Many GPCRs (G-protein-coupled receptors) can activate RTKs (receptor tyrosine kinases) in the absence of RTK ligands, a phenomenon called transactivation. However, the underlying molecular mechanisms remain undefined. In the present study we investigate the molecular basis of GABAB (γ-aminobutyric acid B) receptor-mediated transactivation of IGF-1R (insulin-like growth factor type I receptor) in primary neurons. We take a chemical biology approach by developing an activity-based probe targeting the GABAB receptor. This probe enables us first to lock the GABAB receptor in an inactive state and then activate it with a positive allosteric modulator, thereby permitting monitoring of the dynamic of the protein complex associated with IGF-1R transactivation. We find that activation of the GABAB receptor induces a dynamic assembly and disassembly of a protein complex, including both receptors and their downstream effectors. FAK (focal adhesion kinase), a non-RTK, plays a key role in co-ordinating this dynamic process. Importantly, this dynamic of the GABAB receptor-associated complex is critical for transactivation and transactivation-dependent neuronal survival. The present study has identified an important mechanism underlying GPCR transactivation of RTKs, which was enabled by a new chemical biology tool generally applicable for dissecting GPCR signalling.

INTRODUCTION

GPCRs (G-protein-coupled receptors) represent the largest family of membrane receptors mediating nearly all of the known cellular responses to various ligands, including hormones, neurotransmitters and sensory inputs. Moreover, GPCRs are excellent drug targets [1]. RTKs (receptor tyrosine kinases), another major family of membrane receptors that include the majority of receptors for growth factors, mediate cell proliferation, differentiation, motility and survival [2]. Each cell usually expresses both GPCRs and RTKs, allowing the cell to respond to diverse external signals. Interestingly, it has been shown previously that GPCRs can activate RTKs [e.g. IGF-1R (insulin-like growth factor type I receptor), EGFR (epidermal growth factor receptor), PDGFR (platelet-derived growth factor receptor) and FGFR (fibroblast growth factor receptor)] in the absence of RTK ligands, a phenomenon called transactivation [38]. In other words, RTK machinery can be ‘hijacked’ to transduce signals from GPCRs. By doing so, numerous GPCR ligands, particularly neurotransmitters and neuropeptides which are thought to impinge solely on canonical GPCR signalling, probably exert much of their effects through RTK pathways. This provides an efficient means for cells to mediate the diverse effects of GPCR ligands, particularly those long-term effects involving cell proliferation, differentiation and survival, which cannot be readily explained by canonical GPCR signalling mechanisms. Transactivation appears to involve both phosphorylation of RTKs and recruitment of downstream signalling molecules such as Src family kinases (Src) [9,10]. Nevertheless, whether GPCRs and RTKs can physically associate within the same complexes remains controversial [3]. It is also not known whether such complexes, if present, contribute to transactivation. Consequently, despite their prevalence and importance, the molecular and structural bases underlying transactivation remain poorly defined.

GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in the vertebrate central nervous system and mediates slow prolonged synaptic inhibition through the GABAB receptor, which is involved in numerous types of nociception, cognitive impairment, epilepsy, spasticity and drug addiction [11]. The GABAB receptor belongs to the class C GPCRs and is a heterodimer composed of two subunits, GB1 and GB2 [1214]. The GB1 subunit contains the ligand-binding site that binds to both agonists (e.g. GABA and baclofen) and competitive antagonists (e.g. CGP64213 and CGP54626), whereas the GB2 subunit is responsible for Gi/o-protein activation [15]. CGP7930, a novel PAM (positive allosteric modulator), has been demonstrated to activate the GABAB receptor directly through the GB2 subunit [16,17]. IGF-1 (insulin-like growth factor 1), which is essential for normal brain development and promotes neuronal survival, triggers autophosphorylation of its cognate tyrosine kinase receptor IGF-1R, and activates Src and Akt for neuroprotection [18]. Recently, we have demonstrated that activation of the GABAB receptor by either agonist, such as GABA and baclofen, or a PAM such as CGP7930, induces transactivation of IGF-1R for neuroprotection [19]. We showed that the GABAB receptor transactivates IGF-1R for neuroprotection, and our data also suggested that IGF-1R and the GABAB receptor might physically associate [19]. However, it is unclear whether these two receptors associate with the same complex, and if so, whether other signalling partners in the GABAB receptor and IGF-1R pathways also associate within the same protein complex. It also remains elusive whether such a protein complex, if present, is important for transactivation, and if so, how protein–protein interactions within the complex contribute to transactivation. The difficulty mainly results from the lack of an effective tool that allows us to enrich and isolate protein complexes while preserving the function of the complexes, since several features of GPCRs pose a challenge to studying the mechanisms underlying GPCR-mediated transactivation of RTKs [20]. First, GPCRs are usually expressed at very low levels [21]. Secondly, GPCRs often display poor immmunogenicity, which makes it difficult to develop high-affinity antibodies to enrich these receptors for biochemical characterization [22]. Thirdly, it is difficult to solubilize GPCRs in a functionally active form [22,23].

