Activator of G-protein signaling 3 (AGS3, encoded by GPSM1) was discovered as a one of several receptor-independent activators of G-protein signaling, which are postulated to provide a platform for divergence between canonical and noncanonical G-protein signaling pathways. Similarly, Dishevelled (DVL) proteins serve as a point of divergence for β-catenin-dependent and -independent signaling pathways involving the family of Frizzled (FZD) ligands and cell-surface WNT receptors. We recently discovered the apparent regulated localization of dishevelled-2 (DVL2) and AGS3 to distinct cellular puncta, suggesting that the two proteins interact as part of various cell signaling systems. To address this hypothesis, we asked the following questions: (1) do AGS3 signaling pathways influence the activation of β-catenin (CTNNB1)-regulated transcription through the WNT–Frizzled–Dishevelled axis, and (2) is the AGS3 and DVL2 interaction regulated? The interaction of AGS3 and DVL2 was regulated by protein phosphorylation, subcellular distribution, and a cell-surface G-protein-coupled receptor. These data, and the commonality of functional system impacts observed for AGS3 and DVL2, suggest that the AGS3–DVL2 complex presents an unexpected path for functional integration within the cell.
Activator of G-protein signaling 3 (AGS3; encoded by GPSM1) is a member of the Group II AGS proteins, each of which contain one to four G-protein regulatory region (GPR) motifs that serve as alternative binding partners for specific subtypes of Gα (Gαi, Gαo or Gαt) (Oner et al., 2013a). AGS3 has seven N-terminal tetratricopeptide repeats (TPR) and four C-terminal GPR motifs connected with a linker domain (Blumer and Lanier, 2014). The TPR repeats influence intramolecular dynamics and subcellular distribution of AGS3 through interaction with specific binding partners (Du and Macara, 2004; An et al., 2008; Vural et al., 2010). The GPR motifs in AGS3 act as guanine-nucleotide-dissociation inhibitors stabilizing the Gα subunit in its GDP-bound conformation (Blumer et al., 2012). AGS3 exhibits a tissue-specific expression pattern that is regulated during development and during tissue response and/or adaptation to various physiological or pathophysiological challenges (Blumer and Lanier, 2014).
One key aspect of understanding the role of such proteins in physiological system homeostasis and pathophysiological dysfunction is defining the mechanistic processes involved as a platform for development of diagnostics and therapeutic modalities. However, this often proves to be challenging due to the oft-encountered difficulties in translating genetic and molecular-based discoveries into clear and specific component unit functions within the context of a living organism. Indeed, this is the situation with various protein families, including the AGS proteins. As an example, AGS3 and the Group II AGS protein family play diverse functional roles in specific cell types, tissues and organs (Blumer and Lanier, 2014), including metabolism and cardiovascular function (Blumer et al., 2008), hearing loss (Bhonker et al., 2016; Mauriac et al., 2017), adaptive responses to addiction and craving behavior (Bowers et al., 2004, 2008; Yao et al., 2005; Fan et al., 2009), renal response to injury (Nadella et al., 2010; Regner et al., 2011; Rasmussen et al., 2015; Lenarczyk et al., 2015), polycystic kidney disease (Nadella et al., 2010; Kwon et al., 2012), inflammation (Singh et al., 2014; Xing et al., 2015; Choi et al., 2016; Vural et al., 2019) and prostate cancer (Adekoya et al., 2019). At a cellular level, AGS3 is associated with a broad landscape of biological pathways including asymmetric cell divisions (Sanada and Tsai, 2005; Saadaoui et al., 2017), autophagy (Pattingre et al., 2003; Groves et al., 2010; Garcia-Marcos et al., 2011; Vural et al., 2013, 2016), lysosomal regulation (Vural et al., 2016), phagocytosis (Huang et al., 2014), membrane protein trafficking (Groves et al., 2007; Oner et al., 2013b) and leukocyte migration (Kamakura et al., 2013; Branham-O'Connor et al., 2014; Singh et al., 2014).
To focus on the key question of mechanism and system function, we explored an approach based on image phenotype profile screens. AGS3 oscillates between a noncortical and cortical distribution within the cell, moving among different subcellular compartments (e.g. cytosol, cell membrane, centrosome, trans-Golgi network, punctate structures within the cytosol, aggresomal pathway) in a regulated manner (Pizzinat et al., 2001; Blumer et al., 2002; Sanada and Tsai, 2005; Groves et al., 2007; An et al., 2008; Nadella et al., 2010; Vural et al., 2010, 2018; Oner et al., 2013b; Yeh et al., 2013; Kamakura et al., 2013). The observed cellular distribution of AGS3 to date is likely just a snapshot in time of what is an even more dynamic movement of the protein within the cell, as regulated by various factors.
