Recipient of the American Heart Association Established Investigator Award. To whom correspondence should be addressed: Dept. of Pharmacology, UT-Southwestern, 5323 Harry Hines Blvd., Dallas, TX 75235-9041
* This work was funded by a postdoctoral training fellowship and grants from the National Institutes of Health and by the R. A. Welch Foundation.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 authors contributed equally to this work.
Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Gq and Gi α subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via Gq. Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 104-fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates Gq class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by Gαq and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling.
Heterotrimeric G proteins of the Gq class are mediators of Ca2+ responses in animal cells. Signaling is initiated by agonist binding to heptahelical transmembrane receptors complexed with Gqαβγ and phospholipase C-β (PLCβ)
). Many cells express several Gq-coupled receptors that regulate the location, intensity, and propagation of intracellular Ca2+ waves. For example, pancreatic acini respond to acetylcholine, bombesin, and cholecystokinin by activating the same set of Gq class proteins and mobilizing the same Ca2+ pool, but each receptor evokes distinct patterns of Ca2+ waves (
). In rat pancreatic acinar cells, RGS4 preferentially inhibited Gq/11-mediated signaling evoked by carbachol relative to bombesin and cholecystokinin regardless of the identity of the Gq class α subunit.2 Regulatory specificity was apparently conferred by direct or indirect interaction between RGS4 and the receptor.
In the present study, we used deletion mutations to identify two domains in RGS4 that regulate agonist-dependent Ca2+ signaling. The RGS box accelerates GTP hydrolysis by Gαq whereas the N terminus conveys high affinity and receptor-selective inhibition. These combined activities are required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling.
MATERIALS AND METHODS
Expression and Purification of Recombinant RGS4 Proteins
All recombinant RGS proteins were His6-tagged at the N terminus. Protein expression and GAP assays were performed as described (
were purified by reverse-phase high pressure liquid chromatography and confirmed by amino acid analysis and fast atom bombardment/mass spectroscopy.
Measurement of Ca2+ Release
Measurement of Ca2+-activated Cl− current and Ca2+ release in permeabilized cells was exactly as described.2
The agonist-stimulated steady-state GTPase activity of reconstituted phospholipid vesicles that contained m1 muscarinic acetylcholine receptors and heterotrimeric Gqwas assayed in the presence or absence of RGS proteins or peptides as described (
) using [γ-32P]GTP bound to the R183C mutant of Gαq (chosen to slow hydrolysis and thus facilitate loading).
RESULTS AND DISCUSSION
The N Terminus of RGS4 Is Required for High Potency Inhibition
To determine the domains of RGS4 that conveyed high potency and receptor-selective inhibition, rat pancreatic acinar cells were dialyzed with different recombinant RGS4 proteins through a patch pipette and exposed to 100 μm carbachol, the minimal concentration of carbachol needed to generate a maximal cellular response (
).2 Responses were detected as changes in current carried by a Ca2+-activated Cl−channel. In the control (Fig.1a), carbachol evoked a typical biphasic Ca2+ response consisting of an initial spike caused by Ca2+ release from internal stores followed by a plateau current. Infusion of full-length RGS4 (10 pm) suppressed the initial Ca2+ release and caused subsequent oscillations in response to maximal carbachol stimulation. Increasing the RGS4 concentration to 100 pm further reduced the Ca2+ response to a low frequency oscillation. A similar transition from a sustained to a weak oscillatory response occurred when the carbachol concentration was reduced from 100 to 1 μm (
).2 This indicates that carbachol-evoked signaling in intact cells dialyzed with 100 pm RGS4 is functionally equivalent to a 100-fold decrease in the potency of carbachol stimulation. Exposing cells to higher concentrations of carbachol did not change the inhibitory effect of RGS4 on Ca2+ release. Two other proteins related to RGS4, RGS1, and RGS16 (
), 4Box was approximately 104-fold less potent than full-length RGS4 in inhibiting in vivoCa2+ signaling. For example, 10 pm RGS4 conferred partial inhibition of signaling whereas 100 nm4Box was required before partial inhibition was apparent (Fig. 1,b and d). Increasing the cellular concentration of full-length RGS4 gradually inhibited signaling, decreasing both the amplitude of the initial current spike and the frequency and amplitude of the subsequent current oscillations (Fig. 1, a–c). By contrast, dialysis with as much as 100 nm 4Box did not significantly reduce the initial current spike but caused rapid termination of signaling and obliterated subsequent oscillations in the continued presence of agonist (Fig. 1d). These data also indicated that the mechanisms of inhibition by RGS4 and 4Box were qualitatively different, suggesting that inhibition by intact RGS4 involves more than just Gq GAP activity.
