From the § Department of Molecular Pharmacology and
Biochemistry, Institute for Neuroscience, Northwestern University,
Chicago, Illinois 60611, the The carboxyl terminus of heterotrimeric G protein
Hormones and neurotransmitters that bind to G protein-coupled
receptors control a myriad of physiological functions. Transduction of
these extracellular signals involves receptor-mediated activation of
specific G proteins by catalysis of GDP/GTP exchange. These receptors
are the target for many pharmaceutical products and are the focus of
intense drug discovery efforts. Traditionally, the agonist binding site
is the point of intervention, but in some cases receptor
subtype-selective drugs have been difficult to achieve. Another
possible target for inhibition is the receptor-G protein interface,
which has been defined in some detail and involves the intracellular
loops of the seven-transmembrane helix receptors with several regions
on heterotrimeric G proteins (1-3). It is important to assess whether
inhibitors of this interface can be found or designed and whether
specific inhibition can be achieved.
The carboxyl-terminal region of the G The similarity between the carboxyl terminus of G The extracellular nucleoside adenosine regulates a variety of metabolic
functions through the activation of specific cell membrane receptors.
Adenosine receptors, which exhibit the presumed seven
transmembrane-spanning topography typical of almost all G
protein-coupled receptors, are currently classified into four subtypes,
A1, A2a, A2b and A3,
based on the pharmacological profile for agonist and antagonist ligands
and their effects on intracellular cAMP accumulation (reviewed in Refs.
20-23). The A1 adenosine receptor is widely distributed in
several tissues such as heart, kidney, epididymal fat, and testis, and
it is especially prominent in the central nervous system (24-27). In
the brain, the highest expression is observed in cortex, cerebellum,
hippocampus, and thalamus (25, 26). In the cortex it represents a
primary signaling target for adenosine and is thought to tonically
inhibit neuronal activity. In the hippocampus the highest density is in
the dendritic region of the CA1 area (28-30) where A1
adenosine receptors are located at synaptic and extrasynaptic sites
(31, 32).
Originally, signaling through the A1 adenosine receptor
subtype was linked to inhibition of adenylyl cyclase activity in a pertussis-sensitive manner (33, 34). Since then, A1
adenosine receptors have been shown to modulate phospholipase C
activity in some systems (35, 36), as well as activate K+
currents and inhibit voltage-gated Ca2+ channels (37-40)
through the mediation of pertussis-sensitive G proteins (Gi
family) (41, 42). In reconstituted systems, human and bovine
A1 adenosine receptors appear to interact preferentially with recombinant G Here, we study the effect of synthetic peptides corresponding to the
last 11 residues of G Materials--
[3H]N6-(cyclohexyl)adenosine
(CHA) (27.7 Ci/mmol) and
[3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (80-120
Ci/mmol) were obtained from NEN Life Science Products, and
[125I]N6-(3-iodo,4-amino)benzyladenosine
(ABA) (2000 Ci/mmol) was synthesized by Dr. J. Linden (Department of
Internal Medicine, Cardiovascular Division, University of Virginia,
Charlottesville, VA) (48). Adenosine deaminase (ADA) and
(R)-N6-(phenylisopropyl)adenosine
(R-PIA) were purchased from Sigma, and GTP Synthetic Peptides and MBP Fusion Proteins--
Peptides were
purchased from Peptidogenics (Livermore, CA). The native G Department of
Pharmacology,
Department of
Physiology and Biophysics,
Department of Pharmacology, University of
Colorado, Denver, Colorado 80262
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunits plays an important role in receptor interaction. We
demonstrate that peptides corresponding to the last 11 residues of
G
i1/2 or G
o1 impair agonist binding
to A1 adenosine receptors, whereas G
s or
G
t peptides have no effect. Previously, by using a
combinatorial library we identified a series of G
t
peptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996)
J. Biol. Chem. 271, 361-366). Native
G
i1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine
receptors. Three peptide analogs decreased the Ki,
suggesting that they disrupt the high affinity receptor-G protein
interaction and stabilize an intermediate affinity state. To study the
ability of the peptides to compete with endogenous G
i
proteins and block signal transduction in a native setting, we measured
activation of G protein-coupled K+ channels through
A1 adenosine or
-aminobutyric acid, type B, receptors in
hippocampal CA1 pyramidal neurons. Native G
i1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was
activated. This differential perturbation of receptor-G protein
interaction suggests that receptors that act on the same G protein can
be selectively disrupted.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunits represents an
important site of interaction between heterotrimeric G proteins and
their cognate receptors. Within this region mutations (4-6), covalent
modification by pertussis toxin-catalyzed ADP-ribosylation (7), or
binding of specific antibodies (8) all uncouple G proteins from their
receptors. In particular, the last 4 residues of the G
carboxyl
terminus play an important role in determining the fidelity of receptor
activation (9, 10). Moreover, synthetic peptides from various portions
of the G
s carboxyl terminus inhibit
-adrenergic
receptor-Gs coupling (11, 12). Two of these peptides, G
s-(384-394) and G
s-(354-372), also
stabilize the high affinity state of the receptor (12). A synthetic
peptide corresponding to the last 11 residues of G
t,
G
t-(340-350), both inhibits rhodopsin Gt
coupling and mimics Gt by stabilizing the active
metarhodopsin II conformation (13). By screening a combinatorial
peptide-on-plasmid library based on the carboxyl terminus of
G
t, we previously identified numerous peptides that can
also mimic the effects of heterotrimeric G protein with a much greater
affinity than the native sequence by both binding to rhodopsin and
stabilizing it in its active conformation, metarhodopsin II (14).
