Identification of Rab11a as a Gβ1γ2-interacting Protein Influencing Gβ1γ2 Cellular Distribution
Gβγ can interact with Gα as well as with its diverse effectors at the plasma membrane. It has been recently documented that Gβγ relocates from the plasma membrane to internal membranes upon GPCR activation, via an unknown molecular mechanism (Hynes et al., 2004
; Saini et al., 2007
). To identify novel Gβγ-interacting proteins that might be involved in the intracellular trafficking of Gβγ, we screened a human brain cDNA library by using Gβ1
as the bait in a yeast two-hybrid system. Among the transformants obtained under high-stringency conditions (−Ade/−His/−Leu/−Trp), we identified a clone corresponding to Rab11a. Rab11a has interacted specifically with Gβ1
in the yeast two-hybrid assay (A), as judged by the ability of the pair to support growth under highly stringent conditions and to promote the expression of α-galactosidase from an integrated reporter system. Association of Rab11a and Gβγ was also demonstrable in mammalian cells in pull-down experiments by using His-tagged Gβ1
and a GFP-Rab11a construct both expressed in HEK-293T cells grown in DMEM supplemented with 10% fetal bovine serum (B). To investigate whether the interaction between Gβ1
and Rab11a occurs between endogenously expressed proteins, Rab11a immunoprecipitates were tested for the presence of Gβγ proteins from nontransfected cells grown in DMEM supplemented with 10% fetal bovine serum. C shows that endogenous Gβγ was found in association with endogenous Rab11a.
Figure 1. Gβ1γ2 interacts with Rab11a and they colocalize in HEK-293T cells. (A) A full-length clone of Rab11a was identified as a Gβ1 subunit interactor by yeast two-hybrid. The specificity of the interaction between Rab11a and Gβ (more ...)
To determine the influence of Rab11a on the subcellular distribution of Gβ1
heterodimers were transiently transfected into HEK-293T cells in the presence or absence of EGFP-Rab11a, and the cells were examined by confocal microscopy. When transfected in the absence of Rab11a, Gβ1
was predominantly found at the plasma membrane (D, i), consistent with a previous report (Evanko et al., 2001
). However, expression of EGFP-Rab11a has changed the distribution of Gβ1
that now mostly colocalized with Rab11a at endocytic structures (D, ii–iv).
To investigate whether the interaction between endogenous Gβ1γ2 and Rab11a can be influenced by the activation of LPA receptors, Rab11a was immunoprecipitated from nonstimulated or LPA-stimulated serum-starved cells. A shows that activation of LPA receptors promoted the association of endogenous Gβγ and Rab11a in a time-dependent manner. In addition, we also demonstrated the presence of AKT in the Gβ1γ2:Rab11a complex (A). To determine the effect of GPCR stimulation on the subcellular distribution of endogenous Gβ1γ2, serum-starved HEK-293T cells were stimulated with LPA for the indicated times, and the localization of Gβ and Rab11 was examined by immunofluorescence by using confocal microscopy. In nonstimulated cells, Gβγ was predominantly found at the plasma membrane and in dispersed intracellular vesicles, whereas Rab11 was found in vesicles (B, left, NS). However, after LPA stimulation, both proteins were mostly detected in colocalization at endocytic structures that concentrated in the juxtanuclear area (B, left). Additionally, we analyzed the colocalization area between Gβ1γ2 and Rab11a; in nonstimulated cells, 9.05 ± 2.0% of the proteins colocalized, whereas in LPA-stimulated cells the overlap was extensive, 40.61 ± 2.68% for 5 min LPA-stimulated cells and 42.47 ± 2.59% for 15-min LPA-stimulated cells (B, right). Collectively, these results indicated that the interaction between Gβ1γ2 and Rab11a revealed by the yeast two-hybrid screen and detected by pull-down experiments of transfected epitope-tagged proteins, also occurs between endogenous Gβ1γ2 and Rab11a proteins and is promoted by the activation of LPA receptors. They also suggested that Rab11a might link Gβγ to endosomal compartments regulating the cellular distribution of the heterodimer.
Figure 2. Agonist-dependent interaction between endogenous Gβγ and Rab11 and effect on their subcellular localization. Rab11 was immunoprecipitated from serum-starved HEK-293T cells grown to 80% confluence and stimulated with 10 μM LPA or (more ...)
