J Biol Chem, Vol. 274, Issue 50, 35305-35308, December 10, 1999

COMMUNICATION
A G Protein  Subunit-specific Peptide Inhibits Muscarinic Receptor Signaling^*

Inaki Azpiazu^§, Humberto Cruzblanca^§¶, Ping Li, Maurine Linder^**, Min Zhuo, and N. Gautam

From the  Department of Anesthesiology and the ^** Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^¶ Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein 5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the 5 peptide inhibits modulation of Ca²⁺ current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein  subunit family are significantly less effective. The 5 peptide has no effect on Ca²⁺ current modulation by the 2-adrenergic and somatostatin receptors. In addition, the 5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein  subunit types in signal transduction, most likely at the receptor-G protein interface.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The G protein  subunits are a family of 11 proteins with varying levels of homology to each other and different patterns of expression in mammalian tissues (1). Although the G protein complex has been shown to directly modulate effector function and is required for receptor interaction of the G protein, the individual functions of these  subunits are still unclear. Reconstitution assays with rhodopsin and Gt indicated that G protein coupling with a receptor involves specific contact of the 1 subunit COOH terminal with the receptor (2, 3). To test whether the COOH-terminal domains of other  subunits are involved in receptor interaction we have examined the effect of a peptide from the 5 subunit type on muscarinic receptor signaling. 5 is expressed abundantly in the heart similar to the muscarinic receptor, M2 (4, 5). We examined the effect of the 5 COOH-terminal peptide on the activation of G_i2 reconstituted with the M2 receptor. To examine the effect of the peptide in cells, we injected a peptide specific to the 5 COOH terminus into superior cervical ganglion (SCG)¹ neurons and measured receptor modulation of N-type Ca²⁺ current (I_ca). SCG neurons contain the M1 and M4 muscarinic receptors which inhibit N-type Ca²⁺ channels through G_q and G_o, respectively (6, 7). SCG neurons also contain 2-adrenergic and somatostatin receptors that inhibit I_ca through G_o (6). This variety of receptors modulating the activity of a common effector allowed us to assess the specificity of the 5 peptide action. The effect of 5 peptides as well as peptides from 7 and 12, on these pathways was examined. Finally, to test the effect of the 5 peptide on the central nervous system, we introduced the 5 peptide into postsynaptic neurons in a spinal cord slice and measured the modulation of glutamate receptor mediated synaptic current by muscarinic and 2-adrenergic receptors (8).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cells and Reagents-- [³H]N-methylscopolamine and [³⁵S]GTPS were from NEN Life Science Products. Somatostatin was from Peninsula. Geranylgeranyl bromide was from American Radiolabeled Chemicals. Oxotremorine methiodide (oxo-M), carbachol, and clonidine were from Research Biochemicals. BAPTA and dextran-fluorescein were from Molecular Probes. Leupeptin, ATP, and GTP were from Roche Molecular Biochemicals. Dulbecco's modified Eagle's medium and heat-inactivated horse serum were from Life Technologies, Inc. All other chemicals were from Sigma. Purification of recombinant _i2, 1His-2, and complex from bovine brain were as described before (9, 10). A CHO cell line stably transfected with a vector expressing the M2 receptor has also been described before (11) and was provided by the late Dr. E. G. Peralta (Harvard University). Solid peptide synthesis, mass spectrometry, and amino acid analysis were performed at the Protein and Nucleic Acid Chemistry Laboratory, Washington University School of Medicine. Geranylgeranylation was performed and checked as described (12). Peptide sequences were as follows: 5 peptide, VSSSTNPFRPQKVC or a shorter version, STNPFRPQKVC; 5 scrambled peptide, PSRTPVNFSQVSKC; 7, SENPFKDKKPC; and 12, SENPFKDKKTC. The shorter 5 wild type peptide was used in all electrophysiological assays.

Patch clamp experiments were done on 1-day-cultured SCG neurons from 2- to 4-week-old male Harlan Sprague-Dawley rats. Neurons were dissociated, and plated as described (13).

Preparation of M2 Receptor-containing Membranes-- CHO cell membranes containing M2 were obtained as described (14). To deplete endogenous G protein subunits membranes were washed with 20 mM sodium phosphate buffer, pH 7.4, containing 5 mM MgCl₂, 5 M urea, 100 µM GTPS. Immunoblot analysis with antibodies specific to 1 showed a significant decrease in that subunit after this treatment.