t and
G
i led us to test the G
t peptide analogs,
which bound to rhodopsin, for their ability to bind other
Gi-coupled receptors. In this study we have investigated
whether these peptides can 1) bind to Gi-coupled
A1 adenosine receptors and induce the high affinity binding
of the receptor; 2) block the ability of Gi proteins to stabilize the high affinity state of agonists; or 3) inhibit signal transduction through Gi by two different
Gi-coupled receptors, A1 adenosine and
GABAB1 receptors.
Whereas some receptors activate multiple G proteins (reviewed in Ref.
15), the A1 adenosine receptor (16, 17) and
GABAB receptor (18, 19) are preferentially coupled to Gi/Go proteins in many cellular systems.
i rather than G
o (43,
44). The bovine A1 adenosine receptor couples selectively
to the G
i3 subunit, whereas the human receptor is able
to activate each G
i subtype with similar potency (45).
Other researchers (46), using purified bovine brain G proteins, have
shown that Gi2 is more potent than Gi1 or
Go at restoring high affinity agonist binding to bovine brain A1 adenosine receptors. However, the ability of
A1 adenosine receptors to preferentially interact with a
specific G
subunit does not preclude their ability to interact with
other G protein subunits in an intact system.
i1/2, G
o,
G
t, and G
s or the G
t
peptide analogs on agonist binding to the A1 adenosine
receptor in rat cortical membranes or CHO-K1 cell membranes
overexpressing the human receptor. Our findings indicate that in
contrast to other receptors (12, 13, 47), the carboxyl-terminal region of the G
i1/2 subunit was not capable of stabilizing the
high affinity state of A1 adenosine receptors either in rat
cortex membranes or CHO-K1 cell membranes overexpressing
the human receptor. However, the native peptide
G
i1/2-(344-354) as well as some G
t peptide analogs can negatively modulate agonist binding and compete with heterotrimeric G protein for binding to the A1
adenosine receptor. Moreover, G
i carboxyl-terminal
peptides blocked signal transduction to activation of K+
channels. Depending on whether the activation was through the A1 adenosine or GABAB receptor, different
G
t peptide analogs were most effective. Thus, it appears
that certain G
t peptide analogs can selectively disturb
the molecular interface that occurs between Gi proteins and
Gi protein-coupled receptors.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
S was obtained from
Boehringer Mannheim. Other reagents were from standard commercial
sources.
peptides
tested for their effect on agonist binding to rat cortical membranes
had a free amino terminus, whereas all other peptides were synthesized
with an acetyl group at the amino terminus. All peptides were purified
by reversed-phase high performance liquid chromatography (HPLC), and
their purity was checked by fast atom bombardment-mass spectrometry,
analytical HPLC, and amino acid analysis.
t-(340-350K341R) with the TGGG linker
fused to MBP. All frozen cell stocks were kept in 25% glycerol at
80 °C.
