Gαi2 Signaling Regulates Inflammasome Priming and Cytokine Production by Biasing Macrophage Phenotype Determination.

Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein-coupled receptors are essential in transducing signals from the microenvironment, and heterotrimeric Gα signaling links these receptors to downstream effectors. Several Gαi-coupled G-protein-coupled receptors have been implicated in macrophage polarization. In this study, we use genetically modified mice to investigate the role of Gαi2 on inflammasome activity and macrophage polarization. We report that Gαi2 in murine bone marrow-derived macrophages (BMDMs) regulates IL-1ß release after activation of the NLRP3, AIM2, and NLRC4 inflammasomes. We show this regulation stems from the biased polarity of Gαi2 deficient (Gnai2 -/-) and RGS-insensitive Gαi2 (Gnai2 G184S/G184S) BMDMs. We determined that although Gnai2 G184S/G184S BMDMs (excess Gαi2 signaling) have a tendency toward classically activated proinflammatory (M1) phenotype, Gnai2-/- BMDMs (Gαi2 deficient) are biased toward alternatively activated anti-inflammatory (M2) phenotype. Finally, we find that Gαi2-deficient macrophages have increased Akt activation and IFN-ß production but defects in ERK1/2 and STAT3 activation after LPS stimulation. Gαi2-deficient macrophages also exhibit increased STAT6 activation after IL-4 stimulation. In summary, our data indicates that excess Gαi2 signaling promotes an M1 macrophage phenotype, whereas Gαi2 signaling deficiency promotes an M2 phenotype. Understanding Gαi2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.

Activator of G protein signaling 3 modulates prostate tumor development and progression.

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the USA. Activator of G-protein signaling 3 (AGS3/G-protein signaling modulator 1) was shown to be overexpressed in prostate adenocarcinoma relative to the prostate gland. In this study, we investigated the correlation between AGS3 overexpression and PCa malignancy. Immunoblotting analysis and real-time quantitative-PCR showed increase in AGS3 expression in the metastatic cell lines LNCaP (~3-fold), MDA PCa 2b (~2-fold), DU 145 (~2-fold) and TRAMP-C1 (~20-fold) but not in PC3 (~1-fold), relative to control RWPE-1. Overexpression of AGS3 in PC3, LNCaP and MDA PCa 2b enhanced tumor growth. AGS3 contains seven tetratricopeptide repeats (TPR) and four G-protein regulatory (GPR) motifs. Overexpression of the TPR or the GPR motifs in PC3 cells had no effect in tumor growth. Depletion of AGS3 in the TRAMP-C1 cells (TRAMP-C1-AGS3-/-) decreased cell proliferation and delayed wound healing and tumor growth in both C57BL/6 (~3-fold) and nude mice xenografts, relative to control TRAMP-C1 cells. TRAMP-C1-AGS3-/- tumors also exhibited a marked increase (~5-fold) in both extracellular signal-regulated kinase (ERK) 1/2 and P38 mitogen-activated protein kinase (MAPK) activation, which correlated with a significant increase (~3-fold) in androgen receptor (AR) expression, relative to TRAMP-C1 xenografts. Interestingly, overexpression of AGS3 in TRAMP-C1-AGS3-/- cells inhibited ERK activation and AR overexpression as compared with control TRAMP-C1 cells. Taken together, the data indicate that the effect of AGS3 in prostate cancer development and progression is probably mediated via a MAPK/AR-dependent pathway.

Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Regulation of Chemokine Signal Integration by Activator of G-Protein Signaling 4 (AGS4).