Ca²⁺ Current Recording-- Whole-cell recording of I_Ca used 50-60% compensation of series resistance. I_Ca current records were sampled (25 kHz). Voltage-dependent inhibition of I_Ca was studied with two 10-ms test pulses to +10 mV, from a holding voltage at 80 mV, one before (P1) and other (P2) after a 25-ms prepulse to +125 mV. Facilitation ratio and amplitude of I_Ca were measured as described (19). Agonist-mediated inhibition of I_Ca was quantified only for the P1 test pulse. To avoid one source of systematic bias, control and experimental measurements were alternated in each set of experiments.

Cytoplasmic Injection-- SCG neurons were pressure-injected with G peptides by using an Eppendorf transjector system. Injection pipettes were filled with a solution containing 50 µM geranylgeranylated  subunit-peptides and 0.05% dextran-fluorescein (M_r = 10,000) as injection marker. After injection, cells were returned to the incubator and 1-2 h later were transferred to a 50-µl chamber for I_Ca recording. Experiments were done at 25 °C. To block Na⁺ and L-type Ca²⁺ currents, 0.5 µM tetrodotoxin and 2 µM nifedipine were added to Ringer's solution. External and internal solution compositions were as described (13).

Electrophysiology in Spinal Cord Slices-- Spinal cord slices were prepared and whole-cell recordings performed as described previously (8). Peptide was included in the solution in the recording pipette (see Ref. 8). Carbachol effect was measured 30 min after first EPSC was recorded. Agonists were applied in bath solution (artificial cerebrospinal fluid) for 10 min and then washed out with bath solution (~10 min). 10 µM bicuculline methiodide and 1 µM strychnine hydrochloride were present in the bath solution throughout the experiment. Statistical comparisons were made with the use of one-way analyses of variance (Dunnett test for post-hoc comparison) or Student's t test. p < 0.05 was considered significant.

	RESULTS AND DISCUSSION

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5 Subunit Type-specific Peptide Inhibits G Protein Activation by M2-- Membranes from CHO cells expressing high levels of M2 receptor were depleted of endogenous G protein subunits as described in the methods section and assayed for activity by binding of antagonist. The receptors bound [³H]N-methylscopolamine with a dissociation constant of 0.35 nM. When M2 containing membranes were reconstituted with heterotrimeric G_i2 (under conditions similar to those in Fig. 1B), GTPS binding by the G protein was stimulated 5-fold by the agonist, carbachol, compared with the antagonist, atropine (data not shown). Little or no GTPS was incorporated (i) in the absence of the G protein heterotrimer, (ii) in the presence of the  subunit alone, or (iii) in the presence of the complex alone. These results indicated that the membranes were free of functional G proteins and that the M2 receptors in this preparation were functional with properties similar to those previously reported (17). To examine interaction between the M2 receptor and a  subunit, 5, we synthesized a 14-amino acid peptide specific to the COOH-terminal sequence of 5 and chemically modified it with geranylgeranyl, a C-20 isoprenoid that is post translationally added to the COOH-terminal cysteine of most  subunits (1) (Fig. 1A). The 5 peptide was then tested for its ability to inhibit G protein activation. If the  subunit tail of G_i interacts with M2, the peptide should compete with the heterotrimer for a site on the receptor. Results in Fig. 1B show that the wild type peptide significantly reduced the rate of agonist-stimulated GTPS binding by the G protein. A peptide with the same amino acids scrambled was significantly less effective, indicating that this effect was sequence specific (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   G protein activation by M2 is inhibited by  subunit-specific peptides. A, chromatographic traces of 5 peptide with and without the prenyl moiety. 5 peptides were purified by fast protein liquid chromatography using a PepRPC HR16/10 column. Peptides were eluted with a gradient of water/acetonitrile. The unprenylated peptide (5) was eluted at a concentration of ~25% acetonitrile and the prenylated peptide (5-gg) at ~50% acetonitrile. B, G protein incorporation of GTPS is inhibited by the COOH-terminal 5 peptide. Membrane suspensions of M2 (5 nM) were reconstituted with G proteins (100 nM) (i2/brain ) for 30 min at room temperature in buffer A: 20 mM HEPES, pH 8, 5 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl, 1 µM GDP, and 0.02% sodium cholate in the presence or absence of 10 µM geranylgeranylated wild type 5 (5) or 5 with a scrambled sequence (5 s). Peptides were dried and equilibrated with membranes mixed with G protein. Binding reactions were started by addition (final concentrations) of 0.2 µM [35S]GTPS and 1 mM agonist (carbachol) or antagonist (atropine). Aliquots were taken at the indicated times and quenched by adding ice-cold buffer A containing 500 µM GTP and 1 mM atropine. Samples were filtered on nitrocellullose membranes, washed and quantitated. Representative results from three independent experiments. C, heterotrimer is not disrupted by wild type 5-peptide. 1His-2 was incubated with i2 (200 nM each), for 30 min at 4 °C in buffer A with CHAPS (0.7%). Heterotrimer (100 nM final concentration) was added to buffer with or without geranyl-geranylated peptide (10 µM 5 peptide/5-scrambled final concentration) and incubated for 30 min at room temperature. The protein mix was bound to 5 µl of nickel-nitrilotriacetic acid beads (Qiagen) for 20 min at 4 °C and washed twice with incubation buffer. The beads were then treated as described below or washed with buffer A. Bound proteins were eluted with SDS sample buffer containing imidazole (150 mM). Samples were examined by SDS-gel electrophoresis and immunoblotting using an antibody that recognizes a domain common to  subunits or a 1 subunit-specific antibody. Results from two different immunoblots probed with the  or  subunit antibodies are shown. Lanes: 1, no treatment; 2, treatment for 20 min at 30 °C with buffer A; 3, treatment with buffer A containing 50 µM AlCl and 10 mM NaFl; 4, treatment with buffer A containing 150 mM imidazole; 5, no treatment but sample incubated with wild type 5 peptide; 6, no treatment but sample incubated with 5-scrambled peptide. Representative result from two experiments.