Preparation of Rat Cortical Membranes--
The brain cortices
from young male Sprague-Dawley rats (150-200 g) that had been
subjected to cervical dislocation were rapidly removed and homogenized
in 10 volumes of 0.25 M sucrose prepared in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, containing
protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride,
1 mM benzamidine, and 100 µg/ml bacitracin). The membrane
homogenate was centrifuged at 1,000 × g for 10 min at
4 °C. The supernatant was centrifuged at 46,000 × g
for 20 min at 4 °C. The resulting pellet was resuspended in 10 volumes of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2 (TEM1 buffer) containing protease
inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 100 µg/ml bacitracin), and the homogenate was centrifuged again. The pellet was resuspended in 5 volumes of TEM1 buffer containing protease inhibitors and 2 units/ml
ADA and incubated at 37 °C for 30 min to remove endogenous adenosine. The membrane homogenate was centrifuged, and the final pellet was stored as aliquots at 80 °C until needed. Protein concentration was determined by the method of Lowry et al.
(49) using bovine serum albumin as the standard.
Preparation of Hippocampal Slices-- Young male Sprague-Dawley rats (200-220 g) were decapitated and the brains rapidly removed. The hippocampi were cut into 400-µm thick transverse slices on a Sorvall tissue chopper. Slices were submerged in a recording chamber and continuously perfused with artificial cerebral spinal fluid containing 1.2 mM NaH2PO4, 25.9 mM NaHCO3, 126 mM NaCl, 3 mM KCl, 1.5 mM MgCl2, 2.4 CaCl2, 11 mM glucose, oxygenated with 95% O2, 5% CO2.
Cell Culture-- Chinese hamster ovary (CHO-K1) cells stably overexpressing the human A1 adenosine receptor were grown to confluence in Ham's F12 media (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin (Life Technologies, Inc.), 0.1 mg/ml streptomycin (Life Technologies, Inc.), and 0.5 mg/ml G418 (Sigma) in an atmosphere of 95% air, 5% CO2 at 37 °C. Cells were seeded at 1 × 105 cells/ml and subcultured after detachment with trypsin/EDTA (0.05%, 0.5 mM).
Preparation of CHO-K1 Cell Membranes--
Confluent
CHO-K1 cells were lysed with hypotonic buffer (10 mM HEPES, pH 7.4, 10 mM EDTA) and scraped from
the plate. The lysates were homogenized, centrifuged, and washed twice
in washing buffer (10 mM HEPES, pH 7.4, 5 mM
EDTA). The homogenized lysates were then stored at 80 °C in
storage buffer (10 mM HEPES, pH 7.4, 1 mM EDTA,
10% (w/v) sucrose) until needed. Protein concentration was measured
using the Coomassie Blue binding method (50) (Bio-Rad) with bovine
serum albumin as the standard.
Rat Cortex Binding Assays--
Rat cortical membranes (100-150
µg of proteins) and [3H]CHA (1.3 nM) were
incubated in 0.5 ml of TEM1 buffer containing 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2.7 µg/ml leupeptin, and 1 unit/ml ADA for 60 min at 25 °C. Binding
reactions were terminated by filtration through Whatman GF/C filters
(Hillsboro, OR) under reduced pressure using a Millipore apparatus
(Bedford, MA). Nonspecific binding was defined in the presence of 15 µM R-PIA. Specific binding was 85-90% of total binding
for all experiments. For saturation studies, membranes were incubated
in TEM1 buffer containing protease inhibitors and ADA with eight
different concentrations of [3H]CHA ranging from 0.1 to
46 nM. For studying the effect of GTPS, rat cortical
membranes and [3H]CHA (1.2 nM) were incubated
in the absence and presence of varying concentrations of the guanine
nucleotide ranging from 1 nM to 100 µM.
CHO-K1 Binding Assays--
Membranes (10 µg/ml
proteins) from CHO-K1 cells overexpressing the human
A1 adenosine receptor were added to tubes containing HEM
buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 5 mM MgCl2), 2.5 units/ml ADA,
[125I]ABA (0.5 nM) and either the MBP fusion
proteins (50 µM) or synthetic peptide (100 µM). The Gt peptide analogs were dissolved
in HEM buffer, and their solubility was checked as described by Rarick et al. (51). The reaction was allowed to proceed at 30 °C
for 2 h. The bound and free radioligands were separated by
filtration through Whatman GF/C filter paper (soaked in 0.3%
polyethyleneimine) using a Brandel tissue harvester. Filters were
washed twice with ice-cold TEM2 buffer (20 mM Tris-HCl, pH
7.4, 5 mM MgCl2, 0.5 mM EDTA).
Binding assays were performed in duplicate, and nonspecific binding was
determined by adding 15 µM R-PIA at the same time as the
radioligand to some samples. Specific binding was 85-90% of total
binding for all experiments.
Electrophysiological Recording--
Recording electrodes for
whole cell recording were pulled from borosilicate glass (outer
diameter 1.5 mm and inner diameter 0.86 mm, with filament; Sutter
Instrument Co., Novato, CA) on a Flaming/Brown Micropipette puller
(Sutter) and had tip resistances of 5-10 M when filled with a
solution containing 125 mM potassium gluconate, 11 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 1 mM K-EGTA, 2 mM Mg-ATP, 0.3 mM Tris-GTP, 10 mM
HEPES, pH adjusted to 7.2-7.3 with KOH, osmolarity adjusted to
280-290 mOsm. Peptides were dissolved directly into the electrode
filling solution to obtain a final concentration of 1 mM.
Data Analysis--
A nonlinear multipurpose curve-fitting
computer program (GraphPad Prism, version 2.0, GraphPad Software, San
Diego, CA) was used for analysis of saturation and competition binding
data. A partial F test evaluated whether the data were best fit by a one- or two-site model. The IC50 values calculated from the
competition curves were converted to Ki values by
the Cheng and Prusoff equation (53). The dose-response curves for the
G peptides were fit using nonlinear regression analysis and
IC50 values were derived (GraphPad Prism, version 2.0).
Data are presented as mean ± S.E. of at least three experiments,
unless otherwise noted. The statistical differences were determined
using the unpaired t test (GraphPad Prism, version 2.0).
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RESULTS |
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Table I shows the amino acid sequences of all peptides used for the experiments and indicates the EC50 values of their ability to stabilize rhodopsin in its active conformation, metarhodopsin II. Synthetic or MBP-fused peptides were tested for their ability to modulate agonist binding to A1 adenosine receptors from two different species, rat and human. The human receptor was expressed in stably transfected cells, whereas the rat receptor was in its native cellular background.
|
Effects of Native G Carboxyl-terminal Peptides on Agonist
Binding to Rat A1 Adenosine Receptors--
The
A1 adenosine receptor agonist, [3H]CHA, binds
specifically to a single class of binding sites in rat cortical
membranes with KD and Bmax
values of 1.1 nM and 418 fmol/mg protein, respectively
(data not shown). Specific binding was decreased by the
non-hydrolyzable GTP analog, GTP
S, in a dose-dependent fashion with an approximate IC50 value of 200 nM. At 100 µM GTP
S-specific [3H]CHA binding was inhibited by 95%, indicating that
the majority of A1 adenosine receptors are coupled to G
proteins. The effect of synthetic peptides corresponding to the
carboxyl-terminal sequence of several G
subunits (Table I) on
[3H]CHA binding was evaluated. Peptides
G
i1/2-(344-354) and G
o1-(344-354) inhibited [3H]CHA-specific binding in a
dose-dependent fashion with IC50 values of
31.29 and 30.02 µM, respectively (Fig.
1). In contrast, neither G
s-(384-394) nor G
t-(340-350) inhibited
agonist binding to A1 adenosine receptors at concentrations
of 200 µM (Fig. 1). Therefore, under these conditions,
both G
i1/2 and G
o1 peptides appear to compete with heterotrimeric G proteins for interaction with the A1 adenosine receptors in rat cortical membranes, but they
are unable to stabilize high affinity agonist binding.
|
Effects of Gt Peptide Analogs from the Combinatorial
Library--
By using a combinatorial approach, we previously
identified a series of G
t peptide analogs which can both
bind to rhodopsin and stabilize the receptor in its active
conformation, metarhodopsin II, with higher affinity than the native
G
t carboxyl-terminal peptide (14). There is a high
degree of homology between G
t and G
i
carboxyl-terminal regions, with only one amino acid difference between
G
t and G
i1/2 (Table I) and two amino acid
differences between G
t and G
i3. To
determine whether these peptides could stabilize the high affinity
agonist binding state of Gi-coupled receptors, we tested
MBP-fused peptides or synthetic peptide analogs in agonist binding
assays of human A1 adenosine receptors overexpressed in
CHO-K1 cells. As reported under "Experimental
Procedures," approximately 50% of A1 adenosine receptors
in CHO-K1 cell membranes are coupled to G proteins.
Therefore, if any of the MBP fusion proteins or peptide analogs were
able to mimic the heterotrimeric G protein, we should detect an
increase of agonist binding. None of the MBP fusion proteins or peptide
analogs tested resulted in a significant increase of specific
[125I]ABA binding to CHO-K1 cell membranes
expressing human A1 adenosine receptors (data not shown).
However, a few of the analogs either as MBP fusion proteins or
synthetic peptides inhibited agonist binding to CHO-K1 cell
membranes. MBP fusion proteins 19 and 24 (50 µM)
inhibited [125I]ABA-specific binding by 24 and 23%,
respectively. The corresponding synthetic peptides (100 µM) resulted in 21 and 33% decrease of specific binding,
respectively.
|
|
Functional Effects of Gi- and
G
t-related Synthetic Peptides on Inhibition of Signal
Transduction--
To study the ability of these peptides to compete
with endogenous G proteins in the native setting and block signaling
through Gi, we introduced the peptides into hippocampal
neurons through a patch pipette and subsequently determined the extent
to which G protein-coupled inwardly rectifying K+ channels
(GIRKs) could be activated by either A1 or
GABAB receptors. Superfusion of hippocampal brain slices
with 100 µM adenosine or 50 µM baclofen
elicit outward currents in CA1 pyramidal neurons, a reflection of the
activation of GIRK by A1 adenosine or GABAB receptors, respectively. This effect is mediated via a pertussis toxin-sensitive G protein (54). An example of recording under standard
conditions is shown in Fig.
3A. The maximal outward
current induced by adenosine was 50 ± 5.5 pA (n = 38 cells). Internal dialysis of these neurons with the
carboxyl-terminal G
1/2 peptide completely eliminated the
adenosine response (Fig. 4;
p < 0.0001 versus control). Synthetic
peptides 19 and 24 (Figs. 3C and 4) were also able to
completely block the response to adenosine (p < 0.0001 and p < 0.002 versus control,
respectively), whereas other peptides (8, 9, and 15) appeared to
partially block the adenosine response, although these effects were not
statistically significant. Peptide 23 had no effect on the response to
adenosine (Fig. 3B). Thus, the native G
i1/2
peptide as well as peptides 19 and 24 are effective inhibitors of G
protein-coupled signal transduction through A1 adenosine
receptors.
|
|
Specificity of Functional Blockade of Gi-coupled
Receptors by G Peptides and Analogs--
One would expect that a
peptide corresponding to the carboxyl terminus of G
i
would block signaling through all Gi-coupled receptors. It
is of great interest to determine whether there can be any selectivity
at the receptor-G protein interface. To evaluate whether the
G
t peptide analogs show a pattern of specificity for
different Gi-coupled receptors, we measured the effect of these peptides on GABAB receptor mediated activation of
GIRK in CA1 pyramidal neurons, an effect which is also mediated via a pertussis toxin-sensitive G protein (55). Superfusion of hippocampal brain slices with 50 µM baclofen elicited large outward
currents (Fig. 3A) with an average maximal response of
83 ± 8.8 pA (n = 29). Internal dialysis of CA1
pyramidal neurons with carboxyl-terminal G
peptides either had no
effect upon current response to baclofen (peptide 23; Fig.
3B) or reduced the baclofen response (the native G
i1/2, peptides 8, 9, 15, 19, and 24 (Fig. 4)). No
synthetic peptide completely blocked baclofen-induced activation of
GIRK. However, peptides 8, 15, and 24 produced significant reduction of
the current response compared with control (p < 0.005, p < 0.007, and p < 0.05).
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DISCUSSION |
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Recently, a variety of studies have focused on finding new agents
that selectively uncouple receptors from G proteins (14, 56, 57) and
thus disrupt cellular responses. The carboxyl-terminal region of G
subunits provides the molecular basis for receptor-mediated activation
of G proteins and plays a crucial role in determining the fidelity of
this activation (13, 47). Synthetic peptides corresponding to the last
11 residues of the G
t and G
s subunit are
able to mimic the conformational effects of heterotrimeric G proteins
on their cognate receptors, rhodopsin and
-adrenergic receptors, by
stabilizing their active conformation, although with low potency (12,
13, 47). By using a random "peptides-on-plasmids" library approach
(14), we identified several analogs of the native peptide
G
t-(340-350) that bind with high affinity to rhodopsin and stabilize its active form, metarhodopsin II. As the
carboxyl-terminal sequences of G
i1/2 and
G
i3 diverge just by one and two amino acids from
G
t carboxyl-terminal sequence, respectively, we
evaluated the effects of native G
and analog peptides on receptor
coupling to Gi proteins.
