From the
A conserved helix 2 Asp is required for the proper function of
many G-protein-coupled receptors. To reveal the structural basis for
the role of this residue, the additive effects of mutations at this
locus and at a conserved helix 7 locus were investigated in the
5-HT
Members of the G-protein-coupled receptor (GPCR)
The
functional roles of highly conserved GPCR residues are being explored
with site-directed mutagenesis, and many have been shown to be required
for proper receptor function. However, little is currently understood
about the structural basis for the disruptive effect of mutations at
these loci. For example, the conserved TMH-2 Asp has been studied in a
large number of GPCRs, and mutations of this residue have been found to
alter signal
transduction(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) ;
agonist
affinity(2, 3, 6, 8, 10, 12, 13, 14, 15) ;
and allosteric modulation by
sodium(15, 16, 17, 18) , pH (16), or
guanyl
nucleotides(2, 3, 4, 8, 10, 11) .
When viewed together, the varying functional effects of mutations at
this site in different GPCRs suggest that this residue is involved in
receptor rearrangement among different conformational states. While
this rearrangement is likely to involve interactions of this residue
with side chains in other helices, there is no direct data from any
receptor that conserves this TMH-2 Asp to illuminate the nature of this
interaction or to identify candidate interaction sites in other
helices.
The absence of fine resolution structural data on any GPCR
makes it especially difficult to propose molecular mechanisms that
could explain why specific mutations produce the particular effects
that are observed in various assays of receptor function. Although a
9-Å projection density map showing the helical nature of the
transmembrane domains and their arrangement has recently been obtained
for rhodopsin (19), the low resolution does not permit inferences about
side chain proximities and helix-helix interactions in this or any
other GPCR.
In an attempt to generate the experimental basis for
understanding receptor activity at a detailed molecular level, we have
begun to study the functional effects of mutation of the conserved
TMH-2 locus and the additive effects of concomitant mutations at other
receptor sites. To interpret the results from such studies in a
structural context, we have developed techniques for modeling both
receptor structure and conformational behavior in response to mutation
and/or complexing with ligand, an approach referred to as dynamic
modeling(28) . The evaluation of these models by molecular
dynamics simulations can generate experimentally testable hypotheses
that relate structural details to functional mechanisms. For example,
the reciprocal mutation studies of the gonadotropin-releasing hormone
(GnRH) receptor integrated with computational modeling of GPCRs led to
the proposal of a spatial proximity of the TMH-2 locus, which contains
an Asp in nearly all GPCRs, to a specific locus in TMH-7(20) .
In these studies, a functionally disruptive mutation in one helix is
corrected by the introduction of a second mutation in the other helix.
Restoration of function by the second mutation indicates that the two
residues share a microenvironment. This explanation for their
interrelated functional roles is consistent with the network of
interactions observed in the three-dimensional model of the GnRH
receptor(20) . However, the residues present in the GnRH
receptor at the two loci studied in TMH-2 and TMH-7 diverge from the
conservation pattern found in most other GPCRs. Therefore, we were
interested in the ability of our approach to elucidate the relationship
between the two loci in a neurotransmitter receptor having the pattern
found in >95% of GPCRs, i.e. Asp in TMH-2 and Asn in TMH-7.
In this study, the role of the conserved TMH-2 Asp and its
relationship to the TMH-7 Asn in the activation of the serotonin
5-HT
Specific binding was determined by subtracting the amount of
[
Mutation of
Asp
If two protein loci are independent in their contribution to
a measured property, the effect of a mutation at either locus should be
additive(32, 33) . The effects of mutations at the TMH-2
and TMH-7 positions studied are not additive. Mutation of the conserved
TMH-2 Asp
The altered coupling observed with
these mutations cannot be attributed to disruption of the binding
pocket because the affinities for agonists and antagonists do not vary
significantly among all the constructs studied. Furthermore, the
functional differences caused by the mutations are not due to
differences in the levels of expression obtained because the construct
with the highest level of expression, Asp
The results of molecular dynamics simulations of the effects of
agonist binding to the wild-type and mutant receptors suggest a
possible mechanism for the loss of coupling observed with the TMH-2 Asp
It is
notable that the structure of the mutant receptor model itself is quite
different from that of the wild-type receptor model in the segments of
the two helices that are proximal to the third intracellular loop.