Constitutively Active Rab11a Mutant Promotes the Accumulation of Gβ1γ2 Associated with Large Endosomes
To investigate whether the activation status of Rab11a may affect its interaction with Gβ1γ2, we determined the interaction of His6-Gβ1γ2 with either wild type, constitutively active, or dominant-negative EGFP-tagged Rab11a mutants (Rab11a WT, Q70L, or S25N, respectively) by pull-down assays using lysates from HEK-293T cells transfected with increasing amounts of each Rab11a (A). Although all three forms of Rab11 showed interaction with Gβ1γ2, the constitutively active Rab11a Q70L mutant clearly had a higher affinity for Gβ1γ2 as shown by its ability to interact more efficiently than wild-type Rab11a or Rab11a S25N in cells transfected with reduced amounts of the construct (A). To investigate whether Gβ1γ2 could affect the catalytic status of Rab11a, an in vitro GTPase assay was used to determine the GTPase activity of recombinant His6-Rab11a in the presence or absence of His6-Gβ1γ2 isolated from HEK-293T cells. B shows that Gβ1γ2 did not modify the GTPase activity of Rab11. We then examined the cellular distribution of Gβ1γ2 in the presence or absence of Rab11a mutants (C). The constitutively active Rab11a mutant (Rab11Q70L) caused a prominent accumulation of Gβ1γ2 in large vesicles, in which this Rab11a mutant was also detected (C, i–iii). In contrast, the dominant-negative Rab11a mutant (Rab11S25N) prevented the formation of large vesicles and did not change the cellular distribution of Gβ1γ2, which remained at the plasma membrane (C, iv–vi). These results suggested that Gβ1γ2 trafficking toward endocytic compartments depends on the interaction of this heterodimer with active Rab11a.
Figure 3. Gβ1γ2 shows a higher affinity for a constitutively active Rab11a Q70L mutant but does not affect the GTPase activity of recombinant Rab11a. HEK-293T cells were transfected with His6-Gβ1γ2 and the indicated amounts of GFP-tagged (more ...)
Effect of PhLP1 and Gαi2 on the Interaction between Gβ1γ2 and Rab11a
Next, we wanted to determine whether Gβ1
interaction with Rab11a required the availability of Gβ1
as a free heterodimer, exposing its Gα-interacting interface. For this, we tested whether the interaction between Gβ1
and Rab11a could be competed with Gαi2
or PhLP1, a ubiquitous protein known to interact with Gβγ at the Gα-interacting interface (Gaudet et al., 1996
). In these experiments, either Flag-tagged PhLP1 or Gαi2
were transiently transfected into HEK-293T cells together with His6-Gβ1
and EGFP-Rab11a, and the interaction between His6-Gβ1
and EGFP-Rab11a was analyzed by pull-down assays. Overexpression of PhLP1 completely blocked the interaction between Gβ1
and Rab11a (A, top and bottom). Similarly, overexpression of Gαi2
greatly reduced the interaction between Rab11a and Gβ1
(B, top and bottom), indicating that interaction between Gβ1
and Rab11a occurs when Gβ1
dissociates from Gα, suggesting that it might be part of the signal transduction process initiated with the release of Gβ1
upon GPCR activation.
Figure 4. Expression of Gαi2 or PhLP1 prevent the interaction between Gβ1γ2 and Rab11a. HEK-293T were transfected with His6-Gβ1γ2 and GFP-Rab11a either in the presence or absence of Flag-tagged PhLP1 or Gαi2. The (more ...)
Effect of GPCR Stimulation on Gβ1γ2 Signaling Associated with Rab11a-positive Endosomes
Next, we investigated the effect of G protein-coupled receptor stimulation on the subcellular distribution of Gβ1
and Rab11a. HEK-293T cells were treated with LPA for various times, and then endosomes were isolated on a flotation sucrose gradient (Kobayashi et al., 2002
). The identity of the endosomes was verified by their immunoreactivity to Rab5 or EEA1, and Rab11 recognized as early and recycling endosomal markers, respectively (A); no Rab7 immunoreactivity was detected in this preparation (D). The activation of LPA receptors promoted the association of Gβ1
toward early and recycling endosomes as demonstrated by a significant increase in the presence of Gβ in endosomes obtained from LPA stimulated cells, which increased in a time-dependent manner, being above basal levels at 15 min, reaching a maximum between 30 and 60 min, and starting to decrease at 75 min after LPA stimulation (, A and B).
Figure 5. LPA stimulation increases Gβ1γ2 association to Rab11a-positive endosomes. HEK-293T cells grown to 80% confluence were serum starved for 15 h followed by stimulation with 10 μM LPA for the indicated times. Cells were fractionated (more ...)