To identify the flanking structure in full-length RGS4 that conveys high potency inhibition, we tested the activities of other deletion mutants. C-terminal truncation of RGS4 (RGS4ΔC) reduced its potency by about 100-fold (Fig. 1, g and h). For example, 10 nm RGS4ΔC inhibited the initial current spike to the same extent as did 100 pm full-length RGS4 and caused similar subsequent oscillations in response to 100 μmcarbachol (compare Fig. 1, c and h). This effect was not further characterized because deletion of the N-terminal sequence flanking the RGS box (RGS4ΔN) caused an even more dramatic reduction in potency and altered the mechanism of RGS4 inhibition to resemble that observed with 4Box (Fig. 1, i andj). Hence, the N terminus of RGS4 is essential for high potency inhibition and contributes significantly to RGS4 interaction with the receptor-Gq protein complex.
Besides diminishing the effect and potency of RGS4 action, 4Box also lost selectivity among different receptors. RGS4 inhibits signaling preferentially through the m3 muscarinic receptor compared with the CCK receptor, assayed either separately or sequentially within the same cell (Fig. 2).2 By contrast, 4Box inhibited Ca2+ signaling by carbachol and CCK equivalently over a wide range of concentrations (Fig. 2,c–f). These data suggest that terminal regions of RGS4 both enhance potency and mediate recognition of receptor.
Receptor-selective Inhibition by the RGS4 N-terminal Domain
Next, we tested whether the 33-amino acid N-terminal peptide of RGS4 (P1–33) can itself alter Gq-mediated signaling. P1–33 (100 nm) both blocked the initial spike and converted the following sustained current to an oscillatory response (Fig. 2,g–i). Higher concentrations of P1–33completely blocked signaling by carbachol. In contrast, these concentrations of P1–33 had no effect on CCK signaling. Only at 1 μm P1–33 was the sustained response evoked by CCK converted to an oscillatory current. These data indicate both that P1–33 effectively inhibited Gq-mediated Ca2+ signaling and that inhibition retained the selectivity for the m3 muscarinic receptor over the CCK receptor that is characteristic of RGS4.
RGS4 Flanking Sequences Enhance Gαq-GAP Activity in Vitro
We then tested the relative GAP activity of RGS4 and 4Box toward purified, recombinant Gαq proteins in vitro. In a solution phase, single-turnover Gαq GAP assay, RGS4 was about 10-fold more active than 4Box (Fig.3, a and c), in contrast to their equal potency when assayed with Gαi1(
). We then compared the GAP activity of full-length RGS4 and 4Box on wild-type Gq in a steady-state, receptor-coupled in vitro assay. Phospholipid vesicles were reconstituted with m1 muscarinic cholinergic receptor and trimeric Gqαβγ, wherein steady-state GDP-GTP exchange on Gαq was agonist-dependent (
). In this assay, RGS4 was about 125-fold more potent than 4Box in stimulating steady-state receptor-dependent GTPase activity (Fig. 3, b andc). Phospholipid vesicles reconstituted with m2 muscarinic cholinergic receptor and trimeric Gαi1βγ revealed an even more pronounced difference between the relative activities of full-length RGS4 and 4Box (data not shown). These findings provide additional evidence that sequences flanking the RGS box are essential for optimal GAP activity in receptor-G protein-coupled signaling.