Furthermore, whereas antagonists induce essentially no conformational
change in the wild-type receptor model (Fig. 3C), the
agonist induces a conformational rearrangement of the mutant receptor
model that is different from that of the wild-type receptor (Fig. 3B). Therefore, the results from computational
simulations with these models suggest that agonist still induces a
conformational change in the mutant receptor, but that the specific
helix rearrangements achieved may not support the same mechanism of
coupling to the G-proteins mediating activation of phospholipase C.
These simulation results are interesting in view of the variable
effects on signal transduction obtained with mutation of this locus in
different
receptors(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 34) .
In studies of this locus in the
The modeling and mutagenesis results are consistent
with the hypothesis that a favorable network of hydrogen bonding
involving the side chains at the two loci is required for proper
agonist-induced conformational changes in the receptor. A
hydrogen-bonding network involving Asn
Although the double mutant receptor has improved function
relative to the single mutant Asp
The results obtained with mutation of these loci
have interesting parallels and contrasts relative to those reported for
the GnRH receptor(20) . In both receptors, a single mutation at
the TMH-2 locus disrupts function, and functionality is restored by a
second, interchanging mutation at the TMH-7 locus. The demonstration of
a restoration of function by reciprocal mutations involving these two
loci in both the GnRH receptor and the 5-HT
Mutation of TMH-2 Asp
The
exploration of the structural and pharmacological properties of the
5-HT
Binding
affinities (K
The K
Dr. Alan G. Saltzman is gratefully acknowledged for
providing the human 5-HT
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptor. All mutant receptors studied retained high
affinity agonist and antagonist binding. Whereas an Asp
Asn
mutation in helix 2 eliminated coupling, interchanging the residues at
the two positions by a second mutation of Asn
Asp in helix 7
restored receptor function. These data suggest that these residues are
adjacent in space and interact. The loss of function observed with Ala
at either position is consistent with each side chain forming hydrogen
bonds. Molecular dynamics simulations were performed on
three-dimensional computational models of agonist-receptor complexes of
both the wild-type receptor and the Asp
Asn mutant receptor.
Consonant with the lack of coupling observed for the mutant construct,
introducing the mutation into the computational model produced a
conformational change in a direction opposite to that seen from
computational simulations of activation of the wild-type receptor
model. These results implicate both loci in a common hydrogen-bonding
network underlying receptor activation by agonist.
(
)superfamily contain seven putative transmembrane
helix (TMH) domains and activate intracellular heterotrimeric
G-proteins. The cloning of more than 200 distinct members of this gene
family has revealed a high degree of homology in the TMH regions in
which certain amino acid sequence motifs are highly conserved among the
different receptors (for review, see Ref. 1). Sequences resistant to
alteration during an evolutionary process that has generated receptors
for ligands as diverse as neurotransmitters and glycoprotein hormones
are likely to constitute structural elements that mediate universal
receptor functions. One structural template that is potentially shared
by most, if not all, GPCRs is an interhelical network of side chain
interactions underlying the activation of these receptors. The highly
conserved amino acids are therefore reasonable candidates for having a
key functional role in the agonist-induced rearrangement of the TMHs
that constitutes the mechanism of receptor activation.
receptor was investigated by complementary
site-directed mutagenesis experiments. From the pharmacological
characterization of the mutant receptors, we are able to demonstrate a
function-restoring reciprocal mutation involving these two loci. In
parallel, we have evaluated the effects of mutation of TMH-2 Asp
Asn in a three-dimensional molecular model of the 5-HT
receptor. Computational simulation studies of the wild-type
5-HT
receptor model have characterized the pattern of
structural rearrangements induced by agonist binding in the helix
bundle and have suggested a mechanism of receptor
activation(21, 22, 23) . Similar molecular
dynamics simulations for the mutant receptor are shown here to lead to
a very different structural rearrangement of the receptor regions
presumed to be involved in coupling to G-proteins. Evaluation of the
results of site-directed mutagenesis in the context of these dynamic
simulations of a receptor model provides a structural hypothesis for
the functional effects of these mutations. Our results suggest that a
common hydrogen-bonding network involving the two loci is required for
receptor activation by agonist.
DNA Constructs and Transfection
A cDNA clone
encoding the human 5-HT receptor was generously provided
by Dr. Alan G. Saltzman(24) . The insert was digested with SmaI and ASP700 and ligated into SmaI-digested pALTER
(Promega, Madison, WI). Mutations were introduced with oligonucleotides
following the manufacturer's protocol and were confirmed by
sequencing. For expression, the insert was subcloned into the XbaI and EcoRV sites of pcDNAI/Amp (Invitrogen, San
Diego, CA) for expression. The mutations were reconfirmed by sequencing
the mutation site in the expression vector. COS-1 cells (American Type
Culture Collection, Rockville, MD) were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. Each
100-mm plate, seeded 24 h earlier with 3
10
cells,
was transfected with 8 µg of DNA construct and 48 µl of
Lipofectamine (Life Technologies, Inc.).
Phosphatidylinositol Hydrolysis Assay
24 h
following the transfection in 100-mm plates, the cells were harvested
with trypsin and seeded in 2 12-well plates. 32 h later, the
medium was replaced by serum-free medium containing 0.5 µCi/ml myo-[
H]inositol (DuPont NEN). 16 h
later, the cells were washed and incubated with the desired
concentrations of 5-HT in the presence of 20 mM LiCl for 45
min at 37 °C. Cell extracts, in 10 mM formic acid, were
applied to a Dowex ion-exchange column before elution by buffer
containing 1 M ammonium formate and 0.1 M formic
acid. The procedure essentially followed published
protocols(25) . After scintillation counting, the data were
plotted using Kaleidagraph (Synergy Software, Reading, PA) and fitted
to the following equation: E = E
/(1 + (EC
/D)), where E
is the maximum stimulation, D is the
concentration of the agonist, and EC
represents the
agonist concentration that produces half-maximal stimulation.
Ligand Binding Assay
Three days following
transfection, cells were harvested, and cell pellets were stored at
-70 °C. For preparation of membranes, all procedures were
carried out at 4 °C. Pellets were thawed and homogenized in 20 ml
of 50 mM Tris-HCl (pH 7.4) (buffer A) at 25 °C with a
Polytron homogenizer (setting 6 for 8 s). An additional 20 ml of buffer
A was added, and the homogenates were centrifuged at 35,000 g for 15 min. The membrane pellets were resuspended in buffer
A with a Teflon/glass homogenizer (10 strokes by hand). Each binding
incubation tube contained 70-180 µg of membrane protein,
[
H]Ketanserin (DuPont NEN), unlabeled drug as
required, and buffer A in a final volume of 1.0 ml. For saturation
binding studies, eight concentrations (0.05-10 nM) of
[
H]Ketanserin (specific activity of 85 Ci/mmol)
were tested in duplicate. For competition binding studies, the
concentration of [
H]Ketanserin was 1.7-2.0
nM, and 10 concentrations of competing ligands were tested in
duplicate. Nonspecific binding was determined in the presence of 10
µM methysergide. Incubations were carried out for 1 h at
37 °C and were terminated by rapid filtration through Whatman GF/C
glass fiber filters (Brandel cell harvester) followed by washing with
10 ml of buffer A at 4 °C. The radioactivity retained on the
filters was quantitated by liquid scintillation spectrometry. Protein
content was measured by the method of Lowry et al. (26).
H]Ketanserin bound in the presence of
methysergide from the amount bound in the absence of methysergide. For
saturation studies, B
and K
values for [
H]Ketanserin were obtained
by a fit of the data to the following equation: specific binding
= B
/(1 + (K
/D)), where D is the free
concentration of [
H]Ketanserin. For competition
studies, specific binding was expressed as a percent of specific
binding in the absence of the competing ligands and fit to the
following equation: % binding = 100 - (100/(1 +
(IC
/[5-HT]))), where IC
is the
concentration of competing drug that produces 50% specific binding of
[
H]Ketanserin. IC
values were
converted to K
values by the method of
Cheng and Prusoff (27) by using the K
values for [
H]Ketanserin determined
for each construct from saturation binding studies. Curve fitting of
data was carried out with Kaleidagraph.
Locus Numbering Scheme
The positions of various
amino acids in the receptor sequence and the structural changes in the
various receptor constructs are identified throughout with a numbering
scheme that facilitates the comparison among different receptors. This
consensus numbering scheme has been defined in detail
elsewhere(28) . Briefly, each locus is identified by a TMH
number followed by the position relative to the most conserved residue
in that helix, which is assigned the number 50. The conserved
Asp in TMH-2 of the 5-HT
receptor, for
example, is numbered 2.50, and Asn
, located one residue
before the conserved Pro in TMH-7, is numbered 7.49. The original
numbering is retained in parentheses, and amino acid mutations are
indicated by an arrow. Thus, the 5-HT
construct containing
a mutation of Asn
in TMH-7 to Ala is written
Asn
Ala.
Molecular Modeling of 5-HT
The model of the transmembrane helix bundle of the
5-HTReceptor
Mutants and Computational Simulations of Interactions with
Ligands
receptor and its development have been reported
previously(21, 29) . A primary objective in the early
development of this model was to reflect the pharmacological data on
the structure-activity relations of 5-HT
receptor ligands.
The relative position of the helices in this particular model of the
5-HT
receptor differs from that presented in other
published GPCR models, but it is one of the arrangements compatible
with the information currently available for rhodopsin(30) . In
this model of the 5-HT
receptor, the relative positions of
the TMH-2 Asp locus and the TMH-7 Asn locus proposed in the GnRH
receptor model (20) are preserved. The close agreement between
the results from computational explorations of the effects of ligand
binding to this receptor model and quantitative pharmacological data (21, 22) supports its use to construct the molecular
model of an Asp
Asn mutation in TMH-2.
Asn was introduced in the molecular
model of the 5-HT
receptor described previously (21). The
construct was energy-optimized, and a molecular dynamics run was
carried out for 100 ps with the CHARMM program (31) using the
same protocol as described previously(21) . The equilibrium
structure of the mutant receptor was obtained from the energy-minimized
average structure over the final 60-100-ps period of the
simulation. 5-HT was positioned in the binding site of the mutant
receptor in the same manner and using the same parameters for molecular
dynamic simulations as reported previously(21) . Simulation of
the ligand-receptor complex was carried out for 210 ps, and the
equilibrated structure of the agonist-receptor complex was obtained
from the energy-minimized average over the final 110-210-ps
period of the simulation. The changes produced by ligand binding in the
structure of the receptor model were determined as described (21, 22, 23) by calculating the root mean square
deviations of the positions of the
-carbons in the average
structure of the complexes relative to the equilibrated receptor
structures.
Agonist-stimulated Inositol Phosphate
Accumulation
The wild-type and mutant receptors were expressed
in COS-1 cells, and the concentration-response curves for
5-HT-stimulated inositol 1-phosphate accumulation were determined.
Representative concentration-response curves are shown in Fig. 1,
and all results are summarized in . The EC value measured for 5-HT on the wild-type receptor was 2.2
± 4
10
M, with a maximal PI
accumulation of 5.2 ± 0.7-fold over basal levels. Mutation of
Asp
Asn eliminated detectable PI
accumulation, confirming a report of this mutation in the rat
receptor(10) . Introducing the second mutation in helix 7,
Asn
Asp, interchanges the residues at the
two positions and restores agonist-induced PI hydrolysis. The efficacy
of activation of this reciprocal mutant receptor was apparently
reduced, as indicated by a maximal stimulation of only 3 ±
0.6-fold over basal levels and an increased EC
value for
5-HT (1.94 ± 0.36
10
M)
relative to the wild-type receptor (see ``Discussion'').
Introducing only the single mutation of Asn
Asp caused an intermediate increase in the EC
value for
5-HT relative to the wild-type receptor (9.5 ± 0.4
10
M). The requirement for a polar residue
at either position was explored with substitution by Ala at position
2.50(120) or 7.49(376). No 5-HT-stimulated PI hydrolysis was observed
following expression of either construct ().
Figure 1:
Concentration-response curves of PI
hydrolysis with 5-HT stimulation for the wild-type and mutant
constructs. A concentration-response curve representative of three
experiments is shown. The Asp
Ala and
Asn
Ala receptors showed no response over
basal levels at any 5-HT concentration, and for clarity, these data are
not plotted. Although a lower maximal response was obtained with the
single TMH-7 Asn
Asp mutation in the experiment shown, the E
values with this construct were, on average,
comparable to those obtained with the wild-type receptor (Table
I).
Radioligand Binding of Wild-type and Mutant
Receptors
The wild-type and mutant 5-HT receptors
expressed transiently in COS-1 cells bound
[
H]Ketanserin with high affinity. Representative
saturation isotherms for three of the constructs are shown in Fig. 2A; data for all constructs are shown in . The affinity of the mutant receptors for
[
H]Ketanserin varied by <2-fold compared with
the wild type. In contrast, B
values, calculated
from the maximal specific binding of
[
H]Ketanserin, were dependent on the particular
construct. The lowest levels of expression were obtained with the
Asn
Asp mutant, and highest levels with the
Asp
Asn mutant. The expression level for a
given construct was found to be highly consistent in separate
preparations.
Figure 2:
Saturation
and competition binding to the wild-type and mutant 5-HT receptors. A, saturation binding of
[
H]Ketanserin to the wild-type and mutant
5-HT
receptors. Incubations and analyses were carried out
as described under ``Experimental Procedures.'' At 10
nM [
H]Ketanserin, nonspecific binding
was 39% of total binding for the wild type (WT), 27% for
Asp
Asn, and 59% for the double mutant. Inset, representation of data according to the method of
Scatchard (35). Duplicate points from one experiment are shown. These
results were replicated in one additional experiment. B,
competition by 5-HT and mianserin for
[
H]Ketanserin binding to the wild-type and mutant
5-HT
receptors. Incubations and analyses were carried out
as described under ``Experimental Procedures.'' Opensymbols are competition with mianserin, and closedsymbols are competition with 5-HT.
, wild-type
receptor;
, Asp
Asn receptor;
,
double mutant receptor. The concentrations of
[
H]Ketanserin for 5-HT and mianserin competition
were 0.9 and 1.1 nM, respectively. Nonspecific binding was 15%
of total binding for the wild type, 8% for Asp
Asn, and 27% for the double mutant. Duplicate points from
one experiment are shown. These results were replicated in one
additional experiment.
The affinities of 5-HT for the wild-type and mutant
receptors and of 2,5-dimethoxy-4-iodoamphetamine (DOI) LSD, mianserin,
haloperidol, and 5-methoxygramine for the wild-type, Asp
Asn, and double mutant receptors were determined in
competition binding experiments for sites labeled with
[
H]Ketanserin. Fig. 2B shows
representative competition curves, and all data are summarized in . The affinity of 5-HT for four of the mutant receptors
varied by <2-fold. For the Asp
Ala
receptor mutant, the affinity of 5-HT was
5 times lower than for
the wild-type receptor. The affinities of the other agonists and
antagonists for the receptor mutants studied were close to values
measured for the wild-type receptor.
Molecular Dynamics Simulations of the Asp
The structure of the
Asp Asn Mutant Receptor
Asn receptor mutant obtained from the
modeling and molecular dynamics equilibration procedures differs from
that of the wild-type 5-HT
receptor model reported
previously(21) . The major difference between the models of the
wild-type and mutant receptors is localized in TMH-5 and TMH-6. The
segments of helices 5 and 6 that connect to the third intracellular
loop were found in the previously reported simulations of
ligand-receptor complexes of the wild-type 5-HT
receptor
to be the sites most affected by the binding of
agonists(21, 22, 23) . In particular, the
average structure of the ligand-receptor complex in which 5-HT is
positioned in the binding site of the wild-type receptor showed a large
rearrangement of these segments of TMH-5 and TMH-6, pointing away from
each other in the helix bundle (Fig. 3A). It is
noteworthy, therefore, that the structure of the uncomplexed
Asp
Asn receptor model differs from that of
the wild-type uncomplexed receptor mainly in the same region. However,
the departure is in a direction opposite to that found in the
simulation of wild-type receptor activation by agonist (Fig. 3B). Binding 5-HT to the mutant receptor model
induces a small outward movement in that same region of the helix 5 and
6 segments, but this is insufficient to correct the paradoxical inward
displacement in the uncomplexed Asp
Asn
mutant relative to the wild type. Thus, the outward movement resulting
from 5-HT binding to the mutant does not have the structural
characteristics of the ``activated'' wild-type receptor
model. These results obtained from simulations of a receptor model are
remarkably consonant with the experimental finding that this mutant
does not mediate PI hydrolysis. No significant conformational
rearrangement in the intracellular portions of the other TMH domains
was observed upon binding of agonist to either receptor model. For
comparison, the response of the wild-type receptor model to the binding
of the antagonist 5-hydroxygramine is shown in Fig. 3C.
With antagonist in the binding pocket, no significant displacement is
observed in any of the domains of the 5-HT
receptor model
that were affected by agonist binding (21-23).
Figure 3:
Time-averaged structures from molecular
dynamics simulations of uncomplexed and ligand-bound 5-HT receptor models. The
-carbon trace of the helices is viewed
from the side parallel to the membrane with the outside of the cell at
the top of the figure. A, effects of complexing 5-HT with the
wild-type receptor (see Ref. 21 for details). Significant
conformational changes (see arrows) are observed only in the
segments of helices 5 and 6 toward the intracellular side. Black
lines, uncomplexed wild-type receptor; gray lines,
wild-type receptor complexed with 5-HT; curved arrows,
conformational change induced by 5-HT binding. B, comparison
of the effects of complexing 5-HT with the wild-type receptor and with
the Asp
Asn receptor mutant. Because there
was no significant change in the position of the other helices, only
TMH-5 and TMH-6 are shown. Note that the helices of the unbound mutant
receptor are positioned differently than those of the unbound wild-type
receptor and that the mutant receptor shows a smaller conformational
change upon 5-HT binding (compare black and grayarrows). Black lines and curved arrows,
wild-type receptor uncomplexed and complexed with 5-HT; gray lines and curved arrows, mutant receptor uncomplexed and
complexed with 5-HT. C, effects of complexing the antagonist
5-hydroxygramine with the wild-type receptor (gray lines) (see
Ref. 21 for details). No significant conformational changes are
observed relative to the unbound receptor (black
lines).
Asn in the 5-HT
receptor eliminates
coupling; this effect is reversed by introducing a second mutation of
Asn
Asp in TMH-7. This restoration of function, albeit to a
lower maximal level, by the second mutation cannot be due to any
beneficial effect of this mutation by itself because, in fact, the
TMH-7 mutation alone does not enhance function. Thus, the two loci
cannot be independent, and these results therefore support the
hypothesis that these residues are adjacent in space and have a shared
network of interactions(20) . The finding that the Ala mutations
at each of these loci decrease or eliminate receptor-mediated PI
hydrolysis implicates both side chains of the wild-type receptor in the
process of receptor activation.
Asn, mediates no detectable PI hydrolysis, whereas the receptor with
the lowest expression, Asn
Asp, has an E
value equivalent to that of the wild-type
receptor. Thus, the observed decrease in or absence of agonist-mediated
PI hydrolysis must represent an alteration in the capacity of each
mutant receptor studied to mediate coupling to signal transduction.
Asn mutation. As previously reported, simulation of agonist
binding to a computational model of the wild-type receptor resulted in
a conformational rearrangement affecting only the distal segments of
helices 5 and 6 that connect to the third intracellular
loop(21, 23) . Simulations of complexes of the wild-type
receptor interacting with ligands that are known to produce a range of
responses differing in their pharmacological potencies resulted in a
structural change in the same direction(22) . We find that in a
model in which mutation of Asp
Asn is
introduced, the same segments of helices 5 and 6 are distorted in a
direction opposite to that seen with activation of the wild-type
receptor. Simulation of the effect of agonist binding to this mutant
model produces only a small movement of these helices toward the
positioning achieved by the wild-type receptor-ligand complex (Fig. 3B). Thus, the molecular models of the wild-type
receptor and the Asp
Asn mutant receptor
manifest different conformational rearrangements of the helices
adjacent to a domain implicated in G-protein coupling(1) . These
findings parallel the results of mutagenesis, which show that this
mutation causes a loss of detectable coupling to PI turnover.
-adrenergic receptor,
for example, distinct coupling pathways are differentially affected.
Whereas an
-adrenergic receptor having a mutation of
Asp
Asn was found to be uncoupled from
potassium currents, coupling to calcium currents was
preserved(4) . Our modeling results suggest that the mutant
receptor undergoes an agonist-induced conformational change, albeit
different from that of the wild-type receptor, and may thus sustain
coupling to some G-proteins. Thus, the selective preservation of
coupling reported with mutation of this site in the
-adrenergic receptor may result from the induction of
a different conformational state that has an altered G-protein
selectivity.
in the
wild-type receptor may connect to Asp
. Thus,
substitution by Ala at either position eliminates the capacity of each
locus to form hydrogen bonds and thereby leads to a loss of the
interactions required for receptor activation. In the Asp
Asn mutant receptor, Asn
eliminates the
charge and one of the groups that can serve as a hydrogen-bond
acceptor; these properties are reinstated by the double mutant. The
Asn
Asp receptor is relatively good at
mediating PI hydrolysis, which can be attributed to protonation of one
of the Asp side chains in the vicinity of an already negatively charged
Asp
. This restores the hydrogen-bonding
relationship to that found in the wild-type receptor and thereby allows
the network of interactions involving these loci to persist in this
mutant.