Of particular note, AGS3 possesses an inherent propensity to form well-defined and easily observed punctate structures that are discontinuously distributed in the cytosol of mammalian cells (Vural et al., 2010). The distribution of AGS3 to cytosolic puncta is induced by the introduction of single amino acid substitutions in either the TPR or GPR motifs of AGS3 (Vural et al., 2010, 2018) and this distribution is regulated by AGS3 binding partners and perhaps by phosphorylation of AGS3 (Vural et al., 2010, 2018). Such punctate positioning of AGS3 is predicted to be intimately associated with its multifunctionality as well as occupying an essential nexus in cellular signaling networks. Toward this end, and as a path to understand the mechanism–function axis, we asked what other proteins or functional pathways may exhibit a similar regulated movement in and out of punctate structures in the cell? As a first pass, we identified a number of such entities or signaling systems, including purinosomes, the misfolded protein pathway, P-bodies and Dishevelled signalosomes, which exhibit a subcellular distribution not dissimilar to that observed with AGS3 (Kedersha and Anderson, 2007; Brielle et al., 2015; Wu and Fuxreiter, 2016; Pedley and Benkovic, 2017). As we examined the relationships of these visual snapshots of ‘image-signaling system connectivity’, we focused first on the protein dishevelled-2 (DVL2), which was of particular interest in the context of the biochemistry and functional properties of AGS3.
Dishevelled (DVL) proteins are a key element of the signaling pathway involving the Wingless-INT1 (WNT) family of hormones (Wodarz and Nusse, 1998; Nusse and Varmus, 2012). In mammalian systems, WNT signaling involves at least 19 endogenous Frizzled (FZD) ligands and ten WNT receptors, which have the distinguishing seven-transmembrane-span structure characteristic of G-protein-coupled receptors (Schulte and Wright, 2018). The dynamics of signal processing through the WNT signaling system is complex and involves canonical and noncanonical branching pathways with various roles for G protein subunits, Dishevelled, adaptor proteins, various signal transducers and co-receptors, which presents some challenges to mechanistic understanding of the WNT signaling system overall (Bilic et al., 2007; Bryja et al., 2007a; Schwarz-Romond et al., 2007b; Kilander et al., 2011; Schulte and Shenoy, 2011; Halleskog et al., 2012; Halleskog and Schulte, 2013; Kilander et al., 2014; Wright et al., 2018). Dishevelled proteins have essential roles in both canonical and noncanonical WNT signaling pathways. Canonical WNT signaling includes β-catenin (CTNNB1) stabilization and β-catenin-dependent transcriptional regulation during embryonic development and in adult tissues (Gao and Chen, 2010). On the other hand, noncanonical WNT signaling pathways have effects on cell motility and migration, ciliogenesis, planar cell polarity and morphogenesis (Mlodzik, 2016). Of note is that, similar to AGS3, DVL2 also exhibits regulated distribution in and out of punctate structures (Schwarz-Romond et al., 2005, 2007a,b; Gammons et al., 2016), which is postulated to be a determining factor relative to its functional engagement in the WNT–β-catenin-dependent and -independent signaling pathways and the associated distinct cell responses.
As we pursued this line of study, we previously determined that AGS3 is recruited to DVL2 puncta (Vural et al., 2018), suggesting that the two proteins interact as part of various cellular signaling pathways. To address this hypothesis, in the current study we asked the following questions. (1) Does AGS3 influence canonical WNT signaling pathways? (2) Do AGS3 and DVL2 interact with each other in a regulated manner? (3) Is the distribution of AGS3 to DVL2 puncta regulated by a cell-surface G-protein-coupled receptor?
Influence of AGS3 on the WNT–β-catenin signaling pathway
AGS3, which typically has a non-homogeneous, diffuse distribution in the cytosol, is redistributed to DVL2 cell puncta upon co-expression of the two proteins (Vural et al., 2018). As an initial approach to determine the role of AGS3–DVL2 colocalization within the cell (Vural et al., 2018), we asked whether AGS3 influences WNT-induced activation of β-catenin signaling. This pathway involves cell-surface receptors, co-receptors and Dishevelled, through a signal processing pathway that is not completely defined but that eventually stabilizes β-catenin, with subsequent movement of the protein to the nucleus and interaction with transcription factors for targeted gene regulation (Nusse and Clevers, 2017). The relative degree of involvement of heterotrimeric G proteins in the WNT-induced activation of the β-catenin signaling pathway may be somewhat influenced by cell context and the tissue systems involved (Gao and Chen, 2010; Garcia-Marcos et al., 2011; Halleskog and Schulte, 2013; Bowin et al., 2019). AGS3 clearly interacts with at least two key components of this signaling pathway – Gα and DVL2.