No G Carboxyl-terminal Peptides Stabilize the High Affinity
State of Rat A1 Adenosine Receptors--
The effects of
synthetic peptides G
i1/2-(344-354),
G
o1-(344-354), G
t-(340-350), and
G
s-(384-394) on agonist binding to A1
adenosine receptors were studied using rat cortical membranes. None of
the native peptide sequences increased agonist binding, implying that
they are unable to stabilize the high affinity state of the receptor.
On the contrary, peptide G
i1/2-(344-354) and G
o1-(344-354) inhibited specific binding in a
dose-dependent fashion. The result suggests that synthetic
peptides corresponding to G
i1/2 and G
o1
carboxyl-terminal sequence disrupt the interaction between
A1 adenosine receptors and Gi proteins. Since
in our assay conditions, agonist binding was also sensitive to
inhibition by GTP
S, indicating that most receptors were effectively
interacting with G proteins, probably these peptides compete with
Gi/Go for binding to the receptor. Such an
outcome implies that peptide G
i1/2-(344-354) and
G
o1-(344-354) are not able to mimic heterotrimeric G
protein in stabilizing the high affinity state of the rat
A1 adenosine receptor. The inability of peptide
G
t-(340-350) to inhibit agonist binding was also quite
surprising since there is only one amino acid difference between it and
peptide G
i1/2-(344-354). This difference may be due to
G
t peptide having decreased affinity for the rat
A1 adenosine receptor, which would be critical for its
competition with Gi/Go proteins.
No Gt Peptide Analogs Stabilize the High Affinity
State of Human A1 Adenosine Receptors--
These peptide
analogs were selected for their ability to bind with high affinity to
metarhodopsin II. All analogs stabilized metarhodopsin II with higher
affinity than the native G
t peptide (14). It is possible
that if another receptor is used to screen the combinatorial library,
high affinity peptides that selectively bind and stabilize this
receptor in its active state might be found. The structural basis of
this idea is that receptors have different amino acid sequences and
thus perhaps some differences in structure in their G protein binding
region(s). The combinatorial approach should be able to find such
differences. These considerations motivated us to examine the effects
of G
t-(340-350) analogs on agonist binding to human
A1 adenosine receptors overexpressed in CHO-K1
cell membranes. Under our cell culture and binding assay conditions,
approximately 50% of total receptors appeared to be in the high
affinity state and thus effectively coupled to G proteins. Therefore,
peptides did not need to compete with the heterotrimeric G proteins for
binding to the receptor and modulating agonist affinity. However, none
of the G
t-(340-350) peptide analogs either as MBP
fusion proteins or synthetic peptides were able to increase agonist
binding to human A1 adenosine receptors, suggesting they were unable to mimic the effects of heterotrimeric
Gi/Go proteins.
Modulation of Agonist Affinity by Gt Peptide
Analogs--
Although G
peptides could not mimic heterotrimeric G
proteins and stabilize the high affinity state of the A1
adenosine receptor, we found evidence that the peptides bind to the
receptors and compete with heterotrimeric G proteins, as indicated by a
decrease of agonist high affinity binding. Interestingly, three
G
t-(340-350) analogs, peptides 8, 15 and 19, increased
the Ki value of the high affinity state of the
receptor, indicating that these peptides not only disrupt the high
affinity interaction between agonist-activated receptors and
Gi proteins, but they also stabilize the receptor at an
intermediate affinity state. Thus, these peptides appear to be
affecting the receptor conformation in a subtle way. One might
speculate that there is a continuum of conformations between R and R*,
and these peptides may stabilize an intermediate conformation. The
existence of multiple distinct active receptor states differing in
their G protein-coupling abilities has been suggested for both
rhodopsin-(58-61) and the human thyroid-stimulating hormone receptor
(15).
