RGS-PX1, a GAP for Gαs and Sorting Nexin in Vesicular Trafficking

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Science  30 Nov 2001:
Vol. 294, Issue 5548, pp. 1939-1942
DOI: 10.1126/science.1064757


Heterotrimeric GTP–binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein–mediated signaling through their ability to serve as guanosine triphosphatase–activating proteins (GAPs). We have identified RGS-PX1, a Gαs-specific GAP. The RGS domain of RGS-PX1 specifically interacted with Gαs, accelerated its GTP hydrolysis, and attenuated Gαs-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking.

Heterotrimeric G proteins relay extracellular signals initiated by hormones, neurotransmitters, chemokines, and sensory stimuli through G protein–coupled receptors to intracellular effectors and trigger a variety of physiological responses (1, 2). Receptor activation causes dissociation of Gα subunits from Gβγ dimers and subsequent regulation of downstream effectors. Members of the RGS protein family serve as GAPs that attenuate G protein–mediated signal transduction by binding to Gα subunits through a conserved RGS domain and accelerating GTP hydrolysis of Gα subunits (3).

The RGS proteins characterized to date are GAPs for Gi, Gq, or G12/13 classes of G proteins, but no RGS GAP for Gαs has been found. To identify RGS proteins that might serve as GAPs for Gαs, we searched sequence databases with representative RGS domains from the six known mammalian RGS subfamilies and subsequently isolated a cDNA clone encoding a 957–amino acid protein from a human heart cDNA library (4) (Fig. 1A), which we named RGS-PX1 based on the presence of both an RGS domain (Fig. 1B) and a Phox (PX) domain (5, 6). RGS-PX1 also contains an NH2-terminal hydrophobic region (∼36 amino acids), a PX-associated domain (PXA) of unknown function (5), and several coiled-coil regions (Fig. 1A).

Figure 1

Structure of RGS-PX1. (A) Schematic representation of RGS-PX1. PX, Phox homology domain; PXA, PX-associated domain; CC, coiled-coil regions; φ, hydrophobic regions. (B) The RGS domain of RGS-PX1 is homologous to those of other RGS proteins (27). Conserved residues are shaded in black; similar residues are shaded in gray. The regions containing the alpha helixes (α1 through α9) found in RGS4 are indicated above the sequences. The Gα i1-contacting (asterisks) and hydrophobic core residues (diamonds) of RGS4 are also indicated. p115, p115RhoGEF.

To determine whether RGS-PX1 interacts directly with Gα subunits, bovine brain lysates were incubated with fusion proteins containing glutathione S-transferase (GST) and either the RGS domain of RGS-PX1 or RGS4, immobilized on glutathione-agarose beads in the presence of guanosine diphosphate (GDP) and AlF4 (7), which mimicks the transition state of Gα. RGS-PX1 specifically bound Gαs but not Gαi3, Gαq, or Gα12 in brain lysates (Fig. 2A), whereas RGS4 bound Gαiand Gαq but not Gαs and Gα12as previously reported (8, 9). The specificity of the interaction between RGS-PX1 and Gαs was confirmed by incubating Gαs or Gαi1 proteins with RGS4 or the RGS domain of RGS-PX1 bound to beads in the presence of GDP or GDP and AlF4 (10). RGS-PX1 bound the GDP-AlF4 form of Gαs, whereas RGS4 bound only the GDP-AlF4 form of Gαi1 (Fig. 2B). These data indicate that RGS-PX1 specifically interacts with Gαs.

Figure 2

RGS-PX1 specifically interacts with Gαs and stimulates its GTPase activity. (A) The RGS domain of RGS-PX1 and full-length RGS4 immobilized on glutathione beads were incubated with bovine brain lysates in the presence of GDP/AlF4 . Bound proteins were analyzed by immunoblotting for the indicated Gα subunits. (B) RGS domains of RGS-PX1 and RGS4 immobilized on Ni-NTA beads were incubated with purified recombinant Gαs (lanes 1 through 5) or Gαi1 (lanes 6 through 10) in the presence of GDP/AlF4 (lanes 2, 3, 7, and 8) or GDP alone (lanes 4, 5, 9, and 10) and analyzed as in (A). WB, Western blotting. Lanes 1 and 6 were loaded with 0.1 μg of Gαsor Gαi1. (C) RGS-PX1 (400 nM, circles) but not RGS4 (400 nM, triangles) increases the rate of GTP hydrolysis of Gαs over Gαs alone (squares). (D) RGS4 (250 nM, triangles), but not RGS-PX1 (800 nM, circles), increases the rate of GTP hydrolysis of Gαi1over Gαi1 alone (squares). The hydrolysis reaction contained 80 nM Gαs (C) or 60 nM Gαi1 (D) and was performed on ice. Data shown are representative of at least three independent experiments.

To test whether RGS-PX1 can function as a GAP for Gαs, single turnover GTPase assays were performed (11, 12). RGS-PX1 accelerated the catalytic rate of GTP hydrolysis of Gαs at least 20-fold over that of Gαs alone, whereas RGS4 had no effect (Fig. 2C). In the absence of RGS-PX1 or in the presence of RGS4, the half life (t 1/2) of GTP hydrolysis by Gαs was ∼5 min, whereas in the presence of RGS-PX1 it was <15 s, the earliest time point (Fig. 2D). RGS-PX1 had no effect on Gαi1, whereas RGS4 markedly accelerated the GTP hydrolysis of Gαi1 (Fig. 2D). These results demonstrate that RGS-PX1 is a GAP for Gαs.

To investigate the effects of RGS-PX1 on Gαs-mediated signaling, cAMP production was measured in transfected HEK293 cells expressing the β2-adrenergic receptor (β2AR) (13). Treatment of cells with the β2AR agonist isoproterenol increased the cellular cAMP level. This increase was reduced (∼70%) in cells expressing the RGS domain of RGS-PX1 (Fig. 3A). Additionally, incubation of neonatal rat cardiac membranes with the RGS domain of RGS-PX1 (13) reduced isoproterenol-stimulated adenylyl cyclase (AC) activity by ∼65% (Fig. 3B). No effect was seen on forskolin-induced cAMP production or on AC activation, which does not require Gαs. These data are consistent with the conclusion that RGS-PX1 attenuates Gαs-mediated signaling by functioning as a GAP.

Figure 3

RGS-PX1 attenuates Gαs-mediated signaling. (A) RGS-PX1 inhibits isoproterenol (Iso)– but not forskolin (Fsk)–induced cAMP production. HEK293 cells were transfected with the RGS domain of RGS-PX1 or with empty vector together with β2AR. (B) RGS-PX1 inhibits Iso- but not Fsk-stimulated AC activity in neonatal rat cardiac myocyte membranes. Membranes were incubated for 5 min on ice with 50 nM RGS domain of RGS-PX1 or with vehicle before AC activity was measured. cAMP production over basal production (no agonist) is shown. Data are expressed as the mean ± SEM of three experiments. **P < 0.005 by paired t test.

RGS-PX1 also contains a PX domain followed by a coiled-coil region (Fig. 1A) often found in SNX proteins, which are involved in vesicular trafficking (14–17). To determine whether RGS-PX1 can function as a SNX, we examined the effects of overexpressing a fusion protein containing green fluorescence protein (GFP) and RGS-PX1 (GFP-RGS-PX1) on EGF receptor (EGFR) trafficking (18). Upon ligand stimulation, EGFR is rapidly internalized, sorted in endosomes, and targeted to lysosomes for degradation. Ligand-dependent EGFR degradation was delayed in transfected HEK293 cells expressing GFP-RGS-PX1 (Fig. 4A), which suggests inhibition of lysosomal targeting and/or degradation of EGFR. Because EGFR trafficking to endosomes is important for regulating receptor signaling, we assessed whether expression of GFP-RGS-PX1 influences EGF-dependent mitogen-activated protein kinase (MAPK) activation (18). In controls, phosphorylation of ERK1 and ERK2 increased 5 min after EGF addition and decreased progressively from 30 to 60 min (Fig. 4B), in keeping with the observed rapid degradation of active EGFR (Fig. 4A). In contrast, cells transfected with GFP-RGS-PX1 showed sustained activation of ERK1 and ERK2 at 30 and 60 min (Fig. 4B). This prolonged EGF signaling correlates well with the delay in EGFR degradation, supporting a regulatory role for RGS-PX1 in EGFR trafficking and signaling.

Figure 4

RGS-PX1 is a functional sorting nexin. (A) Expression of GFP-RGS-PX1 causes a delay in the degradation of EGFR in HEK293 cells. Cells transfected with GFP-RGS-PX1 or with empty GFP vector were treated with EGF for the indicated times, followed by immunoblotting with antibodies against EGFR or actin. Data shown are representive of at least three independent experiments. (B) Expression of GFP-RGS-PX1 in HEK293 cells inhibits down-regulation of EGF-dependent MAPK activation. Cells transfected with GFP-RGS-PX1 or with empty GFP vector were treated with EGF for the indicated times, and activation of MAPK (phospho-ERK1/2) was assessed by immunoblotting. Data shown are representive of at least three independent experiments. (C) The PX domain of RGS-PX1 binds strongly to PtdIns(3)P and PtdIns(5)P and weakly to PtdIns(3,5)P2 and PtdIns(4)P. A GST fusion protein containing the PX domain of RGS-PX1 was used in a protein-lipid overlay. Bound proteins were detected by immunoblotting with antibody to GST. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. (D) GFP-RGS-PX1 colocalizes with EEA1 in Cos7 cells. Right panel, merged images. Yellow indicates overlap.

The PX domain has recently been shown to be a phosphoinositide-binding domain involved in membrane targeting (17, 19); and in the case of SNX3, the interaction between the PX domain and phosphoinositides is important for its function (17). To examine its phosphoinositide-binding properties, we performed a protein-lipid binding assay with a GST fusion protein containing the PX domain of RGS-PX1 (18) and found that it bound strongly to PtdIns(3)P and PtdIns(5)P and weakly to PtdIns(3,5)P2 and PtdIns(4)P, but not to other phosphoinositides or other phospholipids (Fig. 4C).

GFP-RGS-PX1 also colocalized with the early endosome marker EEA1 in Cos-7 cells (18) (Fig. 4D). PtdIns(3)P is highly enriched in early endosomes (17), whereas the subcellular localization of PtdIn(5)P has not been established. These results suggest that RGS-PX1 is a functional SNX that could regulate EGFR trafficking and signaling, probably through the interaction of its PX domain with phosphoinositides in endosomes.

As a GAP for Gαs, RGS-PX1 likely contributes to the regulation of cellular responses mediated by Gαs. Gαs stimulates adenylyl cyclases, l-type calcium channels, and Src kinase; inhibits cardiac sodium channels; and is involved in many cellular responses, including cell growth, differentiation and proliferation, membrane trafficking, cardiac contraction and relaxation, hormone secretion, and learning and memory (1, 2, 20). The existence of RGS-PX1 as a GAP for Gαs may explain the difference between the slow rate of GTP hydrolysis of Gαs in vitro and its rapid rate of deactivation under certain physiological conditions (21). The specificity of the interaction between Gα subunits and RGS proteins is very likely determined by the primary sequences of RGS domains and Gα proteins. It has been suggested that the major barrier to Gαs interaction with other RGS proteins is Asp229 of Gαs (3). Substitution of this residue with the corresponding Ser206of Gαi enabled the mutated Gαs to bind to RGS4 and RGS16. It is known from the crystal structure of the Gαi1-RGS4 complex that Ser206 of Gαi1 interacts with Glu126 and Asn128 of RGS4 (3). In RGS-PX1, Arg457 and Thr459 occupy these positions. These two nonconserved amino acid substitutions suggest that Arg457 and Thr459 in RGS-PX1 might contribute to the specificity of Gαs-RGS interaction.

A unique feature of RGS-PX1 is its dual role as both a GAP and a SNX. Whereas the RGS domain of RGS-PX1 is responsible for its GAP activity for Gαs, the PX domain and the COOH-terminal coiled-coil region, which are shared with other SNX proteins, are most likely responsible for its SNX function. The presence of both activities in one molecule makes RGS-PX1 an ideal bridge between G protein signaling and regulation of vesicular trafficking.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: mfarquhar{at}


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