N Terminus and 4Box Act Synergistically to Inhibit Ca2+Signaling
The results in Figs. Figure 1, Figure 2, Figure 3 clearly show that 4Box has Gq-GAP activity but that the N-terminal domain of RGS4 both conferred receptor selectivity upon RGS4 action and increased its potency in intact cells. To address the mechanism of RGS4 action, Ca2+ release from intracellular stores was measured in streptolysin O-permeabilized cells. These cells sequester Ca2+ from the incubation medium into cellular organelles and retain Gq-coupled signaling in response to all agonists that act on acinar cells (
). This experimental approach facilitated addition of GTPγS to reverse inhibition by RGS4 as a test of its Gq GAP activity.
As shown in Fig. 4a, muscarinic stimulation released about 75–80% of the IP3-sensitive Ca2+ pool. Addition of 125 nm RGS4 inhibited more than 95% of the normal carbachol-stimulated Ca2+ release (Fig. 4f). Permeabilized cells were less sensitive to RGS4 than patch-clamped cells, probably because of a slower diffusion of proteins from the extracellular media into the cytosol. RGS4 inhibition was fully reversible by addition of GTPγS (Fig. 4f), suggesting that inhibition of signaling reflects the GAP activity of RGS4 under these conditions. By contrast, addition of up to 1.4 μm 4Box to the incubation medium had no effect on carbachol-stimulated Ca2+ release (Fig. 4b) further demonstrating that full-length RGS4 inhibits Gq-coupled signaling far more potently than does 4Box.
Consistent with its effects in intact cells, P1–33inhibited Ca2+ release in permeabilized cells in a receptor-selective manner (Fig. 4, c–e). Carbachol-evoked Ca2+ release was inhibited completely by 62 μm P1–33, with an IC50 of 25 ± 2 μm (n = 5). Similar measurements with CCK showed an IC50 for P1–33of 62 ± 5 μm (n = 3). Thus, in permeabilized cells, P1–33 inhibited Ca2+release evoked by carbachol 2.5-fold better than that evoked by CCK (Fig. 4e). Several control experiments further indicated that inhibition by P1–33 was specific. First, cells responded normally to addition of IP3, even in the presence of 62 μm P1–33 (Fig. 4, c andd). Second, HT-31, another amphipathic peptide that disrupts protein kinase A anchoring to AKAPs (
), had no effect on signaling in response to carbachol. Third, amino acid substitutions within P1–33 dramatically lowered its inhibitory activity (see legend to Fig. 4). Fourth, P1–33 preferentially inhibited signaling via the muscarinic receptor compared with the CCK receptor.
An important distinction between P1–33 and RGS4 is that the inhibitory effect of P1–33 was not reversed by GTPγS (Fig. 4, c and d), indicating that its effect does not depend on Gq-GAP activity. Sequential addition of P1–33 and RGS4 blocked GTPγS-insensitive inhibition (Fig. 4g), indicating the peptide and full-length RGS4 compete for binding to a subsite of the RGS4 binding site in the receptor-Gq complex. To further investigate P1–33 inhibition of Ca2+ release, we changed the order of addition of inhibitors and activators. If cells were exposed to P1–33, then carbachol, subsequent addition of either RGS4 or 4Box did not restore the ability of GTPγS to stimulate Ca2+ release (similar to Fig. 4d). RGS4 and 4Box only worked when added prior to carbachol stimulation (Fig. 4,f–h). P1–33 does not prevent receptor-catalyzed GTP exchange on Gαq nor does P1–33 act as a Gq GAP (data not shown). This overall pattern of activities suggests that P1–33 binds at a site normally occupied by the N terminus of RGS4, and when added without 4Box, P1–33 blocks interaction of Gqwith its effector protein PLCβ.
A final and distinctive activity of P1–33 was that exposure to both 4Box and P1–33 inhibited Ca2+release with greater potency than either added alone (compare Fig. 4,b, c, and h). Importantly, this inhibition was reversed by GTPγS, indicating that the GAP activity of 4Box was functional in permeabilized cells (Fig. 4, h andi). In cells dialyzed with P1–33 (10 nm) and 4Box (1 nm) through a patch pipette, at concentrations at which neither had activity on its own, the combination fully inhibited carbachol-evoked Ca2+ signaling (data not shown). Thus, the peptide P1–33 and 4Box mutually influenced the activity of each other in cells. We propose the P1–33 peptide facilitates 4Box interaction with the receptor complex (reflected by enhanced potency) and the 4Box alters the P1–33 binding site to relieve GTPγS-resistant inhibition. The analysis in Fig. 4 suggests two separate regions of RGS4 interact with the receptor complex to regulate Ca2+signaling. A combination of the N-terminal peptide P1–33and 4Box reconstituted the essential functions of RGS4, those of receptor specificity and GAP activity. Sequence divergence in the N terminus of different RGS proteins may convey regulatory specificity toward other receptor complexes, and this specificity may be retained in peptides analogous with P1–33.
The N-terminal domain and RGS box of RGS4 cooperate via at least two discrete mechanisms to convey receptor-selective inhibition of G protein signaling. Inhibition of Ca2+ signaling by RGS4 was receptor-selective whereas inhibition by 4Box was not. Receptor selectivity of RGS4 inhibition was not influenced by the identity of the Gq class α subunit.2 Such selectivity suggests intact RGS4 interacts directly or indirectly with receptors, most likely through its N terminus. This is supported by the finding that RGS4 in patch-clamped cells was 10,000-fold more potent than 4Box and RGS4 had 125-fold higher Gq-GAP activity than did 4Box in an agonist-dependent in vitro assay. Finally, partial inhibition by RGS4 simultaneously decreased both the amplitude of the initial Ca2+ signal and the frequency of subsequent oscillations, suggesting recombinant RGS4 protein is active when dialyzed into patch-clamped cells and can preassemble with the receptor-Gq-PLCβ signaling complex. By contrast, 4Box preferentially inhibited signaling following the initial Ca2+ release evoked by carbachol and CCK. This suggests that 4Box is recruited only to active receptor complexes when production of the Gq-GTP substrate would be greatest, and thereby 4Box GAP activity would be most pronounced.
Interaction of the N terminus with components of the receptor-Gq-PLCβ signaling complex is further supported by the ability of the N-terminal peptide P1–33 to inhibit Ca2+ signaling in the absence of 4Box. Inhibition by P1–33 displayed the same selectivity among receptors as displayed by full-length RGS4. In contrast to RGS4, however, inhibition by P1–33 was not overcome by GTPγS, possibly because P1–33 blocked access of Gαq-GTPγS to its effector protein PLCβ. Cooperation between the N terminus and 4Box is most apparent in that a combination of both P1–33 and 4Box restored the regulatory activity of intact RGS4 in cells, including its reversibility by GTPγS. Thus, the N terminus provides anchorage (
) and receptor selectivity whereas the RGS box acts as a GAP, and both domains combine to yield the behavior of the intact protein.
Based on our results, we propose that receptor interactions with the N terminus of RGS4 may help position RGS4 between effector and G protein, where it is poised to inactivate the G protein α subunit via the GAP activity of the RGS box, even in the presence of persistent agonist (Fig. 5). Subcellular co-localization of receptor, RGS proteins, and downstream signaling proteins, including Gq and PLCβ, could provide an additional level of regulatory specificity. Thus, the multiple effects of RGS4 described here may contribute to the temporal and spatial regulation that is a hallmark of intracellular Ca2+ signaling.
We thank Melanie Cobb, David Corey, Rama Ranganathan, Kai Zinn, and members of our laboratories for comments on the manuscript.