Endosomal trafficking of Gβ1
could contribute to the compartmentalization of signaling cascades by the association of effector proteins such as PI3-kinase in endosomal compartments (Seabra and Wasmeier, 2004
). Therefore, we wanted to know whether LPA stimulation promoted the association of PI3-kinase to early endosomes and whether Gβ1
had any influence on this process. To assess this question, the preparation of early endosomes obtained from LPA-stimulated cells was analyzed by Western blotting using antibodies against the catalytic subunit of PI3-kinase-γ, the isoform known to be a target of Gβγ subunits (Stephens et al., 1994
). As shown in A, recruitment of endogenous PI3-kinase-γ to endosomes was induced by LPA in a time-dependent manner, the kinase being present between 15 and 60 min but no longer detectable after 75 min of stimulation. To assess the consequences of the association of PI3-kinase-γ to endosomes, we also examined the recruitment and activation of one of its more relevant downstream effectors, AKT, to this compartment. Unexpectedly, a fraction of AKT was already detected in endosomes obtained from nonstimulated cells and its presence was further increased after LPA stimulation (A). Interestingly, the activation of AKT, determined by monitoring its phosphorylation state with antibodies recognizing AKT phosphorylated at Ser-473, was also increased after 15 min and remained so until 75 min of LPA stimulation (, A and C), following a pattern similar to the association of Gβγ to this endosomal fraction (, A and B). Pretreatment of cells with Wm prevented the association of PI3-kinase-γ and the activation of AKT in response to LPA; however, a fraction of AKT was found associated with endosomes regardless of the inhibition of PI3-kinases with wortmannin (E). A further evidence that wortmannin does not globally disrupt the signaling pathways induced by LPA was the activation of extracellular signal-regulated kinase (ERK) 1/2 detected in whole cell lysates from Wm-pretreated LPA stimulated HEK-293T cells, in which the activation of AKT was also affected by Wm (F).
To test the question of whether the interaction between Gβ1γ2 and Rab11a was necessary for the association of PI3-kinase-γ to endosomes and the ensuing phosphorylation of AKT, endosomal fractions were obtained from LPA-stimulated HEK-293T cells transfected with dominant-negative Rab11a mutant (Rab11a S25N). In agreement with the results obtained by confocal microscopy (C, iv–vi), Gβ1γ2 was barely detectable in endosomes obtained from Rab11a S25N-transfected cells (A) and PI3-kinase-γ was practically absent in these endosomes, being detected only at one time point (1 h) (A). A fraction of AKT, was still associated with early endosomes isolated from Rab11a S25N-transfected cells, but this fraction corresponded to the nonphosphorylated inactive form of the kinase (A). To investigate whether the activation status of Rab11a may affect agonist-dependent Gβγ trafficking to endosomes and the relevant downstream events, we determined the presence of Gβ and its downstream targets in endosomal fractions obtained from control or LPA-stimulated HEK-293T cells expressing either wild-type Rab11a, constitutively active Rab11a mutant (Rab11a Q70L-EGFP), or a dominant-negative Rab11a mutant (Rab11a S25N-EGFP). B shows that both wild-type Rab11a and Rab11a Q70L increased the presence of Gβ in endosomes obtained from LPA-stimulated cells. Yet, dominant-negative Rab11a S25N mutant reduced the presence of Gβ in endosomes. Moreover, endosomal AKT activation was blocked in samples obtained from Rab11a S25N-transfected cells, as observed in A. In addition, we detected a slight increase in the association of PI3-kinase-γ to endosomes from Rab11a Q70L-transfected cells stimulated with LPA (B). Evidence that demonstrated that Rab11 S25N mutant does not globally disrupt the signaling pathways induced by LPA was the activation of ERK 1/2 and AKT detected in whole cell lysates from LPA-stimulated HEK-293T cells expressing Rab11 S25N mutant (Supplemental Figure S1). To investigate whether inhibition of Gi-dependent pathways affects LPA-stimulated trafficking of Gβγ to endosomal compartments and recruitment and activation of AKT, HEK-293T cells were treated with PTX and the effect of LPA on the association of Gβ and AKT to endosomal compartments was determined. C shows that PTX reduced the association of Gβ to endosomes as well as the presence and activation of AKT.
Figure 6. Expression of dominant-negative Rab11a, Gαi2, PhLP1, or PTX treatment attenuated the effect of LPA on endosomal recruitment of Gβγ, PI3-kinase-γ, and phosphorylation of AKT. (A) HEK-293T cells transiently transfected with (more ...)
