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Vol. 61, Issue 5, 1025-1032, May 2002
1b-Adrenergic
Receptor Highlight the Role of the Helix 3/Helix 6 Interface in
Receptor Activation
Institut de Pharmacologie et Toxicologie, Université de Lausanne, Lausanne, Switzerland (P.J.G., O.R., L.A., S.C.); and Dipartimento di Chimica, Università di Modena e Reggio Emilia, Modena, Italy (F.F.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
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Computer simulations on a new model of the 
1b-adrenergic receptor
based on the crystal structure of rhodopsin have been combined with
experimental mutagenesis to investigate the role of residues in the
cytosolic half of helix 6 in receptor activation. Our results support
the hypothesis that a salt bridge between the highly conserved arginine
(R1433.50) of the E/DRY motif of helix 3 and a conserved
glutamate (E2896.30) on helix 6 constrains the 1b-AR in
the inactive state. In fact, mutations of E2896.30 that
weakened the R1433.50-E2896.30 interaction
constitutively activated the receptor. The functional effect of
mutating other amino acids on helix 6 (F2866.27,
A2926.33, L2966.37, V2996.40,
V3006.41, and F3036.44) correlates with the
extent of their interaction with helix 3 and in particular with
R1433.50 of the E/DRY sequence.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
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The
1b-adrenergic receptor (AR) belongs to the rhodopsin family of G
protein-coupled receptors (GPCRs). GPCRs are structurally characterized
by seven transmembrane helices connected by alternating extracellular
and intracellular loops. Whereas ligand binding involves the
extracellular portion of the receptor, the intracellular regions
mediate the interaction of the receptor with G proteins as well as
other signaling and regulatory proteins.
A GPCR-mediated biological response involves a series of events (i.e., receptor activation, receptor-G protein interaction, and receptor-induced G protein activation) for which a detailed mechanism still remains elusive at the molecular level. Whereas residues located in the helical bundle and at the boundary between the membrane and the cytosol may play a role in the "conformational switch" underlying receptor activation [i.e., the transition from the inactive (R) to active (R*) state], amino acids in the intracellular loops are believed to be more directly involved in receptor-G protein interaction and/or receptor induced G protein activation. The combination of these two latter events, which cannot be unequivocally separated experimentally, is generally indicated as receptor-G protein coupling.
Biochemical and biophysical experiments on rhodopsin (Farrens et al.,
1996
; Sheikh et al., 1996) and the 
2-AR
(Jensen et al., 2001) suggest that their activation, i.e., the
transition from the inactive (R) to active (R*) state, involves a
rearrangement of helices 3 and 6 (reviewed by Gether, 2000). Such a
transition would result from the release of constraining interactions
between these two helices.
The recently published structure of rhodopsin in its inactive state
(Palczewski et al., 2000) suggests that a salt bridge between the
highly conserved arginine of the E/DRY motif of helix 3 and a glutamate
on helix 6 represents an important interaction constraining the
positions of helices 3 and 6. The interaction pattern involving this
conserved arginine in the inactive state of rhodopsin is not consistent
with that found in the wild-type model of the
1b-AR previously achieved by following an ab
initio approach (Scheer et al., 1996, 1997, 2000; Fanelli et al.,
1998). In fact, according to this model, the ground state of the
1b-AR would be stabilized by the interactions
between R1433.50 and some amino acids forming a
highly conserved polar pocket (i.e., D912.50 and
Y3487.53).
To advance our understanding of the molecular mechanisms underlying the
1b-AR function, we recently built a homology
model of the receptor based on the crystal structure of rhodopsin and have interpreted a number of experimental results in the context of
this new model (Greasley et al., 2001). In this study, we have challenged the predictions of the 1b-AR
homology model concerning the potential role in receptor activation of
residues in the cytosolic half of helix 6 and in particular of those
that are predicted to be located at the helix 3/helix 6 interface. Our
results provide significant insight into the packing of helices 3 and 6 as well as into some of the structural constraints stabilizing the
inactive state of the 1b-AR.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
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Materials. COS-7 cells were from the American Type Culture Collection (Manassas, VA); DMEM, gentamicin, fetal bovine serum and restriction enzymes were from Invitrogen (Carlsbad, CA). Pwo polymerase was from Roche Applied Science (Mannheim, Germany); [125I]HEAT and [3H]inositol from PerkinElmer Life Sciences (Boston, MA); epinephrine was from Sigma (St. Louis, MO); and prazosin was from RBI/Sigma (Natick, MA).
