inherent in both site-directed mutagenesis and computational modeling, we have integrated mutational studies and the application of computational techniques to the study of structural motifs in the receptor that may constitute functional microdomains. The inferences from studies of these microdomains, whose proposed structure can be substantiated by experimental data, are then evaluated in the context of a whole receptor model. This approach facilitates the elucidation of a structural basis for the phenotypes induced by site-directed mutagenesis. The effect of mutations is tested first in the microdomain models and correlated with the functional effects of site-directed mutant receptors expressed in mammalian cells. Using this approach, we have recently mapped precise interactions in segments of the binding pocket of the serotonin 5HT2A receptor (19).
In the present report, we have applied this approach to study the interaction pattern of the conserved Arg in TMD 3 in the GnRH receptor. The conserved arginine Arg3.50 (see "Experimental Procedures" for locus numbering scheme) has been implicated in the activation of various GPCRs by mutagenesis studies (20, 21) and by computational modeling (21, 22). An understanding of the molecular basis for the functional role of this Arg requires identification of those residues whose specific interactions determine its orientation within the structure of the receptor. Given the great conformational flexibility of the Arg side chain, it is likely that such orienting residues would form a three-dimensional motif to which we refer as the arginine cage.
Specific partners for Arg3.50 have been proposed, such as
the conserved Asp in TMD 2 (Asp2.50) (21, 22), based on the
rationale of a similar conservation pattern and the need to neutralize
a positive charge in a low dielectric environment. However, a complete
exploration of the conformational space of Arg3.50 in the
context of a full molecular model of the receptor is not attainable
with present computational techniques, and several other candidate
interacting residues can be proposed. In particular, analysis of the
conservation pattern centered on Arg3.50 identifies highly
conserved residues that could influence the conformation of
Arg3.50. These residues form the consensus sequence
(I/L)XXDRYXX(I/V) (Fig.
1). Arg3.50,
Asp/Glu3.49, and Ile/Val3.54 are present in all
cloned GPCRs belonging to the rhodopsin family, with the exception of
the platelet-activating factor receptor, which has an asparagine
residue at position 3.49 (23). In an -helical environment, the
conservation pattern described above forms an envelope of conserved
residues surrounding Arg3.50, consisting in the GnRH
receptor of Ile3.46(135), Asp3.49(138), and
Ile3.54(143) (Fig. 2).
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To evaluate the role of these various conserved residues in caging the Arg3.50 side chain in the inactive and active forms of the receptor, we have performed a complete conformational exploration of TMD 3 using Monte Carlo simulations (15). The helical structure and the helix ends of TMD 3 have been experimentally substantiated by Cys scanning of the D2 receptor (24) and by spin-labeling studies of rhodopsin, in which the membrane/aqueous interface has been located between residues 3.52 and 3.53 (25). For the Monte Carlo simulations, a novel biphasic solvent model has been developed that reproduces the interface between the interior of a protein and a water environment.2 Using this Monte Carlo approach, the inferences about the possible caging interactions involving the conserved Arg have been tested by evaluating computationally the structural effects of mutations in this TMD 3 domain model and correlating these results with the functional effects of site-directed mutagenesis. These studies provide insight into the role of Arg3.50(139) in sustaining a pattern of interactions that may occur in the active and inactive forms of the receptor.
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EXPERIMENTAL PROCEDURES |
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Residue Numbering Scheme-- The residues in TMDs are numbered relative to the most conserved residue contained in the helix, as explained previously (14). On the basis of this scheme, the most conserved residue in TMD 3, Arg-139, is designated with the index number 3.50 and is hence referred to throughout as Arg3.50(139); the identification of the preceding Asp is Asp3.49(138).
Computational Methods--
The segment of TMD 3 from
Cys3.25 to Thr3.55 was modeled as an -helix.
These helix boundaries, predicted following methodology described elsewhere (14), are consistent with recent experimental results for
other GPCRs (24, 25). The environment surrounding the Arg residue in
TMD 3 has been investigated in rhodopsin and found to consist of two
distinct phases: a water phase and a membrane-embedded helix (25). The
environment of Arg3.50(139) in the helix was assumed to be
similar to the environment of residues buried inside the protein
interior, consonant with studies on the known structure of the
photosynthetic reaction center (26), and has been modeled by a
distance-dependent dielectric. To simulate the biphasic
environment in our calculations, the novel mixed solvent model includes
a water phase and a distance-dependent dielectric
phase.2 The boundary between the two phases consists of a
plane parallel to the membrane. TMD 3 was initially positioned
perpendicular to the membrane plane at the midpoint between residues
3.52 and 3.53 following experimental observations (23) but was allowed to move ± 2.3 Å vertically in the direction normal to the
membrane plane to prevent arbitrary effects arising from the initial
positioning.
DNA Constructs and Transfection-- Procedures for site-directed mutagenesis of the GnRH receptor, subcloning of the receptor coding region into pcDNA1/Amp and transient receptor expression have been described previously (27). COS-1 cells transfected with plasmid DNA were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. One day after transfection, COS-1 cells were split from 100-mm plates into two 12-well plates for the functional assay or into two 24-well plates for the whole-cell binding assay. Mutations of Asp3.49(138) and Arg3.50(139) were done in the mouse GnRH receptor, whereas mutations of Ile3.46(135) and Ile3.54(143) were generated in the human GnRH receptor.
Binding and Functional Assays--
Binding of GnRH to the
wild-type and mutant receptors was measured at 4 °C in a whole-cell
agonist competition binding assay 72 h after transfection (13).
125I-GnRH-A ((des-Gly10,
D-Ala6, GnRH-ethylamide) was used as the label
and displaced by increasing concentrations of GnRH. The
Kd for GnRH-A was estimated from homologous
competition binding. The radioactivity bound to membranes or cells was
counted in a -counter, and the amount of protein per well was
determined using the Lowry method (28). The phosphatidylinositol
hydrolysis assay was performed as described earlier (27). The binding
and concentration-response curves were fitted using Kaleidagraph
software (27). Ki and Bmax
values were determined by using the program LIGAND (29).
Receptor Efficiency-- We have developed an empirical representation for receptor efficiency (Q) using operational models of occupancy and response (see Equations 1 and 2),
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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RESULTS |
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Computational Simulations of TMD 3 Segment Surrounding Arg3.50(139)
The GPCRs demonstrate a pattern of conservation among several
residues that are in spatial proximity to Arg3.50(139) when
the cytoplasmic side of TMD 3 is modeled as a regular -helix (see
Fig. 2). These potential local sites of interaction for
Arg3.50 include the large hydrophobic residue (Ile/Val/Leu)
at position 3.46, the acidic residue at position 3.49, the Tyr (Ser in
the GnRH receptor) at position 3.51, and the
-branched large
hydrophobic residue (Ile/Val) at position 3.54.
The interaction patterns and rotamer positioning of
Arg3.50(139) with respect to these neighboring TMD 3 residues were explored with Monte Carlo simulations for the wild-type
helix and for various mutant receptors. Many conformations of the
flexible Arg side chain were not attainable due to steric clashes with
the helix backbone. For example, all Arg rotamers whose 1 = g
are unpopulated because of a clash between the Arg
-methyl and the
backbone carbonyl from the preceding turn of the helix (30).
The most striking observation to emerge from the simulations is the
tendency for Arg3.50(159) to form an ionic bond with
Asp3.49(138), as illustrated in Fig. 2B. Nearly
half of the Arg3.50(139) rotamers observed were bound to
this aspartic acid (Table I). As shown in
Fig. 3, a variety of Arg conformations
were identified that form the Arg-Asp interaction. Most other side
chains remained in their original orientations throughout the
simulations, consistent with their preferred rotamer populations in
known -helical structures. Ser and Thr residues were overwhelmingly
(92-99% of rotamers) in the
1 = g+ conformation
due to H-bonding to the backbone carbonyl of the preceding turn (30,
31).
-Branched residues (Val, Ile, Thr), except
Ile3.54(143), were constrained to one single
1
population (83-99% of rotamers) due to clashes with the helix
backbone (30). The structural effects of several mutations of the
residues surrounding Arg3.50(139) were studied
computationally:
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Ile3.46(135)--
The side chain at this position is
not as well conserved as the other residues surrounding
Arg3.50. Although Ile is most commonly found at this site,
Leu or Met also occur in different receptors. For the simulations, we
substituted Ile3.46(135) with Ala, Val, and Leu to test 1)
the functional relevance of the wild-type side chain (by substituting
Ala), 2) the role of the -branched character of the residue (by
substituting Val), and 3) the effect of the large hydrophobic side
chain (by substituting Leu). Monte Carlo simulations of all three
mutant constructs showed that the conformational preferences of the Arg
side chain were similar to those found in the wild-type receptor.
Therefore, according to our results, Ile3.46(135) does not
modulate the orientation of Arg3.50(139).
Asp3.49(138)--
Monte Carlo simulations were
performed for a mutation of the conserved acidic residue to Asn. By
neutralizing the charge at this locus, this Asp3.49(138)
Asn mutant would weaken the ionic bond between
Arg3.50(139) and Asp3.49(138) observed in
simulations of the wild-type receptor. This mutation was found to
significantly affect the conformational preferences of the Arg side
chain (see Table I). In the Asp3.49(138)
Asn mutant,
the Arg side chain rarely interacts with the 3.49 locus (3% of
rotamers). Two new orientations appear populated, as shown in Fig.
4. 1) Arg3.50(139) is
oriented toward positions 3.47-3.51 where it can H-bond to Ser3.47(136) and Ser3.51(140) (37% of
rotamers). 2) Arg3.50(139) is oriented toward positions
3.53-3.54 where it can be solvated by water at the cytoplasmic
boundaries. Because activation of rhodopsin has been shown to involve a
proton uptake by Glu3.49 (32, 33), simulations were also
performed for the protonated form of the aspartic acid, termed
Asp3.49(138)
Asp-H. The results yielded a pattern of
preferred conformations very similar to that of
Asp3.49(138)
Asn (Table
II).
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Ser3.51(140)--
A Tyr residue most commonly occurs
at this position in other GPCRs. We tested the functional role of this
residue with the mutant construct Ser3.51(140) Ala.
Hydrogen-bonding between Arg3.50(139) and
Ser3.51(140) was observed in the Asp3.49(138)
Asn mutant receptor, but the mutation Ser3.51(140)
Ala did not change significantly the orientation of the Arg side chain
relative to the wild-type construct (data not shown). Evidently, this
H-bond is not energetically competitive with the ionic bond
Arg3.50(139)-Asp3.49(138).
Ile3.54(143)--
Only Ile or Val residues appear in
GPCR sequences at this position. Therefore, this locus always contains
a bulky -branched, hydrophobic side chain. To test the structural
implications of these properties, we substituted this residue by Val,
Leu, and Ala. The Val side chain displays similar structural features
as isoleucine, being hydrophobic, bulky, and
-branched. Leu is
hydrophobic and bulky but has a
-branched side chain. Ala is
hydrophobic but neither bulky nor branched. Analysis of all rotamers
populated over 5%, shown in Table II, indicates that the prevailing
interaction for the wild-type receptor and the three mutants is still
the ionic bond between Arg3.50(139) and
Asp3.49(138) (65-80% of rotamers). Although maintaining
the same interaction, the individual rotamer conformations preferred
for this interaction vary among the mutants and with respect to the
wild-type receptor (Table II). Of note, a novel orientation for the Arg
side chain toward residues 3.53-3.54 appears significantly populated
in the Ile3.54(143)
Ala mutant (14.3%) but not in the
wild-type, Ile3.54(143)
Val, or
Ile3.54(143)
Leu mutants. In the Ile
3.54(143)
Ala mutant, the Arg side chain can be
positioned toward the aqueous cytoplasm, as illustrated in Fig.
5.
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Radioligand Binding and Agonist-stimulated Inositol Phosphate Accumulation by the Wild-type and Mutant Receptors-- To correlate the predicted local structural roles of the conserved TMD 3 residues with their effect on receptor function, constructs obtained from the mutation of Arg3.50(139) and its surrounding conserved residues (see Fig. 2) were tested for their effects on ligand binding and inositol phosphate accumulation (Table III). The mutation of the Asp3.49(138), Arg3.50(139), and Ser3.51(140) loci were carried out on the mouse GnRH receptor, whereas the Ile3.46(135) and Ile3.54(143) mutants were generated in the human GnRH receptor. Both human and mouse GnRH receptors have identical sequences in the TMD 3 segment studied. The amino acid substitutions were designed to test the side chain property conserved at each locus and/or a specific functional hypothesis derived from the modeling studies. The results of the radioligand binding and phosphatidylinositol assay and the relative coupling efficiencies of the various receptor constructs are summarized in Table III.
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Ile3.46(135)--
Removal of the Ile side chain by the
Ile3.46(135) Ala mutation abolished binding and
activation. Substitution by another
-branched residue
Ile3.46(135)
Val also eliminated detectable ligand
binding and signal transduction. In contrast, the
Ile3.46(135)
Leu receptor manifested coupling
comparable to that of the wild-type receptor. However, due to poor
expression of the Ile3.46(135)
Leu receptor (13% of
wild-type receptor Bmax), the calculated value
for receptor efficiency for this mutant reveals a 5-fold increase above
the value obtained for the wild-type receptor (Table III). The affinity
of this mutant construct for GnRH is comparable to the wild-type
receptor. The restricted pattern of amino acid substitutions that are
functionally tolerated at the 3.46(135) position is most consistent
with this site being involved in helix-helix packing (see
"Discussion").
Asp3.49(138)--
The Asp3.49(138) Ala
mutant had no detectable agonist binding or activation and could not be
evaluated. The more conservative mutation Asp3.49(138)
Asn behaved like wild type in terms of its Ki,
EC50, and Emax values (Table III).
However, the lower Bmax (56% relative to wild
type) suggests that this construct has a modestly enhanced signaling
efficiency.
Arg3.50(139)--
Mutations of
Arg3.50(139) to His and Lys yielded constructs with no
detectable binding or activation. The Arg3.50(139) Gln
mutant expressed well and had wild-type affinity for GnRH but was very
poorly coupled (Table III).
Ser3.51(140)--
Consistent with the lack of
structural effects observed with mutation of this locus in the
computational simulations, the phenotype of the expressed
Ser3.51(140) Ala mutant receptor was similar to that of
the wild-type receptor (data not shown). This finding is consistent
with results reported previously (34).
Ile3.54(143)--
The effects of mutating this locus
to Val, Leu, and Ala were examined. In comparison with the wild-type
receptor, the affinity of GnRH was modestly decreased for the
Ala-substituted receptor. The Ile3.54(143) Ala receptor
showed a marked reduction in signaling efficiency, whereas the
substitutions with Leu or Val were well tolerated (Table III).
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DISCUSSION |
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Arg3.50 is absolutely conserved in all GPCRs, and its substitution in various GPCRs severely affects signal transduction (20, 21). The pattern of conservation and the effects of mutations make it likely that this side chain serves a key role in conformational changes and interactions underlying receptor activation. The results obtained with mutation of this locus in the GnRH receptor support a crucial role for this Arg in receptor function. Among the mutant receptors studied, only the construct with a Gln substitution at this site shows any detectable signal transduction. Similar results obtained by mutating the Arg3.50 in the mouse GnRH receptor have been recently reported (34).
Oliveira et al. (22) proposed a mechanism for receptor activation in which the change in orientation of this Arg3.50 constitutes an Arg-switch leading to activation of GPCRs, a hypothesis recently expanded by a combination of mutagenesis and computational simulations on adrenergic receptors (21, 35). The present study has focused on the molecular details of the microdomains surrounding Arg3.50 by delineating the neighboring residues that interact with or restrict the positioning of the Arg side chain.
Computational simulations provided structural hypotheses for the phenotypes observed with mutagenesis experiments. When studied mutants display detectable binding and affinities for GnRH similar to that of the wild-type receptor, we assume that the overall structure of the receptor was not perturbed significantly by these mutations. Mutant receptors without detectable binding also did not show detectable GnRH-induced signal transduction, suggesting that either the folding or the intracellular processing of these receptors was disrupted. The role of all potential Arg-cage side chains studied will be discussed separately based on the results from the computational simulations and the measured properties of the mutant constructs.
Asp3.49(138)--
Computational experiments indicate
that the propensity to form an ionic bond between
Arg3.50(139) and Asp3.49(138) constrains the
orientation of the Arg side chain. This constraint is relieved when the
simulations are carried out for the Asp3.49(138) Asn
mutant or for a protonated Asp3.49(138). The results of
mutagenesis in the GnRH receptor and in other receptors suggest that
the interaction between Arg3.50 and Asp3.49
stabilizes the inactive receptor state. In the
-adrenergic receptor, the mutation Asp3.49
Asn leads to constitutive
activation of the receptor (21, 35). We find that mutation of
Asp3.49
Asn leads to a modest increase in the
efficiency of GnRH receptor activation similar to the reported result
of the Glu3.49
Gln mutation in rhodopsin (33).
Ile3.54(143)--
This locus shows a 100%
conservation profile as a -branched, bulky hydrophobic residue (Ile
or Val). Experimentally, the Ile3.54(143)
Val and the
Ile3.54(143)
Leu mutants were similar or more efficient
than the wild-type receptor. The Ile 3.54(143)
Ala
mutant was inefficient in mediating signal transduction and displayed a
much lower receptor efficiency than the wild-type receptor. In
simulations, the preferred orientations of the Arg side chain in the
Ile 3.54(143)
Val and the Ile 3.54(143)
Leu mutants were similar to those observed in the wild-type receptor.
However, in the Ile3.54(143)
Ala mutant, a new
orientation of the Arg side chain was significantly populated (14.3%,
Table II). Analysis of the new rotamer conformations that are populated
after mutation of Ile 3.54(143)
Ala provides a
rationale for the observed phenotype. The bulky side chain of an Ile,
Leu, or Val residue at this position would clash with the Arg side
chain when this adopts the rotamer (t, g
, t, g+), as shown in Fig.
6. In contrast, an Ala at this position lacking the bulky side chain would allow this unfavorable conformation of the Arg residue, as can be seen in Fig. 5. According to the membrane-water boundary determined experimentally for rhodopsin and
located between residues 3.52 and 3.53, the (t, g
, t, g+) rotamer of
the Arg would orient the charged guanidinium group toward the aqueous
cytoplasm. The strong solvation of the charged Arg side chain would
inhibit it from further participation in any intramolecular
interactions. Consequently, the results suggest that the structural
role of Ile3.54 is to restrict the positioning of
Arg3.50 during receptor activation. In the absence of a
bulky side chain at this position, the solvation of Arg3.50
in the cytoplasm may prevent it from forming the interactions most
conducive to establishing an active receptor state.
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Ile3.46(135)-- In the simulations, the wild-type receptor and Ile3.46(135) mutant constructs showed similar orientations of the Arg side chain and the surrounding residues. Mutagenesis experiments show that mutation of Ile3.46(135) to either Ala or Val is not tolerated, whereas substitution by Leu shows increased receptor efficiency as compared with the wild-type receptor. Both the lack of a local helix 3 effect of mutation of this locus in computational experiments and the highly restricted pattern of functionally tolerated mutations suggest that Ile3.46(135) does not form a part of the Arg-cage motif and may have a role in interhelical interactions not considered in this study.
Ser3.51(140)-- Although there is a possible H-bonding interaction between Arg3.50(139) and Ser3.51(140), the interaction energy is not competitive with the strong Arg3.50(139)-Asp3.49(138) interaction that predominates in the wild-type receptor. Removal of the H-bonding group of Ser3.51(140) by substitution to Ala produced no detectable alteration of the functional properties of the GnRH receptor (32), suggesting that Arg3.50(139) does not interact with this site or that the energetic contribution of such an interaction to the receptor activation mechanism is not significant.
Mechanistic Hypothesis for the Transition from an Inactive to an Active State of the Receptor-- The implications of the pattern of interactions (e.g. Arg3.50(139)-Asp3.49(138)) and preferred conformations of the Arg3.50(139) side chain derived from conformational analysis on TMD 3 alone were further analyzed in the context of a seven TMD model of the receptor. Because the strongest interaction of Arg3.50 is an ionic bond, we explored whether an alternative charge counterpart (Asp/Glu) could be found within other TMDs. The Asp or Glu residues present within the transmembrane domains of the GnRH receptor are shown schematically in Fig. 7. An acidic counterpart for the Arg would be expected to share its high degree of conservation and to reside at the cytoplasmic side of a TMD. However, the only conserved acidic group in the TMDs of the GnRH receptor is the Asp3.49(138) in the TMD 3 studied here. There are three nonconserved acidic residues present in the TMDs of the GnRH receptor at positions Glu2.53(90) and Asp2.61(98) in TMD 2 and Asp7.49(319) in TMD 7 (Fig. 7). An interaction of Arg3.50(139) with Glu2.53(90) or Asp2.61(98) is inconsistent with the geometrical constraints of an engineered Zn2+ binding site reported recently between TMD 2 and TMD 3 for the NK-1 receptor (36). Therefore these interaction possibilities were excluded. An interaction of Arg3.50(139) with the nonconserved Asp7.49(319) is possible in the complete model of the transmembrane portion of the GnRH receptor.
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ACKNOWLEDGEMENT |
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We thank Dr. Colleen Flanagan for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 DK46943, 5T32DA07135, and KO5 DA00060.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.
§ The first two authors contributed equally to this work.
Present address: Sarnoff Corp., 201 Washington Rd., Princeton,
NJ 08543.
§§ Fishberg Center for Neurobiology Research, Box 1065, Mount Sinai School of Medicine, One Gustave Levy Place New York, NY 10029. Tel.: 212-241-7075; Fax: 212-996-9785; E-mail: sealfon{at}msvax.mssm.edu.
1 The abbreviations used are: GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; TMD, transmembrane domain.
2 F. Guarnieri, manuscript in preparation.
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REFERENCES |
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