Activator of G-protein signaling 4 (AGS4)/G-protein signaling modulator 3 (Gpsm3) contains three G-protein regulatory (GPR) motifs, each of which can bind Gαi-GDP free of Gßγ We previously demonstrated that the AGS4-Gαi interaction is regulated by seven transmembrane-spanning receptors (7-TMR), which may reflect direct coupling of the GPR-Gαi module to the receptor analogous to canonical Gαßγ heterotrimer. We have demonstrated that the AGS4-Gαi complex is regulated by chemokine receptors in an agonist-dependent manner that is receptor-proximal. As an initial approach to investigate the functional role(s) of this regulated interaction in vivo, we analyzed leukocytes, in which AGS4/Gpsm3 is predominantly expressed, from AGS4/Gpsm3-null mice. Loss of AGS4/Gpsm3 resulted in mild but significant neutropenia and leukocytosis. Dendritic cells, T lymphocytes, and neutrophils from AGS4/Gpsm3-null mice also exhibited significant defects in chemoattractant-directed chemotaxis and extracellular signal-regulated kinase activation. An in vivo peritonitis model revealed a dramatic reduction in the ability of AGS4/Gpsm3-null neutrophils to migrate to primary sites of inflammation. Taken together, these data suggest that AGS4/Gpsm3 is required for proper chemokine signal processing in leukocytes and provide further evidence for the importance of the GPR-Gαi module in the regulation of leukocyte function.

Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex.

Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gßγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαßγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oßγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gßγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oßγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαßγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.

Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

G-protein signaling modulator 1 deficiency accelerates cystic disease in an orthologous mouse model of autosomal dominant polycystic kidney disease.

Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1(V/V) mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1(+/+) and Gpsm1(+/-) mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gßγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease.

Activators of G protein signaling exhibit broad functionality and define a distinct core signaling triad.

Activators of G protein signaling (AGS), initially discovered in the search for receptor-independent activators of G protein signaling, define a broad panel of biologic regulators that influence signal transfer from receptor to G-protein, guanine nucleotide binding and hydrolysis, G protein subunit interactions, and/or serve as alternative binding partners for Gα and Gßγ independently of the classic heterotrimeric Gαßγ. AGS proteins generally fall into three groups based upon their interaction with and regulation of G protein subunits: group I, guanine nucleotide exchange factors (GEF); group II, guanine nucleotide dissociation inhibitors; and group III, entities that bind to Gßγ. Group I AGS proteins can engage all subclasses of G proteins, whereas group II AGS proteins primarily engage the Gi/Go/transducin family of G proteins. A fourth group of AGS proteins with selectivity for Gα16 may be defined by the Mitf-Tfe family of transcription factors. Groups I-III may act in concert, generating a core signaling triad analogous to the core triad for heterotrimeric G proteins (GEF + G proteins + effector). These two core triads may function independently of each other or actually cross-integrate for additional signal processing. AGS proteins have broad functional roles, and their discovery has advanced new concepts in signal processing, cell and tissue biology, receptor pharmacology, and system adaptation, providing unexpected platforms for therapeutic and diagnostic development.

EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer.

The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 messenger RNA and EDD transfection enhanced murine Mcl-1 promoter-driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were 4- to 21-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery [1,2-dioleoyl-sn-glycero-3-phophatidylcholine (DOPC)] and cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, P = 0.004; A2780ip2, 75.9% reduction, P = 0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, P = 0.035), with a trend in A2780ip2 (60.3% reduction, P = 0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest that EDD is a therapeutic target for ovarian cancer.

Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by Gαi1 and Gαi-linked G protein-coupled receptors.

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα(i/o)-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα(i1/3)-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα(i1)-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα(i1)-GDP but not active Gα(i1)-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα(i1) complex in brain lysates, and co-expression of RGS14 and Gα(i1) in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα(i)-linked α(2A)-adrenergic receptor induces a conformational change in the Gα(i1)·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα(i1)-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.

Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors.

Group II activators of G-protein signaling (AGS) serve as binding partners for Gα(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-Gα signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with Gα(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-Gα(i1)-YFP BRET were regulated by Ric-8A but not by Gα-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and Gα(i1)-YFP. The inhibitory regulation of GPR-Gα(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-Gα(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-Gα(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-G-Gα(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-Gα(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore Gα expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, Gα(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.

DPP4-Truncated CXCL12 Alters CXCR4/ACKR3 Signaling, Osteogenic Cell Differentiation, Migration, and Senescence.