Mutagenesis of the 1b-AR.
The cDNA of the
hamster 1b. AR (Cotecchia et al., 1992)
was mutated using polymerase chain reaction-mediated mutagenesis and
Pwo DNA polymerase. The constructs were subcloned in the
pRK5 expression vector and mutations confirmed by automated DNA
sequencing of the entire portion amplified with polymerase chain
reaction (Microsynth GmbH, Balgach, Switzerland).
Cell Culture and Transfection.
COS-7 cells were grown in
DMEM supplemented with 10% fetal bovine serum and gentamicin (100 µg/ml) and transfected using the DEAE-dextran method. For inositol
phosphate determination, COS-7 cells (0.15 × 106) were seeded in 12-well plates. The quantity
of transfected receptor encoding DNA was 0.6 to 3 µg/106 cells. In each experiment, the wild-type
1b-AR was expressed using varying quantities
of DNA, thereby allowing us to directly compare the properties of the
mutated receptors with those of the wild-type receptor expressed at
comparable levels within the same experiment.
Ligand Binding.
Membrane preparations derived from cells
expressing the 1b-AR or its mutants and ligand
binding assays using [125I]HEAT were performed
as described previously (Cotecchia et al., 1992). Prazosin
(10
6M) was used to determine nonspecific
binding. [125I]HEAT at a concentration of 250 pM was used for measuring receptor expression at a single concentration
and 80 pM for competition binding analysis. Saturation analysis and
competition curves were analyzed using Prism 3.02 (GraphPad Software,
San Diego, CA).
Inositol Phosphate Measurements.
Transfected cells were
labeled for 12 h with
[myo-3H]inositol at 4 µCi/ml in
inositol-free DMEM supplemented with 1% fetal bovine serum. Cells were
preincubated for 10 min in PBS containing 20 mM LiCl and then
stimulated for 45 min with different concentrations of epinephrine
ranging from 1010 to
104M. Total inositol phosphates were extracted
and separated as described previously (Cotecchia et al., 1992).
Homology Modeling of the 1b-AR and Its
Mutants.
The homology model of the 1b-AR
was built using the program MODELLER (Sali and Blundell, 1993) and the
structure of rhodopsin (Palczewski et al., 2000) as a template, as
described recently (Greasley et al., 2001).
1b-AR
templates were probed in which the e2 loop, the i3 loop, and, in some
cases, the i2 loop were extracted from the input structure of the ab
initio model described previously (Fanelli et al., 1998). Furthermore, helix 5 was elongated by 10 amino acids in the chimeras using the
1b-AR sequence after deleting the 226-to-235
segment of rhodopsin. For each of the eight different templates,
MODELLER generated 25 models. Among the 200 models finally obtained, 20 models were selected showing low restraint violations and low numbers
of main- and side-chain bad conformations or close contacts. These
models were completed by the addition of the polar hydrogens and
subjected to automatic and manual rotation of the side chain torsion
angles when in bad conformations, as well as to energy minimization and molecular dynamics (MD) simulations according to the
computational protocol employed for simulating the ab initio
1b-AR model (Fanelli et al., 1998). About 450 MD trial runs were performed to select the proper input structure for
the wild-type 1b-AR. This structure was
employed to build the input structure of the receptor mutants by
substituting in turn each of the target amino acids. For each receptor
mutant, different starting conformations of the mutated side chain were
probed by MD simulations. These conformations were assigned by using
different rotamer libraries and checking for the absence of bad
contacts between the mutated side chain and its neighboring amino acid
residues. The structure of the wild-type receptor and its mutants
averaged over the last 100 ps of the 150-ps MD trajectory were
finally minimized and considered for the comparative analysis.
In this study, the amino acids are labeled according to a double
numbering system. In addition to numbering their position in the
receptor sequence, the amino acids in the helical bundle are labeled
with superscript numbers indicating the relative position of each amino
acid in the helix (Ballesteros and Weinstein, 1995). According to this
numbering system, every amino acid identifier starts with the helix
number, followed by the position relative to a reference residue among
the most conserved amino acid in that helix. That reference residue is
arbitrarily assigned the number 50.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
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Expression of Receptor Mutants.
The wild-type and mutated
1b-ARs were expressed in COS-7 cells and
tested for their ability to bind the radioligand
[125I]HEAT, epinephrine, and prazosin.
Saturation binding experiments indicated that the
KD of
[125I]HEAT was approximately 80 pM for all the
receptors studied (results not shown), whereas the
IC50 values for epinephrine varied as indicated
in Table 1. The affinity of prazosin for
the different receptor mutants was similar to that for the wild-type
1b-AR (results not shown). Receptor coupling
to the Gq/PLC pathway was assessed as the ability
of the receptor mutants to mediate epinephrine stimulated inositol
phosphates (IP) accumulation (Table 1).
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1b-AR was
expressed using two quantities of DNA (0.6 and 3 µg of
DNA/1x106 cells), resulting in low (between 60 and 120 fmol/well) and high (between 200 and 350 fmol/well) levels of
expression. This allowed us to directly compare the properties of the
mutated receptors with those of the wild-type
1b-AR expressed at comparable levels within
the same experiment.
Mutagenesis of Helix 6 Amino Acids Directed toward Helix 3.
Fig. 1 shows the average minimized
structure of the 1b-AR built on the recent
crystal structure of rhodopsin. A striking feature of the model is the
interaction of R1433.50 of the E/DRY motif with
both the adjacent D1423.49 and
E2896.30. The
R1433.50-E2896.30
interaction would be expected to constrain the relative positions of
helix 3 and helix 6. E2896.30 can make additional
intrahelix interactions with K2856.26 and/or
K2906.31.
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1b-AR is constrained by the charge reinforced
H-bonding interaction between E2896.30 and
R1433.50 of the E/DRY sequence. However, computer
simulations suggest that the formation of the salt bridge is strongly
dependent on the starting conformation of
R1433.50. In other words, the arginine should
have a folded conformation in the input structure as found in the
rhodopsin structure so as to maintain the salt bridge interaction
during MD simulations. However, this conformation (dictated by the
tight packing of the cytosolic extensions of helices 3 and 6 in the
inactive state of rhodopsin) does not correspond to any of the arginine
conformations most frequently found in protein structures (Dunbrack and
Karplus, 1993). Whether the conformation and the interaction pattern of the arginine belonging to the E/DRY motif are just peculiar to the
available rhodopsin structure will await the structural resolution of
other closely related GPCRs.
To challenge the predictions of the model,
E2896.30 was mutated into several amino acids.
The receptor mutants in which E2896.30 was
mutated into Ala, Phe, Lys, and Arg displayed the typical features
described for the active state (R*) of a GPCR (Samama et al.,
1993), including a marked increase in the constitutive and
agonist dependent activity of the receptor as well as increased affinity and potency of the agonist (Table 1 and Fig.
2a). In agreement with the experimental
findings, the model predicts that mutating
E2896.30 to the neutral amino acids Ala and Phe
or to the positively charged residues Lys and Arg breaks the salt
bridge interactions of R1433.50 and should
therefore constitutively activate the 1b-AR.
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). The results of computer simulations did not provide an
unequivocal explanation of the effects resulting from this mutation. In
fact, it was frequently observed that either the reversible or
irreversible neutralization of E2896.30 resulting
from its protonation or mutation to Gln, respectively, can break both
the salt bridge interactions involving R1433.50
and the residues at positions 1423.49 and
2896.30. However, in some simulations, H-bonding
interactions were observed between the replacing glutamine at position
2896.30 and R1433.50. This
suggests that, despite the neutralization of the charge, the mutation
of E2896.30 to glutamine does not induce a
destabilization between the cytosolic ends of helices 3 and 6 sufficient to trigger constitutive activity.
These findings are overall in good agreement with those of a recent
study reporting that mutations of the equivalent glutamate (E2686.30) of the 2-AR into Ala significantly
increased the constitutive activity of the receptor (Ballesteros et
al., 2001). In contrast, the mutation of the glutamate to Gln resulted
only in a modest increase of constitutive activity compared with the
Ala mutation without any significant change in the affinity of the
agonist. It would be interesting to investigate whether other mutations of E2686.30 in the 2-AR would result in
greater levels of constitutive activation.
We then investigated whether swapping the positively and negatively
charged residues reconstitutes their interaction, resulting in a
functional mutated receptor. However, the E289R/R143E mutant was
totally impaired in its ability to mediate an IP response (Table 1 and
Fig. 2a). These findings agree with the predictions of the model
demonstrating that positions 143 and 289 are not interchangeable. In
fact, all MD simulations of the E289R/R143E mutant failed to
reconstitute the salt bridge between positions 1433.50 and 2896.30,
whereas new links between helices 3 and 6 were formed. In addition, the
impairing effect of the double mutation is probably caused by the
requirement for a positive charge at position
1433.50 of the 1b-AR for
agonist-induced receptor-G protein coupling, as demonstrated previously
(Scheer et al., 2000).
The next amino acid in helix 6 that faces helix 3 is
L2966.37. To test its functional role,
L2966.37 was mutated to Ala or Phe, which reduces
or increases its size, respectively. Both mutations of
L2966.37 to Ala and Phe resulted in a decrease in
IP response of greater than 70% (Table 1). At the same time, the
epinephrine affinity of these mutants was unchanged. Computer
simulations indicated that the L296A mutant displayed increased
proximity of the helix 3 and 6 backbones while conserving both the
interactions involving R1433.50. The mutation of
L2966.37 to Phe introduces additional
interactions between the mutated amino acid and residues on helices 2 and 7 but leaves the interactions of R1433.50
unaltered. Thus, L2966.37 seems to have an
important structural role in controlling the helix 3-helix 6 packing
that is perturbed by mutations of this residue to either Ala or Phe.
The final residue in the cytosolic half of helix 6 to directly face
helix 3 is F3036.44. Previous studies have shown
that mutation of this residue into Asn or Gly totally impairs the IP
response but markedly increases the affinity of epinephrine (Chen et
al., 1999, 2000). To further delineate the role of
F3036.44, we mutated it to Ala, Gly, Leu, and Tyr
(Table 1 and Fig. 2b). Consistent with the previously published
findings, the F303G receptor displayed a 50-fold increase in affinity
for epinephrine and an almost complete loss of receptor mediated
signaling. The F303A mutant was functionally similar to the F303G. The
mutation of F3036.44 to Tyr impaired the receptor
mediated maximal response to epinephrine by about 50%. Whereas the
affinity of epinephrine was not significantly changed at the F303Y
mutant, its potency was decreased by almost 20-fold (Table 1). These
findings strongly suggest that the mutation of
F3036.44 to Tyr, which maintains the aromaticity
of the side chain but introduces a polar functional group, impairs
receptor activation. In contrast, the mutation of
F3036.44 to Leu, which maintains the
hydrophobicity but abolishes the aromaticity, increased the
constitutive as well as agonist-induced activity of the receptor. This
mutation resulted also in a 30-fold increase in affinity and a 20-fold
increase in potency of epinephrine (Table 1).
