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J. Biol. Chem., Vol. 277, Issue 16, 13827-13830, April 19, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/16/13827    most recent M201064200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takesono, A. Articles by Lanier, S. M. Search for Related Content PubMed PubMed Citation Articles by Takesono, A. Articles by Lanier, S. M. Social Bookmarking                      What's this?

Activator of G-protein Signaling 1 Blocks GIRK Channel Activation by a G-protein-coupled Receptor

APPARENT DISRUPTION OF RECEPTOR SIGNALING COMPLEXES^*

Aya Takesono, Mark W. Nowak, Mary Cismowski, Emir Duzic^§, and Stephen M. Lanier^¶

From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425 and ^¶ Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

Received for publication, January 31, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Ras-related protein, activator of G-protein signaling 1 (AGS1) or Dexras1, interacts with G_i/G_o and activates heterotrimeric G-protein signaling systems independent of a G-protein-coupled receptor (GPCR). As an initial approach to further define the cellular role of AGS1 in GPCR signaling, we determined the influence of AGS1 on the regulation of G-regulated inwardly rectifying K⁺ channel (GIRK) current (I_ACh) by M₂-muscarinic receptor (M₂-MR) in Xenopus oocytes. AGS1 expression inhibited receptor-mediated current activation by >80%. Mutation of a key residue (G31V) within the G₁ domain involved in nucleotide binding for Ras-related proteins eliminated the action of AGS1. The inhibition of I_ACh was not overcome by increasing concentrations of the muscarinic agonist acetylcholine but was progressively lost upon injection of increasing amounts of M₂-MR cRNA. These data suggest that AGS1 may antagonize GPCR signaling by altering the pool of heterotrimeric G-proteins available for receptor coupling and/or disruption of a preformed signaling complex. Such regulation would be of particular importance for those receptors that exist precoupled to heterotrimeric G-protein and for receptors operating within signaling complexes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Heterotrimeric G-proteins transduce a variety of extracellular stimuli into intracellular responses. Such stimuli are primarily sensed by the superfamily of G-protein-coupled receptors. In general, the specific intracellular response is determined by the external stimuli itself and cell type-specific expression of receptors, G-proteins, and effectors. However, additional proteins also operate to influence signaling specificity as well as signal magnitude and duration. Such accessory proteins may act as scaffolding proteins within a signal transduction complex and/or directly influence the basal activation state of G-proteins and effectors independent of an activated GPCR¹ (1-8). The latter proteins include both the family of regulators of G-protein signaling (RGS), initially defined based upon their ability to accelerate the GTPase activity of selected G_i proteins, and the recently identified activators of G-protein signaling (AGS) 1-3.

AGS proteins (AGS1-3) were identified in a yeast-based functional screen as receptor-independent activators of G-protein signaling (1, 2). These proteins do not share any sequence homology, and each entity exerts different effects within the context of the G-protein activation/deactivation cycle (1-4). AGS1 (AF069506) selectively activated the G_i/G_o-protein signaling pathway, and it appears to act as a guanine nucleotide exchange factor for G_i, somewhat mimicking a GPCR (1, 4). AGS1 is a member of the Ras superfamily of small G-proteins providing a potential interface between signaling pathways regulated by the two broad classes of G-proteins. AGS1 is the human counterpart of the Ras-related protein DexrasI (NP_033052), which was identified as a dexamethasone-inducible cDNA in AtT-20 mouse corticotroph cells (9), where it may influence cAMP regulation of hormone secretion (10). AGS1 was also implicated in N-methyl-D-aspartate receptor signaling in neuronal cells, where it is an apparent target of neuronal nitric-oxide synthase (11). Thus, AGS1 is clearly involved in cellular signaling events and binds to as well as activates G_i/G_o, but it is not known how this apparent G-protein regulator influences the activation of signaling systems by a GPCR. AGS1 may influence the specificity, magnitude, or duration of GPCR signaling events as it may actually interfere with receptor-effector coupling (12)² by altering the pool of G-protein available for interacting with receptor.