Bone marrow skeletal stem cells (SSCs) secrete many cytokines including stromal derived factor-1 or CXCL12, which influences cell proliferation, migration, and differentiation. All CXCL12 splice variants are rapidly truncated on their N-terminus by dipeptidyl peptidase 4 (DPP4). This includes the common variant CXCL12 alpha (1-68) releasing a much less studied metabolite CXCL12(3-68). Here, we found that CXCL12(3-68) significantly inhibited SSC osteogenic differentiation and RAW-264.7 cell osteoclastogenic differentiation and induced a senescent phenotype in SSCs. Importantly, pre-incubation of SSCs with CXCL12(3-68) significantly diminished their ability to migrate toward CXCL12(1-68) in transwell migration assays. Using a high-throughput G-protein-coupled receptor (GPCR) screen (GPCRome) and bioluminescent resonance energy transfer molecular interaction assays, we revealed that CXCL12(3-68) acts via the atypical cytokine receptor 3-mediated ß-arrestin recruitment and as a competitive antagonist to CXCR4-mediated signaling. Finally, a reverse phase protein array assay revealed that DPP4-cleaved CXCL12 possesses a different downstream signaling profile from that of intact CXCL12 or controls. The data presented herein provides insights into regulation of CXCL12 signaling. Importantly, it demonstrates that DPP4 proteolysis of CXCL12 generates a metabolite with significantly different and previously overlooked bioactivity that helps explain discrepancies in the literature. This also contributes to an understanding of the molecular mechanisms of osteoporosis and bone fracture repair and could potentially significantly affect the interpretation of experimental outcomes with clinical consequences in other fields where CXCL12 is vital, including cancer biology, immunology, cardiovascular biology, neurobiology, and associated pathologies.

AKAP signaling in reinstated cocaine seeking revealed by iTRAQ proteomic analysis.

To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells.

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.

Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short).

mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression.

Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. We show that mPins (mammalian homologue of Drosophila melanogaster partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR-MAGUK (N-methyl-D-aspartate receptor-membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs.

Loss of activator of G-protein signaling 3 impairs renal tubular regeneration following acute kidney injury in rodents.

The intracellular mechanisms underlying renal tubular epithelial cell proliferation and tubular repair following ischemia-reperfusion injury (IRI) remain poorly understood. In this report, we demonstrate that activator of G-protein signaling 3 (AGS3), an unconventional receptor-independent regulator of heterotrimeric G-protein function, influences renal tubular regeneration following IRI. In rat kidneys exposed to IRI, there was a temporal induction in renal AGS3 protein expression that peaked 72 h after reperfusion and corresponded to the repair and recovery phase following ischemic injury. Renal AGS3 expression was localized predominantly to the recovering outer medullary proximal tubular cells and was highly coexpressed with Ki-67, a marker of cell proliferation. Kidneys from mice deficient in the expression of AGS3 exhibited impaired renal tubular recovery 7 d following IRI compared to wild-type AGS3-expressing mice. Mechanistically, genetic knockdown of endogenous AGS3 mRNA and protein in renal tubular epithelial cells reduced cell proliferation in vitro. Similar reductions in renal tubular epithelial cell proliferation were observed following incubation with gallein, a selective inhibitor of Gßγ subunit activity, and lentiviral overexpression of the carboxyl-terminus of G-protein-coupled receptor kinase 2 (GRK2ct), a scavenger of Gßγ subunits. In summary, these data suggest that AGS3 acts through a novel receptor-independent mechanism to facilitate renal tubular epithelial cell proliferation and renal tubular regeneration.

American Society for Pharmacology and Experimental Therapeutics Division for Pharmacology Education at EB2022-Meeting report.

The American Society for Pharmacology and Experimental Therapeutics (ASPET) held its annual meeting at the Experimental Biology 2022 conference in Philadelphia, PA on April 2-5, 2022. The authors provide a synopsis and discussion of each of the four sessions presented at the meeting under the ASPET Division for Pharmacology Education (DPE).

Receptor-regulated interaction of activator of G-protein signaling-4 and Galphai.

Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.