5 Peptide Does Not Disrupt the G Protein Heterotrimer-- Heterotrimerization of a G protein is essential for receptor interaction (16). To rule out the possibility that the inhibition of G protein activation was due to the disruption of the heterotrimer by the 5 peptide, an experiment was performed under conditions similar to the receptor assays (described in Fig. 1C). The hexahistidine-tagged complex was brought down with resin containing Ni²⁺. Although aluminum fluoride disrupted the heterotrimer (Fig. 1C, compare lane 3 with lane 2), both in the absence and in the presence of the wild type 5 peptide similar amounts of _i2 were co-eluted with 1His-2 (Fig. 1C, compare lane 5 with lane 1). This indicated that the heterotrimer was not disrupted by the 5 peptide.

Geranylgeranylated 5 Peptide Selectively Disrupts Muscarinic Modulation of N-type Ca²⁺ Currents-- To test the effect of peptides specific to the COOH-terminal region of the 5 subunit on signaling in cells, cultured SCG neurons were injected with a wild type 5 peptide or the 5 peptide with the amino acid sequence scrambled (5 s) (described under "Materials and Methods"). The effect of maximal concentration of the muscarinic agonist oxo-M was measured on I_Ca amplitude and on the facilitation ratio. As indicated in Fig. 2A, voltage-dependent inhibition was revealed by inserting a depolarizing prepulse. In the cells injected with the 5-scrambled peptide, I_Ca amplitude was little affected by the prepulse in the absence of agonist (C1 compared with C2 and open circles compared with filled circles on plot). Oxo-M produced a large inhibition of I_Ca that could be partially relieved by the prepulse, thereby increasing the facilitation ratio from 1.11 to 2.22. In uninjected cells and in cells injected with the 5-scrambled peptide, oxo-M increased the facilitation ratio and inhibited I_Ca, similarly (Fig. 2, C and D). Thus, cytoplasmic injection by itself did not disrupt muscarinic signaling. In contrast, muscarinic modulation of I_Ca was substantially different in the 5 peptide-injected cell: first, inhibition was smaller (compare C1 and O1 records in Fig. 2B); second, the +125 mV prepulse was less effective in relieving I_Ca suppression (compare O1 and O2 records and open and filled triangles in Fig. 2B). Indeed, in 13 neurons injected with the 5 peptide, oxo-M inhibited I_Ca by only 31.8% (Fig. 2D) and increased facilitation ratio from 1.08 to only 1.33 (Fig. 2C). Thus the 5 peptide blocked the voltage-dependent inhibition of I_Ca mediated by M4 receptors.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Voltage-dependent inhibition of ICa is blocked by a geranylgeranylated 5 peptide. A, plots of ICa amplitude during test pulse P1 (open symbols) and test pulse P2 (filled symbols), every 4 s, from a cell injected with a control 5 scrambled peptide (5s) (A) and from a cell injected with the 5-peptide (5) (B). Oxo-M (10 µM) and Cd2+ (100 µM) were applied as indicated by the horizontal bars. Cd2+-subtracted ICa records before (C1 and C2) and after 16-s oxo-M perfusion (O1 and O2) are shown on the right. Inset in A, command pulse protocol used to test voltage dependence of inhibition of ICa. Dashed lines on records indicate zero current level. C, mean (±S.E.) facilitation ratio before oxo-M treatment (BR and BR-5 peptide) and during oxo-M treatment, from uninjected control cells (C) and from cells injected with scrambled peptide (5s) or 5 peptide (5). D, mean (±S.E.) ICa inhibition by oxo-M from the same groups of cells. Cell capacitance and series resistance: uninjected cells, 77 ± 4 pF, 2.1 ± 0.4 M; 5s injected-cells, 72 ± 7 pF, 2.0 ± 0.2 M; 5 injected-cells, 75 ± 5 pF, 1.9 ± 0.2 M.