ABPP (activity-based protein profiling) is a valuable chemical strategy that uses ABPs (activity-based probes) to profile the functional states of proteins in complex proteomes [24]. ABPs such as [125I]CGP64213, [125I]CGP84963 and [125I]CGP71872 [25,26] have aided the identification of membrane receptors such as GABAB receptors. However extra protection is needed during the synthesis and application of these radioactive ligands with short half-lives, which hinder them from being widely employed. Furthermore, ABPs have not yet been applied to study signal transduction events initiated by membrane receptors. We have previously reported a non-radioactive photoaffinity and fluorescent GABAB receptor ligand, Probe 1 [27]. Although this probe can specifically label the functional GABAB receptor in living cells, it does not permit affinity purification of the receptor or its associated signalling proteins, and thus cannot be used to study signal transduction events associated with transactivation. In the present paper, we report another GABAB receptor-oriented non-radioactive photoaffinity probe utilizing biotin as the tag. Because of its small size compared with antibodies, this probe is able to preserve the functional activity of GPCRs during solublization. As it allows for cross-linking after receptor binding, this ABP shows a very high affinity for the GABAB receptor, thus permitting enrichment of this GPCR and its associated signalling proteins to a level that is unattainable by conventional antibodies. Using this ABP, we dissected the dynamic events accompanying IGF-1R transactivation by the GABAB receptor. The results of the present study show that a dynamic assembly and disassembly of a protein complex, including both receptors and their effectors, is critical for IGF-1R transactivation by the GABAB receptor. These results demonstrate, to our knowledge for the first time, that ABPs represent a powerful means for characterizing GPCR signalling and also reveal a critical mechanism contributing to the transactivation of RTKs by GPCRs.

EXPERIMENTAL

Synthesis of the ABP

Compound 1 was synthesized as reported previously [27]. The chemical synthesis and characterization of Compound 2 (biotin N-hydroxysuccinimidyl ester), Compound 3 [N-(prop-2-ynyl)-biotin amide] and CGP64213B (Figure 1B) are described in the Supplementary Online Data at http://www.BiochemJ.org/bj/443/bj4430627add.htm.

Strategy and design of a chemical biology approach

Figure 1
Strategy and design of a chemical biology approach

(A) A proof-of-principle experiment testing the proposed strategy. CGNs were treated with CGP54626 (10 μM) or PBS for 1 h and then incubated with baclofen (100 μM) or CGP7930 (100 μM) for various times as indicated. Cell lysate was subjected to SDS/PAGE and detected with anti-phospho-Akt (pAkt) or anti-Akt antibody. Shown are representative blots from three independent experiments. (B) Structure and synthesis of the biotinylated photoaffinity probe targeting the GABAB receptor (CGP64213B).

Figure 1
Strategy and design of a chemical biology approach

(A) A proof-of-principle experiment testing the proposed strategy. CGNs were treated with CGP54626 (10 μM) or PBS for 1 h and then incubated with baclofen (100 μM) or CGP7930 (100 μM) for various times as indicated. Cell lysate was subjected to SDS/PAGE and detected with anti-phospho-Akt (pAkt) or anti-Akt antibody. Shown are representative blots from three independent experiments. (B) Structure and synthesis of the biotinylated photoaffinity probe targeting the GABAB receptor (CGP64213B).

Photoaffinity labelling of the functional GABAB receptor with CGP64213B in living cells

Cell cultures from transfected HEK (human embryonic kidney)-293 cells, transfected CHO (Chinese-hamster ovary) cells, transfected MEF (mouse embryonic fibroblast) cells or rat primary CGNs (cerebellar granule neurons) were washed once with Ca2+-free HBS (Hepes-buffered solution) containing 10 mM Hepes (pH 7.4), 140 mM NaCl, 4 mM KCl, 2 mM MgSO4 and 1 mM KH2PO4, pre-incubated at 37°C with CGP64213B (2 μM) for 2 h. After UV (365 nm) irradiation for 1 h, the labelled cells were washed twice with HBS before performing further experiments.

High-affinity pull-down enrichment and Western blot analysis

Ice-cold 1× cell lysis buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM 2-glycerophosphate, 1 mM Na3VO4 and 1 μg/ml leupeptin] was added to the CGP64213B-labelled cells. The cells were collected into a microtube and ultracentrifuged at 12000 rev./min for 15 min at 4°C using an Eppendorf 5804 centrifuge with an F-45-30-11 fixed-angle rotor. The supernatant fraction was then transferred to a new tube. Protein concentrations of total cell lysates were determined using the Bradford reagent (Bio-Rad laboratories). To prepare the total cell lysate sample, equal amounts of protein (20 μg) from cell lysates were transferred to another tube and mixed with 4× SDS sample loading buffer. The cell lysates were then mixed with the streptavidin-coated beads (GE Healthcare) for 4 h with gentle rolling. After three washes with ice-cold 1× cell lysis buffer for immunoprecipitation, the pellets from pull-down enrichment were resuspended in 4× SDS sample loading buffer. Samples from pull-down enrichment and total cell lysates were boiled and subjected to SDS/PAGE (5–12% gel gradient), electroblotting and autoradiography. The density of immunoreactive bands was measured using ImageJ (http://rsbweb.nih.gov/ij/), and all bands were normalized to the control values.

Statistical analysis

Data are presented as the means±S.E.M. for at least three independent experiments. Statistical analysis was performed by ANOVA and P<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Development of a strategy to probe the individual functional states of receptors and signalling molecules during transactivation

In addition to the aforementioned technical difficulties, another intrinsic property of GPCRs poses further challenges to studying the mechanisms underlying GPCR-mediated transactivation of RTKs. Specifically, many GPCRs show constitutive activity in the absence of ligand, even in native systems [2830]. The presence of constitutive activity states of these receptors makes it very difficult to isolate the individual steps associated with their transactivation of RTKs. Current models of activation of GPCRs suggest that inverse agonists preferentially bind to the inactive form of the receptor, whereas the active conformation of the receptor prefers agonists [31]. In addition, it is known that a PAM can switch the receptor from an inactive state to an active state in the presence of an inverse agonist. On the basis of these findings, we sought to design a strategy to ‘synchronize’ all GPCRs in an inactive state by blocking the receptor with an inverse agonist and then activate them through a PAM to initiate transactivation. In so doing, we would be able to monitor the individual functional states of the receptors and signalling molecules during transactivation.

We first performed a proof-of-principle experiment by using rat CGNs as a model to first ‘synchronize’ the GABAB receptor in an inactive state with an inverse agonist CGP54626 and then activate them through a positive allosteric modulator CGP7930. Indeed, these neurons express both functional GABAB receptors and IGF-1Rs [17,18]. Furthermore, the GABAB receptor exhibits ligand-independent (constitutive) activity and a high-affinity antagonist CGP54623 acts as a potent inverse agonist, inhibiting the ligand-independent signalling [32,33]. We have shown previously that CGP7930, by acting directly in the GB2 transmembrane domain, could activate the GABAB receptor even when the ligand-binding domain is locked in an inactive state using either an inverse agonist or through mutation [16,35]. In the present study, we pre-incubated CGNs with CGP54626 to clamp the GABAB receptor in an inactive conformation by repressing its constitutive activity, and then stimulated the receptor with CGP7930. Pretreating CGNs with CGP54626 (10 μM) effectively inhibited Akt phosphorylation induced by the GABAB receptor agonist baclofen, indicating that CGP54626 can keep the GABAB receptor at a relatively inactive state in neurons, even in the presence of agonists (Figure 1A). Importantly, application of CGP7930 to CGP54626-pretreated CGNs could efficiently relieve the inhibition of Akt phosphorylation by CGP54626, leading to robust phosphorylation of Akt (Figure 1A). As Akt resides downstream of IGF-1R [19], these results suggest that the inverse agonist-bound GABAB receptor can be activated by PAM, leading to transactivation of IGF-1R.

We then sought to monitor the dynamics of the protein–protein interactions associated with transactivation. To do so, we set out to determine whether the GABAB receptor, IGF-1R and their downstream signalling molecules form a protein complex and, if so, how proteins in the complex behave during transactivation. One strategy was to trigger transactivation by treating the inverse agonist-bound GABAB receptor complex with CGP7930, a PAM, and then monitor the functional states of each molecule in the complex. This strategy required designing ABPs that could be used to isolate signalling complexes through affinity purification. Such a strategy would provide an opportunity to monitor the individual functional states of receptors and signalling molecules in the complex during transactivation.

Design, synthesis and validation of ABPs targeting the GABAB receptor

We designed and synthesized a biotinylated photoaffinity probe based on the known GABAB receptor antagonist CGP64213, named CGP64213B (Figure 1B). In its structure template: (i) the GABAB receptor-binding group is the main structural moiety for receptor binding; (ii) the photolabile diazirine group in the phenyl ring functions as a photocross-linking group to generate a covalent irreversible linkage between the probe and the labelled receptor after UV irradiation; and (iii) the biotin reporter tag via a PEG [poly(ethylene glycol)]-containing linker allows affinity purification of the labelled receptor.

We performed a series of experiments to assess whether CGP64213B can function in a manner similar to CGP54626, an antagonist/inverse agonist, and whether it can be used to isolate the receptor through affinity purification. We first demonstrated that CGP64213B can selectively label the GB1 subunit, but not the GB2 subunit, of the GABAB receptor expressed on the surface of transfected CHO cells (Figure 2A). We also found that CGP64213B inhibited both the GABA-induced IP3 (inositol trisphosphate) production in transfected HEK-293 cells (Figure 2B) and baclofen-induced Akt phosphorylation in CGNs at a potency similar to that observed for CGP54626 (Figure 2C), demonstrating that CGP64213B functions similarly to CGP54626.

CGP64213B is a selective photoaffinity ABP of the GABAB receptor

Figure 2
CGP64213B is a selective photoaffinity ABP of the GABAB receptor

(A) Selectivity of CGP64213B towards GB1 in intact cells. CHO cells transfected with GB1ASAHA (GB1 mutant was able to reach the cell surface alone) [17] or GB2HA (where HA is haemagglutinin) were pretreated with or without CGP54626 (100 μM) and then incubated with CGP64213B (2 μM) for 2 h, followed by UV irradiation for 1 h and washing. The total cell lysate was subjected to SDS/PAGE and detected with streptavidin–HRP (horseradish peroxidase) or anti-haemagglutinin antibody (anti-HA). CGP64213B labels GB1, but not GB2 [detected by anti- streptavidin antibody (anti-strep)], and this activity can be blocked by pre-treatment with CGP54626. The CHO cells transfected with the empty PRK6 vector were used as a negative control. (B and C) Potency of CGP64213B as a GABAB receptor antagonist. (B) CGP64213B inhibits IP3 formation. HEK-293 cells co-transfected with GB1, GB2 and the chimaera Gqi9 were incubated with the indicated concentrations of CGP64213B or CGP54626 for 15 min before stimulation with GABA (100 μM, 30 min). The IC50 value of CGP64213B is 1.03 μM. Values are the mean of triplicates from one representative of three independent experiments. (C) CGP64213B inhibits Akt phosphorylation. The CGNs were incubated with CGP64213B (10 μM) or CGP54626 (100 μM) for 20 min before stimulation with baclofen (100 μM) for 3 min. Anti-pAkt, anti-phospho-Akt. (D and E) Photolabelling and pull down of the heterodimeric GABAB receptor. (D) CHO cells transfected with GB1HA, GB1ASAHA, with or without GB2FLAG were incubated with CGP64213B (2 μM) for 2 h. PRK6 transfection was used as a negative control. (E) CGNs were incubated with or without CGP64213B (2 μM) for 2 h, followed by UV irradiation and washing. IB, immunoblot. Molecular masses are shown on the gels on the right-hand side.

Figure 2
CGP64213B is a selective photoaffinity ABP of the GABAB receptor

(A) Selectivity of CGP64213B towards GB1 in intact cells. CHO cells transfected with GB1ASAHA (GB1 mutant was able to reach the cell surface alone) [17] or GB2HA (where HA is haemagglutinin) were pretreated with or without CGP54626 (100 μM) and then incubated with CGP64213B (2 μM) for 2 h, followed by UV irradiation for 1 h and washing. The total cell lysate was subjected to SDS/PAGE and detected with streptavidin–HRP (horseradish peroxidase) or anti-haemagglutinin antibody (anti-HA). CGP64213B labels GB1, but not GB2 [detected by anti- streptavidin antibody (anti-strep)], and this activity can be blocked by pre-treatment with CGP54626. The CHO cells transfected with the empty PRK6 vector were used as a negative control. (B and C) Potency of CGP64213B as a GABAB receptor antagonist. (B) CGP64213B inhibits IP3 formation. HEK-293 cells co-transfected with GB1, GB2 and the chimaera Gqi9 were incubated with the indicated concentrations of CGP64213B or CGP54626 for 15 min before stimulation with GABA (100 μM, 30 min). The IC50 value of CGP64213B is 1.03 μM. Values are the mean of triplicates from one representative of three independent experiments. (C) CGP64213B inhibits Akt phosphorylation. The CGNs were incubated with CGP64213B (10 μM) or CGP54626 (100 μM) for 20 min before stimulation with baclofen (100 μM) for 3 min. Anti-pAkt, anti-phospho-Akt. (D and E) Photolabelling and pull down of the heterodimeric GABAB receptor. (D) CHO cells transfected with GB1HA, GB1ASAHA, with or without GB2FLAG were incubated with CGP64213B (2 μM) for 2 h. PRK6 transfection was used as a negative control. (E) CGNs were incubated with or without CGP64213B (2 μM) for 2 h, followed by UV irradiation and washing. IB, immunoblot. Molecular masses are shown on the gels on the right-hand side.

To examine whether CGP64213B can be used to isolate the receptor complex, we performed affinity pull-down experiments using streptavidin-coated beads to detect CGP64213B-labelled protein complexes in both transfected cells and primary neurons. From CGP64213B-treated CHO cells transfected with GB1 and GB2, streptavidin-coated beads can pull down the GABAB receptor complex containing both GB1 and GB2 (Figure 2D). The observation that GB2 can only be isolated in a GB1-dependent manner is consistent with the view that CGP64213B labels GB1, but not GB2, and that GB1 and GB2 form a complex (Figure 2D). To check whether CGP64213B can be used to pull down endogenous GABAB receptor, we performed the same experiments on CGNs and obtained a similar result (Figure 2E). The two GB1 subunit isoforms, GABAB1a and GABAB1b, and GB2 were all isolated by CGP64213B, which showed great specificity compared with anti-GB1 and anti-GB2 antibodies. Taken together, our data indicate that CGP64213B is not only as functional as CGP54626, but also can be used to isolate the GABAB receptor complex through affinity pull-down assays.

GABAB receptor, IGF-1R and their downstream effectors form a signalling complex during transactivation

Having validated the properties of CGP64213B, we utilized this probe to determine whether the GABAB receptor, IGF-1R and downstream signalling molecules form a protein complex and, if so, how they behave during transactivation in CGNs. As Akt phosphorylation is a reliable readout of IGF-1R function, we examined CGP7930-induced Akt phosphorylation in CGP64213B-labelled CGNs. Indeed, CGP7930 induced Akt phosphorylation in CGP64213B-labelled CGNs (Figure 3A), confirming that CGP7930 can efficiently activate the ‘synchronized’ GABAB receptors. We also showed that CGP7930 can efficiently increase the association of Akt with the CGP64213B-labelled receptor complex isolated by affinity pull down (Figure 3B), and that this association cannot be induced by IGF-1 (Figure 3C), suggesting that Akt is part of a CGP7930-transactivated complex. As Akt resides downstream of IGF-1R, this raises the possibility that molecules in IGF-1R signalling could also be in the same protein complex.

Detection of the specific CGP7930-transactivated complex

Figure 3
Detection of the specific CGP7930-transactivated complex

(A) Effect of CGP7930 (100 μM) on Akt phosphorylation (pAkt) in the CGP64213B-labelled CGNs for the indicated times. (B) Comparing the blots of co-immunoprecipitation and pull-down enrichment with CGP64213B. Molecular masses are shown on the right-hand side. (C) Effect of IGF-1 (10 ng/ml) on the interaction between IGF-1R and Akt for 3 min. After stimulation, the CGP64213B-labelled cell lysates were pulled down with streptavidin-coated beads. (D) Detection of specific CGP7930-transactivated complex by pull-down enrichment with CGP64213B. (B and D) The labelled CGNs were stimulated with CGP7930 (100 μM) for 3 min before performing co-immunoprecipitation with anti-GB1 or anti-Akt antibodies or pull-down enrichment with streptavidin-coated beads. (AD) Proteins from total cell lysates and immunoprecipitation and pull-down enrichments were subject to SDS/PAGE and detected with the indicated antibodies. All blots shown in this Figure are representative of three separate experiments. IB, immunoblot; IP, immunprecipitation.

Figure 3
Detection of the specific CGP7930-transactivated complex

(A) Effect of CGP7930 (100 μM) on Akt phosphorylation (pAkt) in the CGP64213B-labelled CGNs for the indicated times. (B) Comparing the blots of co-immunoprecipitation and pull-down enrichment with CGP64213B. Molecular masses are shown on the right-hand side. (C) Effect of IGF-1 (10 ng/ml) on the interaction between IGF-1R and Akt for 3 min. After stimulation, the CGP64213B-labelled cell lysates were pulled down with streptavidin-coated beads. (D) Detection of specific CGP7930-transactivated complex by pull-down enrichment with CGP64213B. (B and D) The labelled CGNs were stimulated with CGP7930 (100 μM) for 3 min before performing co-immunoprecipitation with anti-GB1 or anti-Akt antibodies or pull-down enrichment with streptavidin-coated beads. (AD) Proteins from total cell lysates and immunoprecipitation and pull-down enrichments were subject to SDS/PAGE and detected with the indicated antibodies. All blots shown in this Figure are representative of three separate experiments. IB, immunoblot; IP, immunprecipitation.

Notably, CGP64213B can readily pull down Akt and other signalling molecules (see below), whereas we were unable to do so using the conventional co-immunoprecipitation approach with antibodies against the GABAB receptor or Akt (Figure 3B). This further highlights the advantage of the use of CGP64213B, a high-affinity ABP, than conventional antibodies.

To determine the components of the receptor complex, we probed the complex using antibodies against those signalling proteins implicated in the transactivation pathway, including IGF-1R and its downstream molecules Src and Akt, as well as those functioning downstream of the GABAB receptor, such as Gαi and Gβγ. Remarkably, Gαi, Gβ, IGF-1R, Src and Akt were all present in the complex (Figure 3D). Thus it appears that the GABAB receptor, IGF-1R and their effectors form a signalling complex during transactivation.

Dynamic association of IGF-1R, Src and Akt within the signalling complex

To reveal the temporal dynamics of the molecules in the complex, we performed Western blot analysis on the isolated inactive complex, as well as on the CGP7930-transactivated complex at different time points after CGP7930 treatment. Interestingly, association of IGF-1R, Src and Akt with the complex was transiently increased upon GABAB receptor activation by CGP7930 (Figures 4A, left-hand panel, and 4B). One major functional event during transactivation is GABAB receptor-induced phosphorylation of IGF-1R and its downstream signalling molecules [3]. Indeed, CGP7930 also induced a transient increase in the amount of phosphorylated forms of IGF-1R, Src and Akt proteins in the complex (Figures 4A, left-hand panel, and 4C). The kinetics of the phosphorylated amount and total amount of IGF-1R, Src and Akt proteins in the complex nicely correlated with each other, suggesting that IGF-1R, Src and Akt are phosphorylated upon GABAB receptor activation within the same complex before being released into the cytoplasm (Figures 4A, left-hand panel, and 4C). As a control, we also blotted the total cell lysates and found that CGP7930 treatment did not change the overall level of these proteins (Figure 4A, right-hand panel). These results indicate that, during transactivation, IGF-1R and its downstream molecules Src and Akt exhibit a transient increase, followed by a decrease, in their association with the signalling complex.

Capture of dynamic protein–protein interactions during GABAB receptor-mediated IGF-1R transactivation

Figure 4
Capture of dynamic protein–protein interactions during GABAB receptor-mediated IGF-1R transactivation

(A) Dynamic protein–protein interactions within the GABAB receptor-associated protein complex upon CGP7930 stimulation. CGNs were labelled with CGP64213B (2 μM) for 2 h followed by UV irradiation and washing. The labelled CGNs were stimulated with CGP7930 (100 μM). Cell lysates were pulled down with streptavidin-coated beads and probed with antibodies or phospho (p)-antibodies against various signalling molecules in the pathway. Shown are representative blots from three separate experiments. (B and C) Quantification of protein and phosphorylated protein levels within the complex by scanning the density of the bands on the blots. Results are means±S.E.M.; n=3–6.

Figure 4
Capture of dynamic protein–protein interactions during GABAB receptor-mediated IGF-1R transactivation

(A) Dynamic protein–protein interactions within the GABAB receptor-associated protein complex upon CGP7930 stimulation. CGNs were labelled with CGP64213B (2 μM) for 2 h followed by UV irradiation and washing. The labelled CGNs were stimulated with CGP7930 (100 μM). Cell lysates were pulled down with streptavidin-coated beads and probed with antibodies or phospho (p)-antibodies against various signalling molecules in the pathway. Shown are representative blots from three separate experiments. (B and C) Quantification of protein and phosphorylated protein levels within the complex by scanning the density of the bands on the blots. Results are means±S.E.M.; n=3–6.

Dynamic dissociation of preassembled G-proteins from the signalling complex

Strikingly, G-proteins exhibited temporal dynamics distinct from that of IGF-1R, Src and Akt during transactivation. Specifically, the amount of Gαi and Gβ subunits in the complex decreased upon stimulation in a time-dependent manner (Figures 4A, left-hand panel, and 4B). This observation suggests that Gαi and Gβ subunits may preassociate with the inactive GABAB receptor and are disassociated from the activated receptor upon stimulation. Indeed, co-immunoprecipitation with anti-GB1 antibody revealed an increasing association between the GABAB receptor and Gαi subunits by pretreatment of the antagonist/inverse agonist CGP54626 in a concentration-dependent manner (Supplementary Figure S1A at http://www.BiochemJ.org/bj/443/bj4430627add.htm). Furthermore, a TR-FRET (time-resolved fluorescence resonance energy transfer) assay performed on transfected HEK-293 cells indicates that GABA treatment induced dissociation between Gαi and GB1 subunits (Supplementary Figure S1C), also confirming the existence of preassembled G-proteins with the GABAB receptor and their dissociation from the receptor complex upon receptor activation.

Dynamic protein–protein interactions are critical for transactivation

Are these dynamic events important for transactivation? If so, perturbing these dynamic events should lead to suppression of transactivation. To test this, we first examined the role of G-proteins in this process. The heterotrimeric G-proteins (Gαβγ) transmit signals from GPCRs to intracellular targets [36]. In the present study, G-proteins were found to be present in the same complex as the GABAB receptor, IGF-1R, Src and Akt until 5 min post-stimulation (Figures 4A and 4B). Since the preassembly/disassembly process between the GABAB receptor and G-proteins occurred before the formation of the dynamic complex comprising the GABAB receptor, IGF-1R and its downstream molecules Src and Akt, we hypothesized that activation of G-proteins by the GABAB receptor is important for initiating the dynamic process during transactivation. To test this, we perturbed the G-protein activity with PTX (pertussis toxin), a Gi/o-protein inhibitor, in CGP64213B-labelled CGNs. PTX prevented the dissociation of Gαi from the signalling complex upon CGP7930 treatment and, as a result, IGF-1R, Src and Akt were no longer associated with the GABAB receptor and G-proteins (Figure 5A). These results indicate that dynamic dissociation of preassembled G-proteins from the signalling complex is critical for recruiting IGF-1R and its effectors to the complex, and that perturbing this process blocks transactivation.

G-protein activation and IGF-1R tyrosine kinase activity are important for dynamic protein–protein interactions and transactivation

Figure 5
G-protein activation and IGF-1R tyrosine kinase activity are important for dynamic protein–protein interactions and transactivation

(A) PTX abolishes the formation of the signalling complex and hence transactivation. (B) The IGF-1R inhibitor AG1024 impairs signalling events downstream, but not upstream, of IGF-1R. CGNs were labelled with CGP64213B (2 μM) for 2 h. The labelled CGNs were then treated with either PTX (200 ng/ml, 14 h) (A) or AG1024 (0.1 μM, 1 h) (B) before stimulation with CGP7930 (100 μM, 3 min). The cell lysate was pulled down with streptavidin-coated beads and subjected to SDS/PAGE and detected with different antibodies and phospho (p)-antibodies. Total cell lysates were also subjected to Western blot analysis. Shown are representative blots of three separate experiments.

Figure 5
G-protein activation and IGF-1R tyrosine kinase activity are important for dynamic protein–protein interactions and transactivation

(A) PTX abolishes the formation of the signalling complex and hence transactivation. (B) The IGF-1R inhibitor AG1024 impairs signalling events downstream, but not upstream, of IGF-1R. CGNs were labelled with CGP64213B (2 μM) for 2 h. The labelled CGNs were then treated with either PTX (200 ng/ml, 14 h) (A) or AG1024 (0.1 μM, 1 h) (B) before stimulation with CGP7930 (100 μM, 3 min). The cell lysate was pulled down with streptavidin-coated beads and subjected to SDS/PAGE and detected with different antibodies and phospho (p)-antibodies. Total cell lysates were also subjected to Western blot analysis. Shown are representative blots of three separate experiments.

We then attempted to perturb the dynamic process by interfering with the phosphorylation events that occur during transactivation in the protein complex. We treated CGP64213B-labelled CGNs with AG1024, an IGF-1R tyrosine kinase inhibitor. Two interesting phenomena were observed. First, blockade of IGF-1R phosphorylation not only greatly inhibited its own phosphorylation, but also suppressed the phosphorylation of its downstream signalling molecules Src and Akt (Figure 5B). This indicates that AG1024 treatment suppressed transactivation, as Akt phosphorylation is a reliable readout of IGF-1R function. Secondly, despite the reduction in the amount of phosphorylated IGF-1R, Src and Akt, the total amount of these proteins in the complex was in fact greatly increased (Figure 5A and Supplementary Figure S2B at http://www.BiochemJ.org/bj/443/bj4430627add.htm). As a control, the overall protein levels of these molecules in the total cell lysate were not affected by AG1024 treatment. These results demonstrate that disturbing IGF-1R activity not only blocks GABAB receptor-induced phosphorylation of IGF-1R and its downstream signalling molecules, but also prevents their release from the complex, leading to an accumulation of these proteins in the signalling complex. Although the result of blocking G-protein shows that recruitment of signalling molecules to the complex is important for transactivation, this result suggests that the ensuing release of signalling molecules from the complex is also important for transactivation. Taken together, these observations support the notion that the dynamic assembly and disassembly of the protein complex are critical for transactivation.

Dynamic protein–protein interactions are critical for transactivation-dependent neuronal survival

To illustrate the physiological relevance of the observed dynamic process in transactivation, we perturbed it by treating CGP64213B-labelled CGNs with the IGF-1R inhibitor AG1024. Upon activation, IGF-1R can promote the survival of CGNs, a key function of IGF-1R in these neurons [18,19,37,38]. Although CGP7930 treatment promoted CGN survival, this effect was significantly inhibited by perturbation of IGF-1R with AG1024, providing functional evidence that dynamic protein–protein interactions are important for transactivation (Supplementary Figure S3 at http://www.BiochemJ.org/bj/443/bj4430627add.htm).

FAK (focal adhesion kinase) plays a key role in co-ordinating the formation of the signalling complex

Finally, we investigated how GPCRs and RTKs, two discrete membrane receptors, are assembled into the same signalling complex. We reasoned that an adaptor-like protein must exist to co-ordinate the formation of this signalling complex. To fulfil such a role, this protein must have the capacity to form a complex with both the GABAB receptor and IGF-1R. FAK, a non-RTK, came to our attention. It is known that FAK can form a protein complex with G-proteins and can be activated by GPCRs [39,40]. It has also been reported that FAK can physically interact with IGF-1R [4143]. Thus FAK appears to be a promising candidate for co-ordinating the assembly of the signalling complex. We therefore investigated the role of FAK in transactivation.

We found that CGP7930 treatment recruited FAK to the signalling complex, indicating that FAK is part of the complex (Figure 4A). CGP7930 also triggered the autophosphorylation of FAK at Tyr397 within the complex, an event known to be critical for the function of FAK (Supplementary Figure S4 at http://www.BiochemJ.org/bj/443/bj4430627add.htm). If FAK is important for co-ordinating the formation of the signalling complex, inhibiting its function should inhibit transactivation. Indeed, treatment with PF573228, an FAK antagonist, suppressed the recruitment of IGF-1R and its downstream effectors Src and Akt to the complex, as well as their phosphorylation (Figure 6A). Interestingly, this treatment did not have a notable effect on G-proteins (Figure 6A). This is consistent with the view that FAK acts downstream of G-proteins, but upstream of IGF-1R, Src and Akt.

FAK is crucial for co-ordinating the formation of the signalling complex

Figure 6
FAK is crucial for co-ordinating the formation of the signalling complex

(A) FAK acts downstream of G-proteins, but upstream of IGF-1R. The FAK antagonist PF573228 impaired the association of the downstream IGF-1R and its effectors with the GABAB receptor, but without affecting the upstream G-proteins. CGNs were incubated with CGP64213B (2 μM) followed by UV irradiation and washing. The labelled cells were then treated with PF573228 (10 μM) or PBS for 1 h and then stimulated with CGP7930 (100 μM) for 3 min followed by pull-down enrichment with streptavidin-coated beads. Blots of pull-down samples or total lysates were detected with different antibodies as indicated. Shown are representative blots of three separate experiments. (B) No signalling complex was formed in FAK−/− cells. CGP64213B-labelled MEF WT (wild-type), FAK−/− and FAK−/− cells transfected with wild-type full-length FAK cDNA (FAKFL) were stimulated with CGP7930 (100 μM, 3 min) followed by pull-down enrichment with streptavidin-coated beads. All cells also expressed GB1 and GB2 through co-transfection.

Figure 6
FAK is crucial for co-ordinating the formation of the signalling complex

(A) FAK acts downstream of G-proteins, but upstream of IGF-1R. The FAK antagonist PF573228 impaired the association of the downstream IGF-1R and its effectors with the GABAB receptor, but without affecting the upstream G-proteins. CGNs were incubated with CGP64213B (2 μM) followed by UV irradiation and washing. The labelled cells were then treated with PF573228 (10 μM) or PBS for 1 h and then stimulated with CGP7930 (100 μM) for 3 min followed by pull-down enrichment with streptavidin-coated beads. Blots of pull-down samples or total lysates were detected with different antibodies as indicated. Shown are representative blots of three separate experiments. (B) No signalling complex was formed in FAK−/− cells. CGP64213B-labelled MEF WT (wild-type), FAK−/− and FAK−/− cells transfected with wild-type full-length FAK cDNA (FAKFL) were stimulated with CGP7930 (100 μM, 3 min) followed by pull-down enrichment with streptavidin-coated beads. All cells also expressed GB1 and GB2 through co-transfection.

If FAK truly functions downstream of G-proteins and upstream of IGF-1R, then blocking G-protein function should prevent the recruitment of FAK to the complex. On the other hand, blocking IGF-1R function should have no effect on FAK. Consistent with this prediction, inhibition of G-proteins by PTX prevented FAK from being recruited to the complex (Figure 5A), whereas inhibition of IGF-1R activity by AG1024 did not (Figure 5B and Supplementary Figure S2A). This provides further evidence that FAK acts downstream of G-proteins, but upstream of IGF-1R.

To provide additional evidence, we knocked down FAK by siRNA (small interfering RNA) to further investigate its role in transactivation. In MEF cells expressing GABAB receptors, treatment with FAK siRNA prevented IGF-1R and Akt from being recruited to the complex upon CGP7930 treatment (Supplementary Figure S4B). This observation, together with the pharmacological results described above, provides strong evidence that FAK plays a key role in co-ordinating the formation of the signalling complex.

Lastly, we examined FAK−/− cells. As FAK−/− mice are embryonically lethal at day 8.5 (no CGNs are yet formed), we focused on MEF cells. In FAK−/− cells expressing GABAB receptors, CGP7930 treatment could no longer recruit IGF-1R, Src and Akt to the complex (Figure 6B). Transfection of the wild-type FAK gene into FAK−/− cells can fully rescue this defect (Figure 6B). This provides strong evidence that FAK is essential for the formation of the signalling complex.

In summary, we have designed and synthesized a biotinyated photoaffinity probe that allowed us first to clamp the GABAB receptor in an inactive state and then activate it with a PAM to unveil the functional states of receptors and signalling molecules during transactivation in a temporally controlled manner. Thus far, ABPs mostly target cytosolic proteins [4446]. This type of probe has not been developed to characterize membrane receptor-initiated signal transduction. To our knowledge, the present study represents the first such attempt, showing that ABPs are powerful tools for probing signalling events mediated by membrane receptors. Using such a probe, we found that the GABAB receptor and IGF-1R, as well as their downstream effectors, form a signalling complex, and that the dynamic assembly and disassembly of this complex are essential for transactivation. The present study has developed a powerful tool for probing GABAB receptor signalling and this approach may be extended to reveal the dynamic protein–protein interactions between other GPCRs and their downstream signal molecules.

Abbreviations

     
  • ABP

    activity-based probe

  •  
  • CGN

    cerebellar granule neuron

  •  
  • CHO

    Chinese-hamster ovary

  •  
  • FAK

    focal adhesion kinase

  •  
  • GABA

    γ-aminobutyric acid

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • HBS

    Hepes-buffered solution

  •  
  • HEK

    human embryonic kidney

  •  
  • IGF-1

    insulin-like growth factor 1

  •  
  • IGF-1R

    insulin-like growth factor type I receptor

  •  
  • IP3

    inositol trisphosphate

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • PAM

    positive allosteric modulator

  •  
  • PTX

    pertussis toxin

  •  
  • RTK

    receptor tyrosine kinase

  •  
  • siRNA

    small interfering RNA

AUTHOR CONTRIBUTION

Jianfeng Liu and Fajun Nan conceived the original idea, supervised the research and contributed to editing the paper prior to submission; Xin Lin, Ming Jiang and Jianfeng Liu performed most of the experiments and wrote the paper; Xin Li, Linhai Chen, Fajun Nan performed the photoaffinity probe synthesis; Chanjuan Xu and Wenhua Zhang performed the experiments with IGF-1R and FAK1 inhibitors; Chanjuan Xu and Xin Lin performed TR-FRET experiments; Bing Sun, Han Zhao and Xiaoli Xu prepared the primary neuronal cultures.

We thank Dr Xian-Zhong Xu (University of Michigan, Ann Arbor, MI, U.S.A.), Dr Jean-Philippe Pin (Institut de Génomique Fonctionnelle, Montpellier, France) and Dr P. Rondard (Institut de Génomique Fonctionnelle, Montpellier, France) for their helpful discussions.

FUNDING

This work was supported by the Ministry of Science and Technology [grant numbers 2007CB914200 and 2010DFA32140 (to J.L.)], the National Natural Science Foundation of China (NSFC) [grant numbers 31130028 and 30973514 (to J.L.)], the Program of Introducing Talents of Discipline to Universities of Ministry of Education [grant number B08029 (to J.L.)], the National Basic Research Program of China [grant number 2007CB914200 (to F.N.)] and the National Natural Science Foundation of China [grant numbers 30725049 and 90813037 (to F.N.)].

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Author notes

1

These authors contributed equally to this work.

Supplementary data