To examine whether LPA-induced trafficking of free Gβγ is important for the activation of AKT at endosomes, we tested the effect of PhLP1 or Gαi2, whose overexpression would decrease the availability of free Gβγ. For these experiments, we obtained endosomes from control or LPA-stimulated HEK-293T cells transfected with either Flag-tagged PhdLP1 or Gαi2. As shown in D, both PhLP1 and Gαi2 attenuated the agonist-dependent appearance of Gβγ in early endosomes and the association of PI3-kinase-γ to this fraction. Moreover, they interfered with the association of AKT to endosomes (just the agonist-dependent fraction) and prevented its phosphorylation. To confirm that LPA has a dual effect on endosomal AKT (increases its association and promotes its phosphorylation) endosomal samples from LPA-stimulated cells were diluted to obtain a similar amount of total AKT in fractions from nonstimulated cells and LPA-stimulated cells, in these conditions, it was clear that the increase in the phosphorylation detected in endosomal AKT in LPA-stimulated cells was due to phosphorylation of the kinase and that an increase on its association to the endosomal fraction was also occurring (D, bottom, early endosomes). Together, these results suggest that upon GPCR activation, Gβγ dissociated from Gα gets engaged in a trafficking path to early and perhaps recycling endosomes mediated by a direct interaction with Rab11a. This interaction is necessary to assemble and activate a PI3K/AKT signaling complex associated to Rab11-positive endosomes. These data establish a new paradigm of endosomal activation of a Gβγ-regulated PI3-kinase cascade.
Trafficking of Gβ1γ2 to Rab11a Endosomes Leads to Cell Survival and Proliferation
To explore the functional consequences of Gβγ trafficking to Rab11a-positive endosomes, we evaluated agonist-dependent survival and proliferation of HEK-293T cells transiently transfected with wild-type Rab11a, constitutively active Rab11a mutant (EGFP-Rab11a Q70L) or a dominant-negative Rab11a mutant (EGFP-Rab11a S25N). As shown in A, LPA protected against apoptosis in cells expressing either wild-type Rab11a or Rab11a Q70L; in the Rab11a Q70L-expressing cells an antiapoptotic effect was detected event in the absence of LPA stimulation. However, overexpression of dominant-negative Rab11a S25N mutant blocked the LPA-dependent survival effect, resulting in apoptosis. Additionally, overexpression of Gαi2 or treatment with PTX also interfered with the survival effect promoted by LPA, as shown in A.
Figure 7. Expression of dominant-negative Rab11a, Gαi2, or PTX treatment prevents the antiapoptotic and proliferative effect of LPA. (A) Antiapoptotic effect of 10 μM LPA was assessed by Annexin V binding in HEK-293T cells transfected with wild-type, (more ...)
To examine the effect of Rab11a on LPA-dependent cell proliferation, we transiently transfected HEK-293T cells with wild-type Rab11a, constitutively active Rab11a mutant (EGFP-Rab11a Q70L) or a dominant-negative Rab11a mutant (EGFP-Rab11a S25N). As shown in B, the proliferative effect of LPA was not affected by wild-type or Q70L mutant Rab11a. However, dominant-negative Rab11a S25N mutant blocked the proliferative effect of LPA. Similarly, the proliferative effect of LPA was blocked by overexpression of Gαi2 or PTX treatment, two strategies known to interfere with Gβγ signaling (B). Together, our results indicate that agonist-dependent trafficking of Gβγ to Rab11a endosomes is linked to cell survival and proliferation.
To evaluate the possibility that AKT effectors can be recruited to endosomes and activated as a consequence of Gβγ trafficking and AKT stimulation, we explored the presence of GSK3-β, FKHR-1, and Bad proteins, known substrates of AKT, on endosomes from LPA- and PTX-pretreated cells. As shown in C, we detected that LPA promoted the association of GSK3-β to the endosomal fraction; however, we could not reveal its phosphorylation and the treatment with PTX did not show a significant effect on LPA-stimulated association of GSK3-β to endosomes. The lack of phosphorylation of GSK3-β associated to the endosomal fraction makes it difficult to connect its recruitment to the activation of AKT, which could be detected in total cell lysates and where the treatment with PTX attenuated the phosphorylation of this AKT substrate in LPA-stimulated cells (C). In contrast, we could not detect the presence of FKHR-1 or Bad proteins at the endosomal fraction (data not shown).
To further examine the functional role of endogenous Rab11 in LPA-dependent cell survival, we assessed the effect of short hairpin RNA-induced Rab11 knockdown on the antiapoptotic effect of LPA. As shown in D, Rab11 shRNA blocked the LPA-dependent survival effect, resulting in apoptosis. Additionally, we demonstrated that Rab11 shRNA does not globally disrupt LPA-dependent signaling pathways as evidenced by the ability of LPA to promote the activation of ERK 1/2 and AKT as detected in total lysates from cells transiently transfected with shRNA specific for Rab11 (E). Together, our results indicate that Gβγ trafficking to endosomes, in association to Rab11, is linked to cell survival.