Functional Effects of the Native Gi
Carboxyl-terminal Peptide and G
t Analogs in Intact
Cells--
To test the hypothesis that some peptides that disrupt the
interaction between the A1 adenosine receptor and the
carboxyl-terminal region of Gi proteins are also able to
impair signal transduction, we measured the effects of synthetic
peptides on the activation of GIRKs by A1 adenosine
receptors in rat hippocampal CA1 pyramidal neurons. Except for peptide
23 which did not show any significant effect on receptor-mediated
opening of K+ channels, all other peptides reduced the
K+ current with different activity. Thus, peptides that
modulated the high affinity agonist binding to the receptor also
disrupted signal transduction between A1 adenosine
receptors and Gi proteins. The native peptide
G
i1/2-(344-354) as well as peptide analogs 19 and 24 completely blocked the adenosine-activated response. There did not
appear to be a strict correspondence between the ability of the
peptides to modulate agonist binding or antagonist displacement by an
agonist and their potencies as inhibitors of A1 adenosine
receptor-mediated activation of K+ current. However, the
efficacy of the native peptide G
i1/2-(344-354) was
consistent with its activity as inhibitor of agonist binding to
A1 adenosine receptors in rat cortical membranes.
Specificity of the Functional Disruption of Receptor Gi
Protein Coupling by G Peptides--
One would expect that a peptide
corresponding to the carboxyl terminus of G
i would block
signaling through all Gi-coupled receptors. Although most
receptors show specificity for a particular class of G proteins, it is
much less clear whether drugs that target the receptor-G protein
interface could be highly specific for a particular receptor. Thus, it
was important to evaluate the efficacy of G
peptides on activation
of GIRKs by a different Gi-coupled receptor.
GABAB receptors modulate the activity of several downstream
effectors (64) mainly through the activation of pertussis
toxin-sensitive G proteins (17, 55). However, this receptor differs
structurally from other G protein-coupled neurotransmitter receptors
and forms a separate gene family together with the metabotropic
receptor for L-glutamate (64). In this context, it was of
interest that three of the peptides (the native peptide
G
i1/2-(344-354) and peptide analogs 19 and 24) were
more effective blockers of A1 than GABAB
receptor responses, whereas peptide 15 was more effective in blocking
GABAB than A1 receptor responses. Although both
A1 and GABAB receptors in rat hippocampal CA1
pyramidal neurons activate what appears to be a common population of
GIRKs (65, 66), such that outward current responses to baclofen occlude
responses to adenosine, there is no definitive evidence that the same G
proteins mediate these actions. Thus, the differential effects of these
peptides could reflect either the specificity of the interaction of the
peptides with homologous but not identical regions of the
A1 and GABAB receptors or could reflect
mediation of these responses by different G proteins. In this context,
it should also be noted that these peptides are all analogs of the
carboxyl terminus of G
subunits, whereas it is the
dimer that
is thought to activate GIRKs (67, 68). Thus, although it is possible
that these peptides interact directly with GIRKs to inhibit their
function, as has been shown for G
ai1 (69), this could
not explain their selectivity in blocking actions mediated via
A1 receptors versus GABAB receptors.
The most probable explanation for these results is that the peptides
interact instead with specific elements of the A1 and
GABAB receptors in slightly different ways to disrupt their
interaction with the corresponding G proteins. This is also suggested
by comparing the potency of the peptides for rhodopsin and
A1 adenosine receptors. Peptide 15, which was the most
potent at decreasing A1 adenosine receptor agonist affinity
(Table II), was the least potent of the analogs at stabilizing
metarhodopsin II (Table I).
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FOOTNOTES |
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* This work was supported by Grants HL 37942 (to J. L.), EY 06062 (to H. E. H.), and EY10291 (to H. E. H.) from the National Institutes of Health, a NARSAD Distinguished Investigator award (to H. E. H.), and a Human Capital and Mobility Grant CHRX-CT94-0689 from the European Community (to M. R. M.).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.
§§ To whom correspondence should be addressed: Northwestern University, Institute for Neuroscience, 320 E. Superior 5-555 Searle, Chicago, IL 60611.
1
The abbreviations used are: GABAB,
-aminobutyric acid, type B; ABA,
N6-(3-iodo,4-amino)benzyladenosine; ADA,
adenosine deaminase; CHA, (cyclohexyl)adenosine; CHO, Chinese hamster
ovary; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; GIRK, G
protein-coupled inwardly rectifying K+ channel; GTP
S,
guanosine 5'-O-(3-thio)triphosphate; HPLC, high performance
liquid chromatography; MBP, maltose-binding protein; R-PIA,
(R)-N6-(phenylisopropyl)adenosine.
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