As an initial approach to define the role of AGS1 in GPCR signaling, we determined the influence of AGS1 on the regulation of G-regulated inwardly rectifying K⁺ channel (GIRK) current (I_ACh) by M₂-muscarinic receptors (M₂-MR) in Xenopus oocytes (13, 14). AGS1 expression had little effect on basal levels of current (I_hK) but inhibited the increase in GIRK channel activity elicited by activation of the M₂-MR. The inhibition of I_ACh by AGS1 was progressively lost upon injection of increasing amounts of M₂-MR cRNA. These data suggest that AGS1 antagonized GPCR signaling by altering the pool of heterotrimeric G-proteins available for receptor coupling and/or by disrupting a preformed signal transduction complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

cRNA Synthesis-- GIRK1, GIRK4, and M₂-MR in pcDNA3.1 vector (Invitrogen) were kindly provided by Drs. Paolo Kofuji and H. A. Lester (California Institute of Technology). AGS1 and AGS1G31V were propagated in the pcDNA3.1-HisA vector (Invitrogen). All plasmids were linearized with appropriate enzymes at sites immediately following translational stop codons and used for capped cRNA synthesis using mMESSAGE MACHINE kits (Ambion Inc., Austin, TX).

Oocyte Preparation and Injection-- Xenopus oocytes were surgically extracted and dissociated with defolliculation by collagenase treatment (1 mg/ml collagenase A; Roche Applied Sciences, Indianapolis, IN) (15). Healthy stage V and VI oocytes were selected and maintained at 19 °C in ND96 buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM HEPES, pH 7.5) supplemented with 5% horse serum, 2.5 mM sodium pyruvate, and 50 µg/ml gentamycin. Various combinations of cRNAs (final volume, 50 nl in diethyl pyrocarbonate-H₂O) were injected into stage V and VI oocytes with a Drummond microinjector. Injected oocytes were incubated for 2 days at 19 °C with replacement of culture medium twice daily.

Electrophysiology-- Electrophysiological measurements were carried out using a standard two-electrode voltage clamp technique (16). Oocytes were clamped at 80 mV using a Warner oocyte clamp (Warner Instruments, Hamden, CT). Voltage and current recording electrodes had resistances of 0.5-2 megaohms. Constitutive GIRK-associated current responses (I_hK) were elicited by switching the perfusion medium from ND96 to a high potassium solution (2 mM NaCl, 96 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM Hepes, pH 7.5). Acetylcholine-induced GIRK current responses were elicited by switching the perfusion solution from high potassium to high potassium solution containing acetylcholine. The difference between the acetylcholine-mediated current and I_hK is referred to as I_ACh. Switching the perfusion solution back to ND96 returned the current to base-line values (see Fig. 1A).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The ability of AGS1 to interact with heterotrimeric G-proteins raises immediate questions as to its role in signal processing following activation of a GPCR at the cell surface. To address this question, we investigated the role of AGS1 on M₂-MR coupling to GIRK channels using a Xenopus oocyte expression system. The Xenopus oocyte expression system has been widely utilized to analyze the function and regulation of GIRK channel activities because of the ease of gene expression and functional readouts. In oocytes injected with GIRK1/4 cRNAs and M₂-MR cRNA, an inward K⁺ current (I_hK) was elicited by exchanging ND96 for a high potassium solution. Acetylcholine elicited an additional inward K⁺ current (I_ACh), and this receptor-mediated event was completely blocked by injection of pertussis toxin.³