Asn
receptor, it is significantly impaired in its capacity to mediate
signal transduction. The partial restoration of function observed with
the reciprocal mutation is important in implicating both loci in a
common hydrogen-bonding network mediating receptor activation. However,
the decreased efficacy of 5-HT at the double mutant receptor provides
additional insight into the functional structure of the receptor. This
decreased function in comparison to the wild-type receptor indicates
that the two loci are not functionally interchangeable. The functional
asymmetry of the two loci most likely reflects differences in the
interactions of the two side chains at these loci with other receptor
groups. One additional contact site of the Asp
side
chain suggested by three-dimensional models of GPCRs is the highly
conserved Asn in TMH-1, a hypothesis that is being tested
experimentally.
receptor
suggests that the structures of the two receptors and their mechanisms
of activation are likely to be similar. However, the precise mutations
that are functionally accommodated at each position differ in the two
receptors. For example, having an Asn in TMH-2 and TMH-7 of the GnRH
receptor is tolerated, whereas having an Asp in both positions leads to
a loss of high affinity binding and coupling. In the 5-HT
receptor, the opposite pattern is seen, with Asp in both
positions being well tolerated, whereas Asn in both positions causing
elimination of detectable coupling. The reason for the differences in
the residues preferred at each position across receptors must reside in
differences in the microenvironments of the two loci in different
receptors. With the structural basis for the adjacencies of these loci
in the receptor molecule clarified by the results of reciprocal
mutations and dynamic models, the specific side chains contributing to
the unique microenvironment of each locus in the two receptors can be
putatively identified. The hypotheses generated can be probed
experimentally to elucidate other contacts made by each locus in each
wild-type receptor.
Asn has previously
been studied in the rat 5-HT receptor, and a loss of allosteric
modulation by GTP was observed (10). In that study,
10-fold
decreases in the affinity of this mutant receptor for several agonists
and antagonists was observed, although no change was found in the
affinities for iodo-LSD or spiperone. In our present study, we find no
significant change in the affinity of any of the ligands studied for
this mutant. This discrepancy may reflect differences in the effect of
this mutation in the rat and human receptors or differences in the
radioligand binding assay utilized in the two studies.
receptor by mutagenesis and computational studies has
demonstrated a functional and spatial relationship of the conserved
TMH-2 Asp and TMH-7 Asn found in most GPCRs. The results for the
functional double revertant mutant implicate both residues in a common
hydrogen-bonding network that is involved in the conformational changes
produced in the receptor as a consequence of agonist binding. This
approach provides insight into the arrangement of the transmembrane
helix domains of GPCRs, and computational simulations with the
three-dimensional molecular model suggest conformational changes that
occur during receptor activation. The causes for differences in the
effects of single substitutions at the two positions in the 5-HT
receptor and in the GnRH receptor should be addressable in a
similar manner through combined experimental explorations and
computational modeling.
Table: Analysis of saturation binding and PI hydrolysis
data for 5-HT receptor constructs
values) were obtained from
saturation binding with [
H]Ketanserin. EC
values were obtained from concentration-response curves for 5-HT.
The E
values are expressed as a percent of the
maximal stimulation above basal levels obtained with activation of the
wild-type receptor, which, in these experiments, was 4.2 ±
0.7-fold above basal levels. Values represent means ± S.E. of
two experiments for the binding data and three experiments for the PI
assays.
Table: Affinity for
[H]Ketanserin-labeled sites in
5-HT
receptor constructs
values were derived from two
competition experiments. A representative experiment for two of the
competing ligands is shown in Fig. 2B. Competition with
unlabeled 5-HT was also performed for the Asp
Ala, Asn
Asp, and
Asn
Ala receptor mutants, and K
values of 1638 ± 553, 249
± 28, and 549 ± 15, respectively, were obtained.
receptor cDNA. We thank Dr. Saul
Maayani for many helpful suggestions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.