5 Peptide Does Not Disrupt 2-Adrenergic, Somatostatin, or M1 Muscarinic Signaling in Sympathetic Neurons-- We wanted to assess in the same neurons whether the 5 peptide disrupted modulation of I_Ca by other G_o-coupled receptors, namely ₂-adrenergic or somatostatin receptors. Therefore, after I_Ca recovered upon oxo-M and Cd²⁺ treatment, neurons were challenged with norepinephrine or somatostatin. Table I summarizes the results. Here, because there were no statistically significant differences between uninjected cells and cells injected with 5 scrambled peptide, we pooled together both samples (control) to facilitate comparison with the 5 peptide-injected cells. Neither voltage-dependent inhibition of I_Ca by norepinephrine nor by somatostatin were affected by the 5 peptide. In SCG neurons I_Ca is also suppressed by M1 muscarinic receptors in a voltage-independent and pertussis toxin-insensitive manner (13). Inhibition by M1 receptors occurs through the G protein G_q (7). Hence, it is possible that disruption of the M1-mediated signaling pathway might contribute to the smaller muscarinic inhibition of I_Ca in 5 peptide-injected neurons (Fig. 2D). This hypothesis was tested by injecting 5 peptide into pertussis toxin-treated cells. Pertussis toxin blocks the voltage-dependent, M4-mediated component of I_Ca modulation leaving intact the voltage-independent component (17). Furthermore, because M1 receptors also use the same signaling pathway to suppress the K⁺ current named M-current (13), we tested the effect of the 5 peptide on suppression of M-current. The 5 peptide did not block M₁-mediated inhibition of I_Ca or M-current (not shown). Furthermore, the 5 peptide lacking the geranylgeranyl moiety failed to prevent voltage-dependent inhibition of I_Ca (Table II). Thus both the amino acid sequence of the 5 subunit COOH terminus and the prenyl group are essential for activity.

View this table: [in this window] [in a new window]   Table I 5 peptide does not prevent 2-adrenergic and somatostatin-induced inhibition of ICa

View this table: [in this window] [in a new window]   Table II Muscarinic inhibition of ICa is not disrupted by other  subunit-peptides

7 and 12 Peptides Do Not Block Muscarinic Voltage-dependent Inhibition of I_Ca-- Since the 5 peptide reduces only M4-mediated voltage-dependent inhibition of I_Ca and not that stimulated by the adrenergic or somatostatin receptors, we wanted to test whether this selective action is shared by other carboxyl-terminal  subunit type peptides. Table II summarizes data from neurons injected with geranylgeranylated 7 or 12 peptides. The 7 and 12 subunit types are distinct and are grouped in a different subfamily from 5 (1). Neither the 7 nor the 12 peptide affect voltage-dependent inhibition of I_Ca by oxo-M.