The β-catenin signaling pathway activity was monitored using a dual-luciferase-based transcriptional reporter assay in HEK-293 cells. HEK-293 cells were selected for system assays based upon the robustness and reproducibility of the activity readout, as determined in an initial series of experiments with various cell types and parameter optimizations. DVL2 expression in HEK-293 cells increased β-catenin-mediated transcriptional activity by 3.4±0.1-fold (mean±s.e.m.) and this increase was not altered by co-expression of AGS3 (Fig. 1A). We next asked whether increased AGS3 expression altered the responsiveness of the β-catenin signaling pathway subsequent to system activation by WNT-3A or by lithium chloride (LiCl) as upstream and downstream signaling pathway activators, respectively. Agonist-induced activation of the signaling pathway by WNT-3A increased β-catenin-mediated transcriptional activity by 3.6±0.3-fold (mean±s.e.m.) and this activation was not altered by increasing expression of AGS3 in the cell (Fig. 1B). Similarly, AGS3 expression did not alter the increase in β-catenin-mediated transcriptional activity (5.9±0.6-fold, mean±s.e.m.) observed after cell treatment with LiCl, which inhibits glycogen synthase kinase-3β (GSK-3β), stabilizing β-catenin and increasing subsequent transcriptional activity (Fig. 1C). The expression of endogenous DVL2 in HEK-293 cells was not altered by expression of increasing amounts of AGS3 (Vural et al., 2018; A.V. and S.M.L., unpublished)
Interaction of AGS3 and dishevelled-2
Dishevelled and GPR proteins such as AGS3 serve as nexus points in various signaling pathways and are implicated in several similar cellular events (Blumer and Lanier, 2014; Mlodzik, 2016). We thus conducted a series of immunoprecipitation experiments to further define the postulated interaction of the two proteins. Immunoprecipation of AGS3 following expression of AGS3 and DVL2 in HEK-293 cells effectively co-immunopreciptated DVL2 (Fig. 2A). DVL2 was not observed in immunoprecipitates with non-specific rabbit antisera, confirming the specificity of the AGS3–DVL2 co-immunoprecipation (Fig. 2A upper panel, right). We also conducted the reverse set of experiments by first immunoprecipitating DVL2 from cell lysates followed by immunoblots using AGS3 antibody. A similar interaction between DVL2 and AGS3 was observed, and AGS3 was co-immunipreciptated with DVL2 (MA-10B5) antibody (Fig. 2B). The co-immunoprecipitation of AGS3 with DVL2 was not observed in parallel immunoprecipitations using normal mouse IgG (Fig. 2B, right panels) obtained from non-immunized mice, further validating the specificity of the AGS3–DVL2 interaction and the results generated. Thus, the apparent interaction of AGS3 and DVL2 has been observed by both immunofluorescence imaging and co-immunoprecipitation (Vural et al., 2018).
Influence of DVL2 phosphorylation on AGS3 puncta and the WNT–β-catenin signaling pathway
As noted above, we recently reported the localization of AGS3 to cell puncta upon co-expression with DVL2 (Vural et al., 2018). The distribution of DVL2 within the cell is dependent upon its phosphorylation status, and the phosphorylation of DVL2 is increased by WNT-mediated activation of cell-surface receptors (Bryja et al., 2007b; Bernatík et al., 2011). The phosphorylation status of DVL2 is a determining factor for the integration of the protein into selective signaling pathways in the cell (Bryja et al., 2007b; Bernatík et al., 2011). Thus we asked whether the colocalization of AGS3 and DVL2 in cell puncta is influenced by the phosphorylation status of DVL2.
To this end, we first established a platform to track altered phosphorylation status of DVL2 using specific DVL2 antibodies and targeted cellular interventions. The phosphorylation status of DVL2 is revealed through its pattern of migration, subsequent to protein separation by SDS-PAGE, as detected by the DVL2 monoclonal antibodies MA-10B5 and MA-D6 (Fig. 3A). DVL2 MA-10B5 was generated against amino acids 594–736 at the C terminus of DVL2 and primarily recognizes dephosphorylated DVL2, which typically exhibits a faster migration during SDS-PAGE (González-Sancho et al., 2013). DVL2 MA-D6 was generated against amino acids 2–29 of DVL2 and recognizes phosphorylated and unphosphorylated DVL2. The phosphorylation status of DVL2 is regulated by WNT ligands and casein kinase-1ε (CK1ε), providing an additional set of tools for system analysis (Bryja et al., 2007a,b; González-Sancho et al., 2013; Bernatík et al., 2014). The utility of these tools and this experimental approach is illustrated in Fig. 3.
In HEK-293 cells expressing AGS3 and DVL2, the DVL2 antibody MA-10B5 recognizes two predominant distinct species with apparent molecular weights of ∼90,000–93,000, whereas the DVL2 antibody MA-D6 recognizes one predominant immunoreactive species (Fig. 3A). Treatment of cells with the CK1 inhibitor D4476 resulted in a loss of the slower migrating protein identified using MA-10B5 and an intensity reduction for the species identified by MA-D6. These data indicate that the slower migrating species identified using MA-10B5 represents phosphorylated DVL2 and that the faster migrating species primarily represents DVL2 that is not phosphorylated by CK1ε. Consistent with this interpretation is the finding that the slower migrating species detected by MA-10B5 was the predominant form of DVL2 observed in cells transfected with wild-type CK1ε (lane 4) and that the faster migrating species observed with MA-10B5 was the predominant species in cells transfected with the dominant-negative construct CK1ε-K38R (lane 6). Expression of AGS3 did not alter the apparent phosphorylation status of DVL2 under these conditions. Interventions expected to decrease DVL2 phosphorylation (i.e. treatment with CK1 inhibitor or use of CK1ε-K38R) tended to decrease the intensity of the primary species identified using MA-D6, whereas transfection of cells with wild-type CK1ε increased signal intensity of this same species (Fig. 3A).
With this ability to manipulate the phosphorylation status of DVL2, we asked whether alteration of DVL2 phosphorylation influenced the interaction of AGS3 and DVL2 identified by immunofluorescence imaging in COS-7 cells. Approximately 60% of cells expressing DVL2 exhibited a well-defined punctate distribution of the protein, with the remainder of the DVL2-expressing cells exhibiting a more ‘even’ non-homogeneous distribution through the cytosol that was characterized by less well-defined visual puncta. The percentage of cells exhibiting the DVL2 punctate distribution or the non-homogeneous ‘even’ distribution within the cell was not altered by co-expression of AGS3. As indicated in Fig. 3B, the colocalization of DVL2 and AGS3 in cell puncta was disrupted by co-expression of wild-type CK1ε, resulting in an increase in the percentage of DVL2-expressing cells exhibiting a non-homogeneous, ‘even’ distribution through the cytosol (Fig. 3B). The loss of the apparent AGS3–DVL2 colocalization in the cell puncta observed upon co-expression of CK1ε, which results in phosphorylation of DVL2 (Fig. 3A) (Bryja et al., 2007b; Bernatík et al., 2011), was reversed by incubation of cells with the kinase inhibitor D4476. In contrast, co-expression of the dominant-negative kinase CK1ε-K38R did not alter the colocalization of DVL2 and AGS3 in cell puncta (Fig. 3B) and did not obviously influence the non-homogeneous ‘even’ distribution of DVL2 within the cell (A.V. and S.M.L., unpublished). It was previously hypothesized that DVL2 cell puncta represent the dephosphorylated form of DVL2 (Bryja et al., 2007b; Bernatík et al., 2011), consistent with the reduced intensity of the DVL2 puncta observed in cells expressing CK1ε (Fig. 3B). These data indicate that the apparent colocalization of AGS3 and DVL2 in the cell puncta is influenced by the phosphorylation status of DVL2.
We also evaluated the impact of altered phosphorylation of DVL2 on β-catenin-mediated transcriptional activity (Fig. 3C). Expression of CK1ε increased β-catenin-mediated transcriptional activity by 7.9±0.6-fold (mean±s.e.m.). Co-expression of DVL2 and CK1ε resulted in a greater than additive increase in β-catenin-mediated transcriptional activity (27.3±0.1-fold, mean±s.e.m.) as compared to the system activation observed with either protein alone (DVL2, 2.5±0.3; CK1ε, 7.9±0.6; mean±s.e.m.). Expression of the dominant-negative kinase CK1ε-K38R did not alter β-catenin-mediated transcriptional activity in the presence or absence of elevated DVL2 (Fig. 3C). Co-expression of AGS3 did not have any effect on the increase in β-catenin-mediated transcriptional activity induced by CK1ε or the synergistic increase observed with CK1ε and DVL2 co-expression (Fig. 3C). These data are consistent with the hypothesis that the role of DVL2 in the WNT-induced increase in β-catenin-mediated transcriptional activity requires phosphorylation of DVL2. In addition, the lack of an effect of AGS3 expression on β-catenin-mediated transcriptional activity is consistent with an apparent preferred interaction of AGS3 with non-phosphorylated DVL2, and this hypothesis was further explored with the next series of experiments.
Regulated interaction of AGS3 and DVL2
The data presented above suggest that the interaction of AGS3 and DVL2 is regulated by cell stimuli that influence the phosphorylation status and/or subcellular location of the two proteins. We tested this hypothesis using two distinct experimental approaches. First, we took advantage of the toolkit assembled to alter the phosphorylation status of DVL2 (Fig. 3A) and the ability to co-immunoprecipitate DVL2 and AGS3 (Fig. 2). Second, we asked whether the colocalization of AGS3 with DVL2 puncta was altered by G-protein signaling systems involving agonist-induced activation of a cell-surface receptor.
The results of the first series of experiments are indicated in Fig. 4. The co-immunoprecipation of AGS3 and DVL2 from HEK-293 cell lysates was not observed upon co-expression of CK1ε (Fig. 4A, lane 2), which increased the apparent phosphorylation of DVL2, as reflected by the migration pattern of the protein observed on immunoblots (Fig. 4A, input) and as discussed above (Fig. 3A). However, the interaction of the two proteins was not altered by kinase inhibition using the dominant-negative kinase CK1ε-K38R and the CK1 inhibitor D4476 (Fig. 4A). Furthermore, the loss of AGS3–DVL2 co-immunoprecipitation observed upon co-expression of CK1ε was reversed by treatment of cell lysates with alkaline phosphatase, which also increased the migration of immunoreactive DVL2 through SDS-polyacrylamide gels in a manner consistent with protein dephosphorylation (Fig. 4B). Finally, we asked whether WNT-3A, which activates the canonical β-catenin signaling pathway through cell-surface FZD receptors (Fig. 1C) (Bryja et al., 2007b), influenced the interaction of DVL2 and endogenous AGS3. As indicated in Fig. 4C, WNT-3A treatment of cells expressing DVL2 disrupted the subsequent co-immunoprecipitation of DVL2 with endogenous AGS3, and this was associated with an altered migration pattern of immunoreactive DVL2 through SDS-polyacrylamide gels consistent with changes in the degree of protein phosphorylation (Fig. 4C).
These data indicate that the interaction of AGS3 and DVL2 observed by immunofluorescence imaging and co-immunoprecipitation is regulated by phosphorylation and dephosphorylation of DVL2. The shifts in gel migration of DVL2 presented in Figs 3 and 4 are consistent with changes in the phosphorylation status of the protein, and such gel migration shifts are a common feature of serine/threonine-phosphorylated proteins as a result of altered binding of the protein with sodium dodecyl sulfate in the denaturing polyacrylamide gel system. However, such gel migration shifts do not typically provide insight as to which specific phosphorylation sites might be involved, which usually requires the use of additional tools such as phosphospecific antisera and/or targeted mutational strategies.
To further explore the regulation of the interaction between the two proteins, we asked whether their apparent colocalization in cell puncta was influenced by a cell-surface G-protein-coupled receptor. We previously established a signaling system based upon fluorescence resonance energy transfer to monitor coupling of a G-protein-coupled receptor to AGS3–Gαi (Oner et al., 2010a,b). In this platform, AGS3 is complexed with Gαi at the cell surface, and this interaction is disrupted by activation of the α2A/D-adrenergic receptor, which typically couples to the pertussis toxin-sensitive G proteins Gi/Go (Oner et al., 2013b). Subsequent to receptor activation, AGS3 is released from the cell cortex and is enriched at the Golgi (Oner et al., 2013b). AGS3 actually appears to cycle back and forth between the Golgi and the cell cortex in a manner dependent upon receptor activation by agonist. The agonist-induced disruption of the AGS3–Gαi complex at the cell cortex was blocked by receptor antagonist and by cell pre-treatment with pertussis toxin, which modifies Gαi/o/t by ADP-ribosylation, uncoupling the G protein complex from receptor activation. These data are consistent with the direct coupling of a G-protein-coupled receptor to AGS3–Gαi in a manner analogous to receptor coupling to the typical heterotrimer Gαβγ (Robichaux et al., 2015).
We thus used this platform to ask whether the dynamics of the receptor–AGS3–Gαi coupling process is influenced by DVL2. In cells co-expressing Gαi, AGS3 is localized to the cell cortex (Fig. 5A), and this localization reflects the interaction of up to four Gαi subunits with the GPR motifs in AGS3 (Oner et al., 2013b). In contrast, cell expression of Gαi did not alter the appearance of DVL2 puncta. Upon agonist-induced activation of the α2A/D-adrenergic receptor, AGS3 was ‘released’ from the cell cortex, as previously reported (Oner et al., 2013b) and localized to DVL2 puncta and at a perinuclear site, as observed by immunofluorescence imaging (Fig. 5A). Both the ‘release’ of AGS3 from the cell cortex and its localization to DVL2 puncta and/or a perinuclear site subsequent to receptor activation was blocked by the receptor antagonist rauwolscine and by cell treatment with pertussis toxin (Fig. 5A). In the absence of DVL2, upon agonist-induced activation of the α2A/D-adrenergic receptor AGS3 was enriched exclusively at the perinuclear site where it colocalized with the trans-Golgi network protein sialyltransferase (Fig. 5B). These data indicate that the interaction of DVL2 and AGS3 is regulated by the phosphorylation–dephosphorylation dynamics of DVL2 and a cell-surface G-protein-coupled receptor signaling system.
AGS proteins are a broad panel of biological regulators that influence signal transfer from receptor to G protein, guanine nucleotide binding and hydrolysis, and G-protein subunit interactions, as well as serving as alternative binding partners for Gα and Gβγ independently of the classic heterotrimeric Gαβγ (Blumer and Lanier, 2014). The concepts advanced following the discovery and characterization of AGS proteins over the past 20 years have markedly altered the landscape with respect to our understanding of G-protein signaling systems and have provided surprising insight into cell and tissue function as well as disease mechanisms (Blumer et al., 2012; Blumer and Lanier, 2014). This is particularly true for AGS3 and related Group II AGS proteins containing GPR motifs as docking sites for Gαi/o. However, the precise signaling mechanisms involved in the cell and tissue responses associated with various members of the AGS protein family is not fully defined.
The course of discovery, functional characterization, and development of conceptual perspectives and mechanistic insight for the AGS family of proteins is somewhat analogous to studies of the WNT signaling system over the years. The discovery of the WNT signaling system and of AGS proteins involved a number of intersecting observations from model organisms, biochemical studies, and functional and protein interaction screens over a period of many years. As is the case with AGS proteins, WNT ligands are implicated in a broad range of tissue and cell functions, including cell and tissue polarity, neurite outgrowth and cell differentiation during development (Nusse and Clevers, 2017). The WNT signaling pathway involves multiple WNT ligands, a family of cell-surface seven-transmembrane receptors, co-receptor proteins and a series of protein interactions and protein phosphorylation–dephosphorylation events subsequent to interaction of WNT ligands with a FZD receptor (Schulte and Wright, 2018). G-protein signaling mechanisms are implicated in various aspects of cell and tissue responses to WNT agonists, with a large body of work focused on canonical and noncanonical pathways for the WNT signaling system (Schulte and Wright, 2018). DVL is a central element in all aspects of WNT-induced cell and tissue effects (Gao and Chen, 2010; Sharma et al., 2018). However, as is the case for AGS3 and AGS family members, the precise signaling mechanisms involved in the various pathways in which DVL acts are not fully defined and remain somewhat elusive (Mlodzik, 2016).
The range of observations and hypothesis testing involved with the study of the WNT signaling system and the AGS proteins have indeed led to new concepts in the field, and thus are of great interest relative to the role of these systems in tissue development and overall system homeostasis and dis-homeostasis in health and disease. The discovery of the apparent intersection of these two signaling platforms through the regulated interaction of AGS3 and DVL2 within the cell is thus of particular note. AGS3 and DVL2 are involved to varying degrees with similar tissue and cell functions and adaptations including cilia formation, cell polarity and system adaptations associated with addiction (Endo and Rubin, 2007; Vladar and Axelrod, 2008; Blumer and Lanier, 2014; Dias et al., 2015).
The first suggestion of a potential interaction between AGS3 and GPR proteins and the DVL2 pathway was the noted similarity of the subcellular distribution of DVL and of point-mutated AGS3 in cytosolic puncta in various cell types. Subsequent studies indicated that the movement of DVL into and out of these puncta was perhaps regulated and played a role in the pivot of WNT–FZD-induced signaling events between canonical and noncanonical pathways. In parallel studies, it was determined that the localization of AGS3 into cell puncta was also regulated by selected binding partners and phosphorylation (Vural et al., 2010, 2018). With this background, we recently reported that expression of DVL2 in the cell actually induced the distribution of AGS3 to cell puncta (Vural et al., 2018), which led to the current study.
In this study, we focused on the interaction of AGS3 and DVL2 in the cell and the regulation of this interaction by cell stimuli. Utilizing a set of tools, we firstly established that the interaction between AGS3 and DVL2 observed by immunofluorescence imaging was also detectable biochemically through co-immunoprecipitation of the two proteins from cell lysates. Secondly, we established that this interaction between AGS3 and DVL2 is regulated by DVL2 phosphorylation status, as assayed by both immunofluorescence imaging in cell puncta and co-immunoprecipitation. Finally, we also noted that the colocalization of AGS3 to the DVL2 puncta is influenced by G-protein-coupled receptor signaling components. Upon expression of Gαi as another AGS3 binding partner, the presence of AGS3 in DVL2 puncta was lost and AGS3 was redistributed to the cell cortex. In addition, activation of a cell surface receptor known to interact with an apparent AGS3–Gαi complex resulted in the ‘release’ of AGS3 from the cell cortex and its redistribution to DVL2 puncta and to the Golgi, as previously reported (Oner et al., 2013b). Although the agonist-induced translocation of AGS3 to the Golgi is suggested to play a role in vesicle dynamics and secretion, the functional role of the agonist-induced translocation of AGS3 to DVL2 puncta will be an important area for further investigation. The regulated distribution of AGS3 to DVL2 puncta may influence the dynamics of receptor desensitization and/or the temporal and location-dependent regulation of specific signaling pathways. The lack of influence of AGS3 on basal and WNT-induced elevation of β-catenin signaling in this specific cell system suggest that the interaction of AGS3 and DVL2 is more directly involved with β-catenin-independent signaling pathways involving the WNT family of ligands.
This report establishes strong evidence for a direct and regulated interaction of two key signaling proteins involved in a broad range of cell and tissue functions. Several observations suggested that the AGS3–DVL2 interaction is enhanced with interventions that decrease the phosphorylation state of DVL2. These observations include the following: (1) the colocalization of AGS3 and DVL2 in cell punctate structures is lost upon co-expression of CK1ε, and this loss is reversed by cell treatment with the CK1ε inhibitor D4476 (Fig. 3B); (2) the shifts in the gel migration pattern for DVL2 observed with co-expression of CK1ε and cell treatment with the inhibitor D4476 are consistent with changes in the phosphorylation status of DVL2 (Figs 3A, 4A–C, input); and (3) the co-immunoprecipitation of DVL2 with AGS3 is diminished by interventions that apparently increase the phosphorylation status of DVL2 (i.e. CK1ε expression; Fig. 4A, lane 1 versus lane 2; Fig. 4B, lane 1; Fig. 4C, lane 1 versus lane 2) and enhanced by interventions that apparently decrease the phosphorylation status of DVL2 (i.e. CK1ε-K38R expression; Fig. 4A, lane 2 versus lane 3; Fig. 4B, lane 1 versus lane 2). AGS3 thus prefers to interact with a population of DVL2 exhibiting an apparent reduction in phosphorylation, which is of particular note given that: (1) the phosphorylation status of DVL2 is regulated by WNT ligands; (2) the distribution of DVL2 within the cell is regulated by its phosphorylation status, with the dephosphorylated form preferentially localized to cell puncta; and (3) the phosphorylation status of DVL2 may be a key determining factor in the activation of β-catenin dependent versus β-catenin independent WNT signaling pathways.
The observations reported herein are consistent with the hypothesis advanced previously that a key element of the functional diversity associated with AGS3 involves its ability to oscillate through various signaling hot spots and compartments within the cell (Vural et al., 2018), which is also a distinguishing characteristic of the Dishevelled proteins. In addition, both DVL2 and GPR proteins such as AGS3, and the related protein LGN (GPSM2), serve as ‘assemblers’ of signal switches that respond to intra- and extra-cellular stimuli, with functional consequences (Zhang et al., 2007; Turm et al., 2010; Blumer et al., 2012; Wynshaw-Boris, 2012; Yuzawa et al., 2011; Kamakura et al., 2013; Žigman et al., 2005; Culurgioni et al., 2011; Zhu et al., 2011). Similarly, the GPR protein RGS14, through interaction with Gαi and H-Ras, also serves as an assembler of such regulatable signal switches (Vellano et al., 2013; Brown et al., 2015). One of the first reported assemblies of such a signalling switch for the family of proteins with a GPR motif and associated G protein α subunits was the polarity complex signaling module involving the AGS3 and LGN ortholog Pins in Drosophila melanogaster (Schaefer et al., 2000, Bellaiche et al., 2001). DVL is involved in similar assembly roles for signaling switches, including the polarity complex signaling module and as a binding partner for the guanine-nucleotide-exchange factor daple (dishevelled-associating protein with a high frequency of leucine residues) in Xenopus leavis (Oshita et al., 2003; Kobayashi et al., 2005; Ishida-Takagishi et al., 2012; Aznar et al., 2015, 2018). These data, and the commonality of functional system effects observed for AGS3 and DVL2, suggest a role for the AGS3–DVL2 complex as a key integrator of signal switches that provides an unexpected path for functional integration of signals within the cell.
MATERIALS AND METHODS
Antibodies and/or antisera to AGS3 (ABS1531), Giα3 and Goα (371726), and β-actin (A3854) as well as the casein kinase I inhibitor D4476 (218705), phosphatase inhibitor cocktail 2 (P5726), transfection grade T-cell factor (TCF) reporter plasmid (21-170; TOPflash assay system), alkaline phosphatase from bovine intestinal mucosa (P0114) and normal rabbit IgG (12-370) were purchased from Millipore Sigma (Burlington, MA). A second polyclonal AGS3 antiserum (referred to here as GPR antibody) was generated in the laboratory of Dr Dzwokai Zach Ma (University of California, Santa Barbara, CA) by immunization of rabbits with a glutathione S-transferase–AGS3 fusion protein consisting of the AGS3-GPR domain (amino acids A461 to S650) (Groves et al., 2010). DVL2 antibodies [mouse monoclonal (10B5), sc-8026 and mouse monoclonal (D-6), sc-390303], casein kinase Iε [mouse monoclonal (A-6), sc-374069] and normal mouse IgG antibody (sc-2025) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyethyenimine (PEI; linear, Mr∼25,000) was obtained from Polysciences, Inc. (Warrington, PA). DC Protein Assay Kit II (5000112) was purchased from Bio-Rad (Hercules, CA). The Dual-Luciferase Reporter Assay System (E1910) was purchased from Promega Corporation (Madison, WI). Dynabeads™ Protein G immunoprecipitation kit (10007D), Prolong Diamond Antifade (P36961) reagent and Spectra Multicolor Broad Range Protein Ladder (26623) were purchased from Thermo Fisher Scientific (Waltham, MA). Recombinant Human WNT-3A protein (5036-WN-010) was purchased from R & D Systems (Minneapolis, MN). pRC/CMV::DVL-2-Myc was obtained from Addgene (Cambridge, MA; plasmid #42194), as deposited by Robert Lefkowitz and Shin-ichi Yanagawa (Lee et al., 1999). pCEP4::V405 4HA-CKIε (plasmid #13724) and pCEP4::V406 4HA-CKIε-K38R (plasmid #13725) were obtained from Addgene (Cambridge, MA), as deposited by David Virshup. mCherry-SiT-N-15 was from Addgene (deposited by Michael Davidson; Addgene plasmid #55133) (Cambridge, MA). All other materials were obtained as previously described.
Cell culture, transfection and dual luciferase reporter assays
HEK-293 (CRL-1573) and COS-7 (CRL-1651) cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA). Both cell lines were cultured in Dulbecco's Modified Eagle Medium (high glucose) supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum. HEK-293 and COS-7 cells were transfected using PEI as described previously (Vural et al., 2018). For the reporter assays, HEK-293 cells were seeded at a density of 8×105 cells per well of 6-well plates and cultured for 12–15 h. Cells were transiently transfected with a total of 1.8 µg of DNA with 4 µg of PEI per ml of cell culture medium. The 1.8 µg of DNA included 350 ng of the TCF reporter plasmid, 35 ng of pRLuc-N3 and varying concentrations of expression plasmids encoding AGS3, DVL2 and/or CK1ε totaling 1415 ng, as specified in the relevant figure legends. 24 h after transfection, cells were washed twice with cell washing solution (137 mM NaCl, 2.6 mM KCl, 1.8 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) and then incubated with 500 ng/ml WNT-3A or vehicle (0.75% bovine serum albumin in phosphate buffered saline) in serum-free medium, or with 20 mM LiCl in full medium, for 24 h. After treatments, cells were processed using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions, and activity was measured on a CLARIOstar multiplate reader. Each data point was run in triplicate, and five independent experiments were performed. Data were normalized to control HEK-293 cells and analyzed by ANOVA and Tukey's multiple comparison post-tests.
Immunoprecipitation and immunoblotting
Cells at 90% confluency in 100-mm dishes were rinsed twice with cell washing solution and disrupted with 1.0 ml of cell lysis buffer [25 mM HEPES (pH 7.4), 4% glycerol, 0.5% (v/v) Nonidet P-40, 150 mM NaCl, 2 mM CaCl2, protease inhibitor cocktail (P8340, Millipore Sigma) and a PhosSTOP inhibitor tablet (Roche Applied Sciences, Indianapolis, IN)]. For treatment with alkaline phosphatase, cells were lysed in cell lysis buffer without added PhosSTOP inhibitor. Lysates were shaken on ice for 15–20 min followed by centrifugation at 371 g for 10 min at 4°C. For immunoprecipitation, the cell lysates were incubated with corresponding primary antibodies for 15–18 h at 4°C with gentle rotation. Lysates were then incubated with Protein G Dynabeads™ for 2 h at 4°C with gentle rotation, followed by washing and elution steps performed using volumes in accordance with the manufacturer's suggestions. 2× Laemmli sample buffer (4% SDS, 20% glycerol, 10% 400 mM DTT, 0.004% Bromophenol Blue and 0.125 M Tris-HCl, pH ∼6.8) was added to an equivalent volume of the eluates, and tubes were then heated in a water bath at 70°C for 10 min. An aliquot of cell lysates used for immunoprecipitation was processed for immunoblotting by addition of 10× protein loading buffer and sample incubation in a boiling water bath for 10 min. To facilitate resolution of multiple DVL2 and AGS3 species by gel electrophoresis, the eluted immunoprecipitation lysates and input cell lysates were processed by denaturing polyacrylamide gel electrophoresis using Novex 4–20% gradient gels (Thermo Fisher Scientific, Waltham, MA) and generally electrophoresed for a longer period of time (5–6 h) at 100V. The gels were then processed for immunoblotting as previously described (Vural et al., 2018).
Fluorescence confocal microscopy and image analysis
HEK-293 and COS-7 cells were processed for immunofluorescence microscopy as described (Vural et al., 2018) and cell images captured with a 40× or 63× oil immersion objective on a Zeiss LSM 800 confocal microscope (Microscopy, Imaging & Cytometry Resources Core at Wayne State University, School of Medicine). All images were obtained from approximately the middle z plane of the cells, and images were visualized and evaluated using the Adobe Photoshop CC 2018 platform.
Data are expressed as mean±s.e.m. as determined from at least five independent experiments. COS-7 cells containing >20 puncta in the cytosol were defined as punctate for quantitation and comparison. At least 200 cells in each individual experiment were counted to determine the percentage of cells containing DVL2 puncta. Data were analyzed with Prism for Mac OS X (Version 7.0a) software (GraphPad Software, San Diego, CA) using either the two-tailed Student's t-test or one-way ANOVA, where significant differences between groups were determined using Tukey's multiple comparison test. P-values <0.05 were considered statistically significant.
The authors are greatly appreciative of the kind gift of AGS3 antisera from Dr Dzwokai Zach Ma (University of California, Santa Barbara, CA). A.V. gratefully acknowledges the support of Dr Raymond Mattingly (Chair, Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI) for his support and encouragement. S.M.L. acknowledges and is greatly appreciative of the opportunity and support provided by M. Roy Wilson (President, Wayne State University) during his tenure at Wayne State University in Detroit, MI. S.M.L. also appreciates the valued suggestions and gracious engagement of the many students, fellows, colleagues and collaborators that have contributed to the body of work involving activators of G-protein signaling.
Conceptualization: A.V., S.M.L.; Methodology: A.V., S.M.L.; Validation: A.V., S.M.L.; Formal analysis: A.V., S.M.L.; Investigation: A.V., S.M.L.; Resources: S.M.L.; Data curation: A.V., S.M.L.; Writing - original draft: A.V., S.M.L.; Writing - review & editing: A.V., S.M.L.; Visualization: A.V., S.M.L.; Supervision: S.M.L.; Project administration: A.V., S.M.L.; Funding acquisition: S.M.L.
Over many years, the research that led to the current publication was supported, in part, by National Institutes of Health grants DA025896 and NS24821 awarded to S.M.L. Deposited in PMC for release after 12 months.
The authors declare no competing or financial interests.