Co-injection of AGS1 with GIRK1/4 and M₂-MR cRNAs elicited little change in I_hk but markedly diminished I_ACh (Fig. 1).⁴ Mutation of residues in the G₁ domain of AGS1 (G31V) rendered AGS1 inactive (Fig. 1, A and B), as was the case for AGS1 in the yeast assay system and for AGS1 regulation of ERK activity in COS-7 cells (1, 4). The inhibition of I_ACh by expression of AGS1 was progressively enhanced by injection of increasing amounts of AGS1 cRNAs (Fig. 2). At the lower expression levels of GIRK channels (0.01 ng/oocyte), AGS1 inhibited I_ACh by 76 ± 4.2% (Fig. 2A). In oocytes expressing higher levels of GIRK channels, AGS1 inhibited I_ACh by 47 ± 7.7% (Fig. 2B). Expression of AGS1G31V did not alter I_ACh at either expression level of GIRK channels (Fig. 2), indicating that the inhibition of I_ACh was not because of altered expression of receptor or channels, per se. This thought is further supported by the absence of a decrease in I_hk when AGS1 was coexpressed with GIRK channels. Thus, AGS1 likely inhibits I_ACh by interfering with the transfer of signal from receptor to G-protein or perhaps from G-protein to the channels. Similar conclusions regarding the action of AGS1 on GPCR signaling were reported by Graham et al. (12) for formyl peptide receptor activation of ERK1/2 kinases in COS-7 cells.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Influence of AGS1 and AGS1G31V on GIRK channel currents in Xenopus oocytes. Oocytes were injected with cRNAs encoding GIRK1/4 (2 ng), M2-MR (0.1 ng), and AGS1 or AGS1G31V (5 ng) and processed for channel recordings as described under "Experimental Procedures." A, representative tracings of IhK elicited by high potassium solution and IACh elicited by application of acetylcholine (5 µM). The stippled portion of the bar indicates oocyte perfusion with high potassium solution. B, averaged data from an individual batch of six oocytes. Data are presented as the percent of control IACh (2572 ± 234 nA) observed in the absence of AGS1 or AGS1G31V. Data values represent the means ± S.E. Similar results were obtained in three to five batches of oocytes. IhK in B were 972, 1028, and 1222 nA for control, AGS1, and AGS1G31V, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Influence of increasing amounts of AGS1 on receptor-mediated activation of GIRK. IACh were recorded from oocytes injected with cRNA encoding GIRK1/4 (0.01 ng in A, 5 ng in B), M2-MR (0.1 ng), and AGS1 or AGS1G31V. Data are presented as percent of control IACh. Control IACh were 120 ± 20 nA and 2900 ± 480 nA for A and B, respectively. The results shown represent the means ± S.E. from 10-14 oocytes from the same batch. Similar results were obtained in three to five batches of oocytes. Acetylcholine concentration was 5 µM. IhK in A were 340 ± 22 (control), 284 ± 26 (AGS1, 0.1 ng), 245 ± 18 (AGS1, 1 ng), 272 ± 21 (AGS1, 5 ng), 263 ± 30 (AGS1G31V, 0.1 ng), 235 ± 36 (AGS1G31V, 1 ng), and 228 ± 30 (AGS1G31V, 5 ng) nA. IhK in B were 3066 ± 315 (control), 2761 ± 379 (AGS1, 0.1 ng), 2855 ± 262 (AGS1, 1 ng), 2773 ± 309 (AGS1, 5 ng), 3340 ± 549 (AGS1G31V, 0.1 ng), 3627 ± 287 (AGS1G31V, 1 ng), and 2965 ± 374 (AGS1G31V, 5 ng) nA.

As AGS1 clearly interacts with G_i/G_o  subunits (1, 4), AGS1 may inhibit I_ACh by competing with M₂-MR for the available pool of G-proteins. This possibility was addressed by examining the dose-response curve for acetylcholine in the presence and absence of AGS1 and by determining the ability of increasing amounts of M₂-MR to overcome the AGS1-mediated I_ACh inhibition. Under standard experimental conditions (cRNA/oocyte: M₂-MR, 0.1 ng; GIRK1/4, 2 ng for each; AGS1, 5 ng) increasing concentrations of acetylcholine did not overcome the inhibitory effect of AGS1 on I_ACh (Fig. 3A). In contrast to the influence of increasing amounts of agonist, the inhibitory action of AGS1 on I_ACh was overcome by increasing the levels of expressed receptor. Increased levels of receptor increased the amount of I_Ach at fixed amounts of GIRK channels, suggesting that under these experimental conditions receptor was somewhat rate-limiting or that at least G-proteins and effectors were not saturated (17). At the lowest expression levels of M₂-MR (0.1 ng of cRNA/oocyte), AGS1 inhibited I_ACh by 80 ± 3.9% I_ACh (Fig. 3B). At the higher levels of M₂-MR expression, AGS1 inhibited I_ACh by only 17 ± 5.9% I_ACh (Fig. 3B). AGS1G31V did not inhibit I_ACh at any levels of M₂-MR expression (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Influence of increasing amounts of agonist or M2-MR on the inhibitory action of AGS1. A, effect of increasing concentrations of acetylcholine on IACh in the presence and absence of AGS1. IACh were recorded from oocytes injected with cRNA encoding GIRK1/4 (2 ng), M2-MR cRNA (0.2 ng), and AGS1 (5 ng). Data were generated as cumulative dose-response curves with random mixing of acetylcholine concentrations. Data are presented as the percent of control IACh at 10 µM acetylcholine (3320 ± 290 nA) (n = 6). The results represent the means ± S.E. using 6-9 oocytes from the same batch. Similar results were obtained with at least three different batches of oocytes. B, IACh at different levels of receptor in the presence and absence of AGS1. IACh induced by acetylcholine (5 µM) were recorded from oocytes injected with cRNA encoding GIRK1/4 (0.01 ng), AGS1 (5 ng), AGS1G31V (5 ng), and M2-MR. The results shown represent the means ± S.E. from 10-14 oocytes from the same batch. Similar results were obtained in three batches of oocytes. IhK in A were 1167 ± 176 (control) and 1289 ± 102 (AGS1) nA. IhK in nA for B: 312 ± 20 (M2-MR, 0.1 ng), 291 ± 47 (M2-MR, 1 ng), and 255 ± 32 (M2-MR, 10 ng) for control series; 178 ± 17 (M2-MR, 0.1 ng), 158 ± 17 (M2-MR, 1 ng), and 205 ± 22 (M2-MR, 10 ng) for AGS1 series; 174 ± 13 (M2-MR, 0.1 ng), 173 ± 15 (M2-MR, 1 ng), and 202 ± 26 (M2-MR, 10 ng) for AGS1G31V series.

These observations raise several interesting points relative to signal processing through this system. The ability of increased amounts of receptor to overcome the inhibitory effect of AGS1 suggests that, indeed, AGS1 competes with the M₂-MR for the pool of available heterotrimeric G_i/G_o to activate GIRK channel. This may reflect an action of AGS1 to disrupt a preconfigured signal transduction complex that is required for agonist activity. Thus increasing concentrations of agonist could not overcome the inhibitory affect of AGS1 action. Such a signal transduction complex may consist of receptor and G-protein, G-protein and GIRK, or perhaps all three entities (17-19). By complexing with a defined population of G-proteins, AGS1 may limit the ability of a receptor to act catalytically as it cannot access multiple G-proteins.

These thoughts likely have important implications for G_i/G_o-coupled receptors that exist in a precoupled state where the receptor is complexed with G-protein and "stabilized" in a conformation exhibiting high affinity for agonist. AGS1 may block the formation of this precoupled complex, and as such, even high concentrations of agonist would not be able to activate downstream effectors. Increasing the amount of receptors in the presence of AGS1 as opposed to agonist itself would allow more of the receptor population to exist in a precoupled state and thus effectively override the inhibitory effect of AGS1 as was indeed the case in the present study.

A flurry of recent reports indicates that multiple proteins interact with and/or regulate the activation state of heterotrimeric G-proteins. Although the role of these proteins in GPCR processing can be varied, they certainly offer unexpected avenues for manipulating the signaling system. Controlling the population of receptors precoupled with G-protein or the generation of signaling complexes may be a key mechanism for regulating the action of hormones and as such provide new pathways for therapeutics that mimic or disrupt specific signaling systems.

	ACKNOWLEDGEMENTS

We appreciate the suggestions and input of Drs. Motohiko Sato and Joe Blumer in the Lanier laboratory.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants RO1-NS24821 and MH 59331 (to S. M. L.) and K01 AA00287 (to M. W. N.).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.

Present address: The Guthrie Foundation, Sayre, PA 18840.

^§ Present address: Millennium Pharmaceuticals, Inc., 640 Memorial Dr., Cambridge, MA 02139.

To whom correspondence should be addressed: Dept. of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70118. Tel.: 504-568-4744; Fax: 504-568-2361; E-mail: slanie@lsuhsc.edu.

Published, JBC Papers in Press, February 12, 2002, DOI 10.1074/jbc.M201064200

² M. Cismowski and E. Duzic, unpublished observations.

³ A. Takesono, M. W. Nowak, and S. M. Lanier, unpublished observations.

⁴ A. Takesono, M. W. Nowak, and S. M. Lanier, unpublished observations. I_hK were slightly (~2-fold) but consistently increased by co-injection of AGS1 cRNA with GIRK1/4 cRNAs, but this effect was variable and of lesser magnitude when oocytes were also injected with M₂-MR cRNA. A more robust regulation of G-protein signaling by AGS1 may require stimulus input to AGS1 to activate the protein. This signal input is likely absent in the oocyte perfusion system. G-protein activation of AGS1 may also be more robust in an assay system involving a cumulative response readout (i.e. growth or luciferase reporter assays as in yeast and COS7 cells, respectively) (1, 4).

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; AGS, activator of G-protein signaling; GIRK, G-regulated inwardly rectifying K⁺ channel; M₂-MR, M₂-muscarinic receptors.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/16/13827    most recent M201064200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takesono, A. Articles by Lanier, S. M. Search for Related Content PubMed PubMed Citation Articles by Takesono, A. Articles by Lanier, S. M. Social Bookmarking                      What's this?