In the average minimized structure of the wild-type
1b-AR, F3036.44 together
with W3076.48 and F3106.51
forms a cluster of aromatic amino acids on helix 6, making a chain of
intrahelical interactions. The most extracellular residue in this
chain, F3106.51, may be directly involved in the
agonist binding as suggested by both the computational and experimental
results (Chen et al., 1999). In addition,
F3036.44 interacts with
S1323.39 and I1333.40 as
well as with N3447.49 of the NPXXY motif. The
results of computer simulations suggest that the F303A and F303G
mutations enhance the packing of helices 3 and 6 while retaining both
the R1433.50-D1423.49 and
R1433.50-E2896.30
interactions found in the wild-type receptor. Substituting
F3036.44 with Tyr does not disrupt the
interaction pattern of R1433.50. This is
consistent with experimental findings showing that the F303Y is not
constitutively active. The decreased ability of epinephrine to activate
this mutant might be caused, at least in part, by the fact that the
phenolic hydroxy group of Y3036.44 might perform
H-bonds with N3447.49 of the NPXXY motif, which
is involved in the activation of the receptor (Scheer et al., 1996).
This interaction might induce changes in the side chain fluctuations at
either or both of these functionally important positions, compared with
the wild-type.
Interestingly, the mutation of F3036.44 to Leu
perturbs the helix 3/helix 6 and the helix 6/helix 7 packing
interactions in the vicinity of the mutated residue. This change is
propagated along the helix 3/helix 6 interface, destabilizing both the
R1433.50-D1423.49 and
R1433.50-E2896.30
interactions. Therefore, computer simulations suggest that
F3036.44 lies in a position important for the
transfer of information from the binding site of the agonist to the
cytosolic portions of the helix 3-helix 6 interface.
Mutagenesis of Amino Acids on Helix 6 That Face Helices 2, 5, or 7. In this study, we also considered other amino acids in the cytosolic half of helix 6 that do not directly face helix 3 but rather are directed toward helix 2 (A2926.33), helix 5 (V3006.41), or helix 7 (V2996.40).
A2926.33 lies in the proximity to R1433.50 of the E/DRY motif. Consequently, the mutation of A2926.33 to Glu may be expected to reinforce the link between helices 3 and 6, thereby stabilizing the inactive state of the receptor. In agreement with this prediction, the A292E mutant displays a profoundly impaired IP response without any significant change in the affinity of epinephrine (Table 1). As for V2996.40 and V3006.41, their positions in the wild-type receptor suggests that V2996.40 rather than V3006.41 plays a potential structural role. In fact, V2996.40 lies between I3477.52 and Y3487.53 of the NPXXY highly conserved motif in helix 7. In contrast, V3006.41 lies between I2195.54 and L2205.55 in helix 5. To test the functional effect of decreasing and increasing the size at these interhelical positions, V2996.40 and V3006.41 were mutated to Ala and Phe, respectively. Computer simulations of V299F, V300A, and V300F produce average arrangements retaining the wild-type interaction patterns of R1433.50. In contrast, the V299A mutant retains only the R1433.50-E2896.30 interaction. Thus, simulations suggest that mutations at position 3006.41 have higher propensity to behave like the wild-type than mutations at position 2996.40. In agreement with this, experimental mutagenesis indicated that the mutations of V2996.40 and V3006.41 into either Ala or Phe did not impair receptor activation (Table 1). However, the mutation of V2996.40 into Phe increased the affinity for epinephrine by 100-fold. The potency of epinephrine was also significantly increased at the V299F mutant, but only by 5-fold (Table 1). These findings suggest that neither V299 nor V300 plays a crucial role in receptor activation. However, the structural change induced by the mutation of V2996.40 to Phe has a marked effect on agonist binding resulting in a large increase in its affinity.| |
Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
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Using predictions from a model of the
1b-AR based on the crystal structure of
rhodopsin and site-directed mutagenesis, we have investigated the role
in receptor activation of residues on the cytosolic half of helix 6 either facing helix 3 (F2866.27,
E2896.30, L2966.37 and
F3036.44) or other helices
(A2926.33, V2996.40, and
V3006.41). Our findings support the hypothesis
that receptor activation involves the weakening or breaking of
interhelical interactions between the cytosolic halves of helices 3 and
6. On the opposite, mutations in helix 6 that strengthen these
interactions stabilize the inactive state of the receptor resulting in
impaired receptor activation.
The majority of the mutations reported in this study resulted in
important changes in the basal and/or agonist-induced IP response of
the receptor as well as in the affinity and/or potency of epinephrine.
Because the mutated residues are not predicted to be part of the
1b-AR-Gq interface (Fanelli et al., 1999), a
direct effect of the mutations on receptor-G protein coupling could be
excluded. The computational and pharmacological analysis of the
receptor mutants support the hypothesis that the mutations herein
reported can either have a direct effect on the process of receptor
activation [i.e., the transition from the inactive (R) to active (R*)
state] or induce other structural changes having an impact on receptor function.
The Role of E2896.30 on Helix 6 in Receptor
Activation.
A novel finding of our study is that mutations of
E2896.30 on the cytosolic end of helix 6 of the
1b-AR can markedly increase the constitutive
activity of the receptor (Table 1). All the E2896.30 mutants (with the exception of E289Q)
displayed all the properties previously considered the hallmark of the
active state of the receptor (R*): increased constitutive activity,
increased efficacy of the agonist, increased affinity and potency of
the agonist (Samama et al., 1993). Altogether, these findings
identify E2896.30 as an important player in the
activation process of the 1b-AR.
1b-AR, that is different, at
least in part, from that proposed in our previous studies (Scheer et
al., 1996, 1997). The differences mainly concern the structural
features of the inactive states of the 1b-AR.
In fact, in the ab initio model of the 1b-AR
described previously, the arginine of the E/DRY motif was directed
toward helix 2 and we predicted that its interaction with a conserved
aspartate (D912.50) on this helix was an
important constraint in maintaining the receptor in its inactive state.
However, the crystal structure of rhodopsin in its inactive state
(Palczewski et al., 2000) suggests that the arginine of E/DRY sequence
is too far apart for interacting with the conserved aspartate on helix
2, given that it is involved in interactions with both the adjacent
E1363.49 and a glutamate in helix 6, E2476.30. Thus, the interaction between
R1353.50 and E2476.30 is
suggested to be one of the critical constraints keeping rhodopsin in
the inactive state.
Similar to the rhodopsin structure, in the homology model of the
wild-type 1b-AR, D912.50
is too far to interact with R1433.50, because the
distance between the 
-carbon atom of D912.50
and the 
-carbon atom of R1433.50 is 22.8 Å (Fig. 1). Instead, in the inactive state of the receptor R1433.50 makes a salt bridge with both the
adjacent D1423.49 and
E2896.30 on helix 6. The experimental findings
described herein seem to favor the predictions of the homology model,
because mutations of both D1423.49 on helix 3 (Scheer et al., 1996) and of E2896.30 on helix 6 markedly increase the constitutive activity of the receptor. Therefore,
whereas the
D1423.49-R1433.50 and
R1433.50-E2896.30
interactions constrain the receptor in the inactive states, their breakage would contribute to receptor activation.
The functional role of the glutamate homologous to
E2896.30 in rhodopsin remains to be elucidated by
site-directed mutagenesis. However, very recently it has been reported
that mutations of the equivalent glutamate
(E2686.30) of the 2-AR into Gln and A
significantly increased the constitutive activity of the receptor
(Ballesteros et al., 2001). Moreover, spontaneous mutations of
D5646.30 in the human lutropin/choriogonadotropin
receptor result in constitutive activation of the receptor and are
associated with precocious puberty in males (Yano et al., 1995; Shenker
et al., 1993). Altogether these findings strongly suggest that in the
rhodopsin family of GPCRs where the arginine is almost ubiquitous and
the glutamate/aspartate is highly conserved, these two residues are
likely to play an important role in receptor activation.
The Role of Other Amino Acids at the Helix3/Helix 6 Interface. Our results suggest that the integrity of F2866.27, that is the most solvent accessible residue on helix 6, is important in receptor activation contributing to maintain the inactive state of the receptor. In fact, our results suggest that the mutation of F2866.27 to Ala or to Glu could favor or impair receptor activation weakening or reinforcing the link between the cytosolic halves of helices 3 and 6, respectively.
We have found that structural variability at position 2966.37 of the1b-AR on
the cytosolic half of helix 6 is poorly tolerated. In fact, the
substitution of L2966.37 with Ala or Phe
profoundly impaired the receptor-mediated IP response. Leucine,
isoleucine, or valine is generally found at the equivalent position in
GPCRs belonging to the rhodopsin family. A previous random mutagenesis
study on the C5a receptor demonstrated that a conservation of
hydrophobicity at this position is necessary to maintain productive
receptor-G protein coupling (Baranski et al., 1999). Another study has
suggested that a leucine at this position contributes to the
selectivity of the M1, M3, and M5 muscarinic cholinergic receptors for
Gq (Blin et al., 1995). Computer simulations
performed in this study suggested that the integrity of
L2966.37 plays a fundamental role in maintaining
the helix 3/helix 6 packing around R1433.50.
Our experimental findings also suggest that structural variability at
position 3036.44 of the
1b-AR is not well tolerated, in accordance
with the high degree of conservation of this residue in the rhodopsin
family of GPCRs. Interestingly, mutations of
F3036.44 had different effects on the activation
and agonist binding properties of the 1b-AR.
Because F3036.44 belongs to a chain of aromatic
amino acids near the binding site of epinephrine, it is not surprising
that mutations of F3036.44 can have an effect on
the affinity of the agonist. In fact, mutations of
F3036.44 to Gly, Ala, and Leu markedly increased
the affinity of epinephrine (Table 1). However, this increase in
agonist affinity was correlated with increased constitutive activity
only for the F303L mutant, in which the interaction pattern of
R1433.50 was perturbed by the mutation. In
contrast, the nonconstitutively active F303A and F303G mutants, in
which the packing of helices 3 and 6 was enhanced and the interaction
pattern of R1433.50 was conserved, were
profoundly impaired in their activation properties. Altogether, our
findings suggest that F3036.44 is involved in the
transfer of the agonist-induced conformational change along the helix
3/helix 6 interface. The analysis of the structural features of the
constitutively active F303L mutant might provide insight into the
conformational changes triggered by the agonist that probably can
modify the helix3/helix6 and helix 6/helix7 packing.
The Role of Amino Acids on Helix 6 Facing Helices 2, 5, and 7.
The mutational analysis of other residues in the cytosolic half of
helix 6 suggests that in contrast to those residues facing helix 3, the
majority of amino acids facing helix 2 (A2926.33), helix 5 (K2906.31, A2936.34, and
V3006.41) or helix 7 (R2886.29 and V2996.40) do
not play a prominent structure-functional role. In fact, we have
reported recently that mutations of R2886.29 and
K2906.31 to Ala or Glu did not significantly
change the functional properties of the 1b-AR
(Greasley et al., 2001). In this study, we have found that mutating
V2996.40 and V3006.41 to
Ala or Phe did not markedly change the receptor-mediated IP response
(Table 1). The only exceptions are residues that lie at almost the same
level as R1433.50 with respect to the membrane
(i.e., A2926.33 and
A2936.34). In fact, it has been shown previously
that all mutations of A2936.34 increased the
constitutive activity of the 1b-AR (Kjelsberg et al., 1992) Mutation of A293 to Glu induces the formation of an
intrahelix salt bridge between the replacing glutamate and K2906.31. The latter residue loses its
interaction with E2896.30, which consequently
loses its charge reinforced H-bonding interaction with
R1433.50, gaining new intrahelix interactions
with both K2856.26 and the adjacent
R2886.29. Thus, the constitutive activity of the
A293E mutant seems to be correlated with a rearrangement of charged
amino acid side chains that results in the loss of the helix 3-helix 6 interactions. In contrast, the impairing effect of mutating
A2926.33 to Glu might be correlated with the
possible formation of a salt bridge between the replacing glutamate and
R1433.50, thereby reinforcing the link between
helices 3 and 6.
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Conclusions |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
|
|---|
Taken together, the results of this study suggest that the role in
activation of the amino acids in the cytosolic half of helix 6 of the
1b-AR is strongly correlated to the extent of their structural/dynamic connection with helix 3 and the arginine of
the E/DRY sequence. Consistent with the hypothesis previously inferred
from the ab initio model, the homology model of the
1b-AR suggests that the active receptor states
would be characterized by the release or the weakening of the
interactions that, in the inactive state, constrain the motion of the
fully conserved arginine of the E/DRY motif. In general, we have
observed that the weakening or breakage of the
R1433.50-E2896.30
interaction is associated with the breakage of the
R1433.50-D1423.49
interaction in those mutants displaying a high degree of constitutive activity (e.g., A293E, F303L, E289A, E289R, E289K). In contrast, in
some weakly constitutively active mutants (e.g., F286A), only the
breakage of the
R1433.50-D1423.49
interaction was found, suggesting that this is one of the early events
in the transition of the 1b-AR from its
inactive to active states.
It is worth noting that a dramatic detachment between the cytosolic
halves of helices 3 and 6, as observed in the light-induced active
state of rhodopsin (Farrens et al., 1996), has never been observed in
our simulations. This agrees with the finding that in rhodopsin, the
constitutively activating mutation of E134 to glutamine induces helix
motions different from those caused by photoactivation (Kim et al.,
1997). Indeed, the E134Q active mutant did not display the dramatic
increase in distance between helices C and Phe as observed upon
photoactivation of the wild-type rhodopsin (Farrens et al., 1996).
According to both the ab initio and homology models of the
1b-AR, in the most active mutants, the
perturbations in the interactions of R1433.50 are
associated with the increase in solvent accessibility of amino acids in
the cytosolic extension of helix 6 and, for the ab initio model only,
in the N-terminal portion of the second intracellular loop (Fanelli et
al., 1999). However, this increase in solvent accessibility very rarely
involves R1433.50, suggesting that the main role
of this amino acid is to mediate the allosteric transition between the
inactive ad active receptor states rather than directly binding the
G-protein. The hypothesis suggested by the theoretical models is that
the change in the interaction pattern of R143 would be instrumental to
allow amino acids in the third intracellular loop, including R254 and
K258 to assume the proper configuration for binding and/or activating the G protein (Fanelli et al., 1999; Greasley et al., 2001). However, the role of the highly conserved arginine of the E/DRY in the activation process of GPCRs awaits full elucidation.
| |
Acknowledgments |
|---|
We acknowledge the contribution to the experiments of undergraduate students Nguyên Duc-Quang and Bondollaz Percy. We are grateful to Monique Nenniger-Tosato for her excellent technical assistance.
| |
Footnotes |
|---|
Received July 26, 2001; Accepted February 12, 2002
This work was supported by the Fonds National Suisse de la Recherche Scientifique (grant 31-51043.97) and by the European Community (grant BMH4-CT98-3566). F.F. is supported by Telethon-Italy (grant n. 68/cp) and is an Assistant Telethon Scientist.
The receptor coordinates are available upon request from fanelli{at}unimo.it.
Address correspondence to: Susanna Cotecchia, M.D., Institut de Pharmacologie et de Toxicologie, Faculté de Médecine, 27 Rue du Bugnon, 1005 Lausanne, Switzerland. E-mail: susanna.cotecchia{at}ipharm.unil.ch
| |
Abbreviations |
|---|
AR, adrenergic receptor(s);
GPCR, G
protein-coupled receptor;
DMEM, Dulbecco's modified Eagle's medium;
[125I]HEAT, [125I]iodo-2-[-(4-hydroxyphenyl)-ethylaminomethyl]tetralone;
MD, molecular dynamics;
IP, inositol phosphates.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
Conclusions
References
|
|---|
2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6.
J Biol Chem
276:
29171-291771B-adrenergic receptor is a key switch residue involved in activation and catecholamine ring aromatic bonding.
J Biol Chem
274:
16320-163301-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis.
J Biol Chem
267:
1633-16391b-adrenergic receptor: involvement of basic and hydrophobic residues in receptor activation and G protein coupling.
J Biol Chem
276:
46485-464942 adrenergic receptor mapped by site-selective fluorescent labeling.
J Biol Chem
276:
9279-92902-adrenergic receptor. Extending the ternary complex model.
J Biol Chem
268:
4625-46361b-adrenergic receptor: effects on receptor isomerization and activation.
Mol Pharmacol
57:
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