The 5 Peptide Specifically Disrupts Muscarinic Receptor Signaling in Spinal Cord Slices-- In spinal cord slices, electrical stimulation of the dorsal root entry zone evokes EPSCs which are mediated by -amino-3-hydroxy-5-methylisoxozole propionic acid and kainate receptors (18) (Fig. 3A). Bath application of carbachol or clonidine, an agonist of the 2-adrenergic receptor, significantly decreased EPSCs (Fig. 3, A and B). Postsynaptic application of the 5 scrambled peptide had no effect on the inhibition of EPSCs by carbachol (Fig. 3, A and C). However, the wild type 5 peptide significantly relieved this inhibition by carbachol but not by clonidine (Fig. 3). These results confirm the ability of the 5 peptide to specifically disrupt signaling from muscarinic receptors in the spinal cord.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Muscarinic receptor inhibition of glutamate-induced EPSCs are relieved by 5 peptide. A, EPSCs evoked by electrical stimulation of the dorsal root entry zone. The traces show EPSCs before drug application (Pre), 10 min after application of carbachol (20 µM), and then wash out for 10 min. Results from three experiments are shown. Recording electrodes contained normal solution alone (Control) or peptides added (3 µM wild type (5) or scrambled 5 (5 s)). Peptides were allowed to diffuse into cells for 30 min before carbachol was applied through bath solution. B, similar experiment as above with 10 µM clonidine and 3 µM wild type 5 peptide in recording pipette. C, bars represent pooled data from traces. They are shown as a percent of the EPSC evoked before agonist application (Pre), which was taken to be 100%. Error bars represent S.E. **, EPSC amplitude in the presence of the 5 wild type peptide (5) was significantly higher (p < 0.01) than in the absence of the peptide (C). Amplitude was not significantly different in the presence of the scrambled 5 peptide (5 s). Wild type peptide did not significantly affect the amplitude of EPSCs in the presence of clonidine (5 compared with C).

The receptors that use G_o to modulate Ca²⁺ channels in SCG neurons are known to act through the G protein complex (19, 20). One possible explanation for the effect of the 5 peptide is that it competes for a site on the Ca²⁺ channel with the complex released on receptor activation. However, in the experiments presented here, the 5 peptide inhibits G protein activation by muscarinic receptors in a reconstituted system and also selectively disrupts signaling from the same class of receptors in intact cells. It seems more likely therefore, that the peptide competes with the complex for a site on the receptor rather than the effector.

Among members of the muscarinic receptor family it is known that M2 and M4 share similar properties in terms of G protein coupling (21, 22). The ability of the 5 peptide to inhibit M2 activation of a G protein in a reconstituted system and also inhibit signaling from M4 receptors in intact cells implies that the 5 peptide interacts with this class of muscarinic receptors. The inability of the 7 and 12 peptides to affect signaling from M4 in combination with the inability of 5 to affect signaling from receptors other than M4 indicate a high degree of specificity in the action of the peptide. Past findings where antisense oligonucleotides specific to two different  subunits inhibited the action of the muscarinic and somatostatin receptor signaling indicated that G protein  subunit types may have a specific role in signaling (23). Results from the analysis of rhodopsin coupling to Gt with different  subunit types indicated specificity between  subunit types and a receptor at the protein level (24). The results here indicate that there may be selectivity in the interaction between  subunit types and receptors. Furthermore, the indication of such specificity in intact cells raises the possibility that peptides from the  subunits and their more potent analogues can be used to selectively disrupt individual pathways in a signaling network.

	ACKNOWLEDGEMENT

H. C. thanks Dr. Bertil Hille for research facilities and helpful discussions.

	FOOTNOTES

^* This work was supported by Consejo Nacional de Ciencia y Tecnologia Grant 4113P-N9607 and Pew Charitable trusts (to H. C.), by National Institutes of Health Grants GM51466 (to M. L.), NS08174 (to Dr. Bertil Hille), and GM46963 (to N. G.), and by the NINDS and NIDA (to M. Z.).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.

^§ These two authors contributed equally to this work.

Present address: Centro Universitario de Investigaciones Biomedicas, Universidad de Colima Col. Villa San Sebastian Colima, Col. 28000, Mexico.

To whom correspondence should be addressed: Box 8054, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8568; Fax: 314-362-8571; E-mail: gautam@morpheus.wustl.edu.

	ABBREVIATIONS

The abbreviations used are: SCG, superior cervical ganglion; CHO, Chinese hamster ovary; EPSC, excitatory postsynaptic current; GTPS, 5'-O-(thiotriphosphate); oxo-M, oxotremorine methiodide; BAPTA, 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; , ohm(s); CHAPS, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; F, farad(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES