rapsynoid/partner of inscuteable
The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Two closely related proteins, GPR-1 and GPR-2 (G protein regulator), associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Galphai/o. A number of proteins in other metazoans contain GoLoco motifs in various numbers, and several GoLoco motif proteins, including mammalian AGS3 and Drosophila Pins, have been shown to interact with Galphai/o subunits of heterotrimeric G proteins. Inactivation of lin-5, gpr-1/gpr-2, or the Galphai/o genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Galpha locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Galphai/o signaling (Srinivasan, 2003).
In both Drosophila and C. elegans, a conserved PAR protein complex establishes cell polarity and spindle position but is not required for chromosome movements. This PAR-determined polarity directs spindle positioning possibly through activation of G protein signaling mediated by Pins/Inscuteable (Insc) in Drosophila neuroblasts, Pins/Discs large (Dlg) in Drosophila SOP cells, and GPR/LIN-5 in C. elegans embryos. Although Insc, Dlg, and LIN-5 all act to localize GoLoco proteins, their functions and localizations differ. LIN-5, GPR-1/GPR-2, and Galphai/o interactions appear to be required for cell division and chromosome segregation, whereas no such role has been shown for Drosophila Galphai or Pins. Consistent with a role in chromosome movements, GPR-1/GPR-2 proteins localize to the spindle apparatus, whereas Pins does not. This may indicate that an additional spindle-associated GoLoco protein exists, and/or possibly that in Drosophila multiple Galpha subunits act redundantly in mitosis, as in C. elegans. Consistent with the former hypothesis, a mammalian homolog of Pins, LGN, is required for spindle assembly and localizes to spindle asters (Srinivasan, 2003 and references therein).
G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2, homologs of Drosophila PINS), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. The asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99 (a novel protein that acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle). Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that the signaling of LET-99 and Galpha/GPR-1/2 functions in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division (Tsou, 2003).
Forces must be polarized in response to PAR polarity cues in order to achieve proper spindle positioning. The localization of GPR-1/2 has led to the model that the enrichment of GPR-1/2 at the posterior provides higher pulling forces on the posterior spindle pole, thus mediating anaphase spindle positioning. This model does not address a role for GPR in nuclear rotation, however. Posterior enrichment of GPR-1/2 was seen in only some embryos during nuclear rotation. Such asymmetry at this time is actually predicted to be counter-productive, as it would potentially hold the nucleus at the posterior and prevent centration and rotation (Tsou, 2003).
It has been proposed that the asymmetric enrichment of LET-99 in a cortical band provides the asymmetric cue to polarize forces during both rotation and anaphase. Loss of LET-99 results in an absence of nuclear rotation and an absence of the normal asymmetric spindle pole movements during anaphase. Based on the hyperactive movements of nuclei and metaphase spindles, it is proposed that the ultimate effect of LET-99 activity is a downregulation of cortical forces that act on centrosomes. Because LET-99 is enriched in a cortical band that encircles P lineage cells, downregulation of cortical forces in this region during prophase would result in higher net anterior and posterior forces that would produce a rotational movement of the nuclear-centrosome complex. After rotation, the posterior centrosome/spindle pole lies partially underneath the LET-99 band. Downregulation of cortical forces in the LET-99 band region at this stage would affect lateral astral microtubule interactions, producing higher net forces directed towards the posterior and thus asymmetric anaphase spindle elongation. The results reported here on the genetic interactions between LET-99 and Galpha/GPR signaling are consistent with this model. Loss of LET-99 causes gain of Galpha/GPR-1/2-like phenotypes, hyperactive nuclear and spindle movements. These hyperactive movements are completely suppressed in Galpha(RNAi); let-99 or gpr-1/2(RNAi); let-99 mutant embryos, suggesting that LET-99 opposes Galpha/GPR-1/2 signaling. The antagonistic role of let-99 to Galpha/GPR-1/2 signaling is further supported by the observation that partially reducing let-99 activity suppresses the lethality caused by loss of gpa-16 activity alone. Finally, the weak asymmetry of spindle positioning observed in gpr-1/2(RNAi) embryos is no longer observed in gpr-1/2(RNAi); let-99 double mutant embryos. These results suggest that let-99 not only functions oppositely to Galpha/GPR-1/2 signaling, but also indeed provides an asymmetric cue. Based on these results and the pattern of cortical LET-99 localization, it is proposed that LET-99 antagonizes Galpha/GPR-1/2 signaling, thus downregulating cortical forces asymmetrically during both rotation and anaphase spindle elongation (Tsou, 2003).
Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric G? subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. The Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). The GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. These results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins (Gotta, 2003).
Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage C. elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. The GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, are essential for generation of proper pulling forces. GPR-1/2 interact with guanosine diphosphate-bound GOA-1 and are enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2 (Colombo, 2003).
The Purkinje cell protein-2 (Pcp2, also known as L7) gene is abundantly expressed only in Purkinje cells of the cerebellum and bipolar neurons of the retina. The yspatio-temporal expression pattern of this gene suggests a role for PCP2 in Purkinje cell development or normal cell physiology. A PCP2-deficient mouse was created by gene targeting to test the hypothesis that it is required for Purkinje cell development or function. Although normally present in abundance, the absence of PCP2 in null animals causes no observable cerebellar abnormalities. Behavioral analysis reveals normal abilities for balance and coordination. Null cerebellum has normal Purkinje cell numbers, morphology, and ultrastructure. Retinal bipolar neurons appear similarly unaffected. Aged null animals (22 months) were also examined and no deficits were detected using the same behavioral and histologic analyses. Although the null animal does not reveal the function of PCP2, it does rule out an essential role for PCP2 in Purkinje cell development, in Purkinje cell survival, and in at least some aspects of cerebellar function (Mohn, 1997).
The yeast two-hybrid system has been used to identify proteins that interact with the alpha-subunit of the heterotrimeric GTP-binding protein, Gi2. A human B cell cDNA library was screened with full-length G alpha i2 and four positive colonies were isolated, one of which expresses the 44-kDa COOH terminus of a previously unrecognized 677-amino acid (aa) protein. A full-length clone was isolated from a HeLa cell cDNA library. The deduced protein contains 10 Leu-Gly-Asn repeats, and thus it has been named LGN. Computer analysis indicates that LGN is a mosaic protein with seven repeated sequences of about 40 aa in length at its N-terminal end, and four repeated sequences of about 34 aa at its C-terminal end. Each of the two repeat regions shows substantial similarity to proteins found in other organisms. RT-PCR analysis of human tissues shows that the mRNA of LGN is ubiquitously expressed. The specificity of interaction between G alpha i2 and LGN was confirmed by an in vitro binding assay using recombinant proteins. These data indicate that the yeast two-hybrid system can identify novel proteins, such as LGN, that interact with G alpha proteins. As a mosaic protein, LGN shows similarity with portions of proteins from many species and thus may define a new protein family (Mochizuki, 1997).
The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice, but its function remains unknown because Pcp2 knockout mice are normal. Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 are cotransfected into COS cells, Galphao is detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function (Luo, 1999).
The G-protein regulatory (GPR) motif in AGS3 is a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 20-amino acid motif, a GPR consensus peptide was designed and its influence on the activation state of G-protein and receptor coupling to G-protein was determined. The GPR peptide sequence (28 amino acids) encompasses the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevents the binding of AGS3 to Gialpha1,2 in protein interaction assays, inhibits guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilizes the GDP-bound conformation of Gialpha. The GPR peptide has little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocks receptor coupling to Gialphabetagamma, indicating that although the AGS3-GPR peptide stabilizes the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) is not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems (Peterson, 2000).
Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). A human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. LGN binds the nuclear mitotic apparatus protein NuMA (Drosophila homolog: Mushroom body defect), which tethers spindles at the poles, and this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopus egg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, a mammalian Pins homolog has been identified as a key regulator of spindle organization during mitosis (Du, 2001).
LGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), that is required for polarity establishment and asymmetric cell divisions during embryonic development. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis. LGN also regulates spindle organization, possibly through inhibition of NuMA function, but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, it is shown that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. It is therefore proposed that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization (Du, 2002).
Mammalian LGN/AGS3 proteins and their Drosophila Pins ortholog are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in neuroblast asymmetric division. Using overexpression studies in different cell line systems, it has been demonstrated that like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. In WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression (Kaushik, 2003).
Asymmetric cell division is a fundamental mechanism used to generate cellular diversity in invertebrates and vertebrates. In Drosophila, asymmetric division of neuroblasts is achieved by the asymmetric segregation of cell fate determinants Prospero and Numb into the basal daughter cell. Asymmetric segregation of cell fate determinants requires an apically localized protein complex that includes Inscuteable, Pins, Bazooka, DmPar-6, DaPKC and Galphai. Pins acts to stabilize the apical complex during neuroblast divisions. Pins interacts and colocalizes with Inscuteable, as well as maintaining its apical localization. A mouse homolog of pins (Pins) has been isolated and its expression profile has been characterized. Mouse PINS shares high similarity in sequence and structure with Pins and other Pins-like proteins from mammals. Pins is expressed in many mouse tissues but its expression is enriched in the ventricular zone of the developing central nervous systems. PINS localizes asymmetrically to the apical cortex of mitotic neuroblasts when ectopically expressed in Drosophila embryos. Like Pins, its N-terminal tetratricopeptide repeats can directly interact with the asymmetric localization domain of Insc, and its C-terminal GoLoco-containing region can direct localization to the neuroblast cortex. Pins can fulfill all aspects of pins function in Drosophila neuroblast asymmetric cell divisions. These results suggest a conservation of function between the fly and mammalian Pins homologues (Yu, 2003a).
Database searches of the mouse genome with the fly Pins amino acid sequence identified EST clones that encode two Pins-like proteins with varying homologies to Pins. The mouse protein showing a higher percentage of homology to Drosophila Pins is referred to as PINS. PINS shows a higher level of homology to human LGN than to rat AGS3. The second mouse protein is more closely related to AGS3 than to LGN and is therefore referred to as mouse AGS3. Hence, there are at least two homologues of Drosophila Pins in mouse, PINS and mouse AGS3. Similarly, the human genome project also identifies two Pins-like sequences, LGN and AGS3. Hence, PINS/LGN and mouse AGS3/AGS3 appear to be paralogues, formed by duplication after divergence of mammals and flies. The two Pins-like proteins identified in the mammalian genomes have different features. In situ hybridization of mouse Pins and Ags3 shows a distinct distribution in the neural tube: Pins is enriched in a layer of cortical precursors, whereas Ags3 is uniformly distributed in the neural tube, suggesting distinct roles for these proteins during neurogenesis. This is reminiscent of the localization profiles of mouse numb and numb-like in the neural tube of the mouse embryo (Yu, 2003a).
During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai (see Drosophila Gαi) and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, it was found that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. It is proposed that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode (Du, 2004).
Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in C. elegans and Drosophila have placed RIC-8 (Ric8a in Drosophila) in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the G alphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of G alphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and G alphai regulate a similar process. Mammalian Ric-8A dissociates G alphai-GDP/LGN/NuMA complexes catalytically, releasing activated G alphai-GTP in vitro. Ric-8A-stimulated activation of G alphai causes concomitant liberation of NuMA from LGN. It is concluded that Ric-8A efficiently utilizes GoLoco/G alphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Galphai-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division (Tall, 2005).
Models are envisioned in which one cellular function of Ric-8A is to dissociate Galphai-GDP/GoLoco complexes by stimulation of nucleotide exchange. G protein control of asymmetric cell division involves cycling of Galphai between its GDP- and GTP-bound forms, as evidenced by the fact that (in C. elegans) both RIC-8 and RGS7 influence the pathway in opposed fashion. It remains speculative whether Galphai-GDP/GoLoco or the production of Galphai-GTP from a GoLoco scaffold activates signaling. It stands to reason that Galphai-GTP must dissociate from GoLoco at some point during signaling. If multiple rounds of cycling between Galphai-GDP/GoLoco and liberated Galphai-GTP are required to complete cell division, then Ric-8A-stimulated dissociation of a Galphai/GoLoco complex could be responsible for either terminating or activating the signal. In either context, RGS-facilitated hydrolysis of GTP by Galpha ensues. The resultant Galphai-GDP could rebind to GoLoco (and not betagamma) to complete one round of the cycle. Rapid cycling of this process may be necessary to regulate the pulling forces on microtubules appropriately during a round of chromosome segregation (supporting information on the PNAS web site, for these proposed models). Regulation of other Galpha or Galpha/GoLoco-mediated signaling pathways by Ric-8A is also worth considering, given the number of distinct Galpha binding partners of mammalian Ric-8A and Ric-8B and the many processes that appear to be regulated by RIC-8 in C. elegans (Tall, 2005).
In cell polarization of Drosophila neuroblasts, Inscuteable (Insc) functions via tethering Partner of Insc (Pins) to Bazooka, homologous to human cell polarity protein Par3. However, little has been known about mammalian homologues of Insc. Two distinct cDNAs have been cloned from human Insc gene, which is differentially expressed from alternative first exons: one encodes 579 amino acids, whereas the other lacks the N-terminal 47 amino acids. In contrast to human homologues for Pins and Par3, human Insc exhibits a weak homology with the Drosophila counterpart. Nevertheless, human Insc proteins bind to the human Pins homologues LGN and AGS3, and also to human Par3 and its related protein Par3beta. Although LGN by itself is incapable of interacting with Par3, coexpression of human Insc leads to the interaction between LGN and Par3, indicating that human Insc plays an evolutionarily conserved role as an adaptor protein that links Pins to Par3 (Izaki, 2006).
Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. mPins (mammalian homologue of Drosophila 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 (Sans, 2005).
mPins is a ubiquitously expressed protein that is critical for the regulation of mitotic spindle organization in dividing cells. mPins interacts with several functionally distinct proteins, including NuMA, Ras, LKB1 and Galphai. The finding that mPins interacts with the PSD-95 family adds another group of important proteins to those whose trafficking depends on mPins. Drosophila Pins is required for asymmetric division of sensory organ precursor cells (pI) and dividing neuroblasts. Whereas the roles of Pins in cell division are relatively well-characterized, the function of mPins in the mature mammalian central nervous system remains enigmatic. The related protein, AGS3, may affect cocaine-induced plasticity by regulating G-protein signalling in the prefrontal cortex. The data show that mPins and AGS3 are both expressed in the developing hippocampus but have different subcellular localizations, perhaps because mPins, but not AGS3, interacts with SAP102. Moreover, AGS3 is down-regulated in adult hippocampus and seems to be absent from the PSD, whereas mPins is expressed throughout development and is enriched in synaptic membranes. mPins and AGS3 are found in different domains throughout the cell body and dendrites in primary cultures of hippocampal neurons. mPins, but not AGS3, redistributes into punctate structures after ionomycin or NMDA treatment, suggesting that calcium signalling functions in trafficking of mPins complexes. These findings strongly suggest that these two orthologues of Drosophila Pins have different functions in neurons (Sans, 2005).
The MAGUKs do not compete with the other known interacting proteins of mPins suggesting that the association of these other interacting proteins may indirectly influence the trafficking of the MAGUK and its associated proteins, such as NMDARs. Both Ras and Galphai are particularly interesting in this context. Ras has been implicated in the trafficking of GluRs. Characterized as molecular switches that alternate between GTP-bound ('on') and GDP-bound ('off') forms, these proteins are involved in the reorganization of synaptic structure. G-proteins, such as Galphai, influence NMDAR trafficking through metabotropic GluRs. In this study, it is shown that Galphai proteins function in NMDAR trafficking through a direct interaction with the mPins-SAP102 complex. mPins mediates G-protein signalling through binding to Galphai1-3GDP, thereby inhibiting binding of Galphai to Gßγ (and consequently enhancing Gßγ signalling in the absence of a G-protein-coupled receptor). mPins shifts between a closed state, when the N- and C-terminal halves of the protein bind to one another, and an open state when NuMA binds to mPins to switch it open, allowing the binding of Galphai. SAP102, similarly to SAP97, may exist in the cytoplasm as a folded molecule in which the GK domain is folded onto the SH3 domain. The data suggest that SAP102 binds to mPins in its closed state, as the two proteins localized in ring-like structures in COS cells. Therefore, mPins could be required upstream of, or in parallel to, the NR2B-SAP102 interaction. It is also shown that SAP102-mPins complexes have a different fate from that of NR2B-SAP102-mPins complexes, since the three proteins form clusters in COS cells and synaptic clusters in spines. These data suggest that NMDARs can open the SAP102-mPins complexes. Interestingly, cotransfection of the linker region of mPins with NR2B and SAP102 results in the formation of ternary complexes that are rapidly degraded, suggesting that interaction of Galphai with GoLoco domains (or an unidentified protein with TPR domains) is important for stabilization of the complex. mPins can bind four Galphai molecules, and it is unclear at present whether all of the sites need to be occupied for proper folding and targeting of mPins. As a modulator of G-protein signalling, the possibility cannot be excluded that Galphai binds to the NMDAR-MAGUK-mPins complex at synapses after activation of a G-protein-coupled receptor. Studies have suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) can exhibit some of their effects through interactions with heterotrimeric G-proteins in addition to their ionic channel function. For instance, it has been shown that AMPA can induce dissociation of Galphai1 from the Galphai1/ß heterotrimeric complex and its association with GluR1 through an adaptor protein. In light of the current data, the possibility exists that Galphai signalling proteins may also be recruited to certain MAGUK-mPins complexes through simultaneous dissociation from AMPARs (Sans, 2005).
The results suggest that the NMDAR associates indirectly through SAP102 with two molecular complexes -- the exocyst and mPins-Galphai complexes -- and that these associations are necessary for proper trafficking of receptors in neurons. The results also suggest that this complex is formed in the ER in heterologous cells and early in the secretory pathway in neurons. Although this has not been demonstrated directly for native proteins, an association of MAGUK with AMPARs in the ER (or cis-Golgi) has been shown for native AMPARs in brain by using the endo-H sensitivity of immature AMPARs, so such an association is not unprecedented. These results suggest that NMDARs are trafficked as part of a large complex from their site of synthesis in the cell body to the postsynaptic membrane, presumably in a transport vesicle. The identification of other components of the SAP102 cargo complex (containing NMDARs, the exocyst and mPins-Galphai complexes) will undoubtedly help to clarify the steps involved in trafficking of NMDARs from assembly and ER exit to transport in dendrites and spines in normal and disease states (Sans, 2005).
G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). This study reports that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, an essential role was demonstrated of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability (Wiser, 2006).
In Drosophila, the GDI proteins Partner of Inscuteable (PINS) and Locomotion Defects (LOCO) act synergistically in neuroblasts to release Gβγ subunits, thereby ensuring asymmetric division. The mammalian homologs of these proteins are LGN, also known as mPINS, and Activator of G protein Signaling 3 (AGS3). These proteins contain three or four GPR motifs in the C-terminal GPR domain, seven tetratricopeptide (TPR) motifs in the N-terminal TPR domain, and a linker peptide in between; the linker as well as the TPR domain interact with other proteins. Both LGN and AGS3 bind Gαi more strongly than Gαo, and exhibit much stronger GDI activity for Gαi than Gαo. Both LGN and AGS3 are expressed in neurons, though likely in different subcellular compartments; these GDI proteins may also be subjected to different regulation (Wiser, 2006).
The importance of GDI proteins in neuronal signaling is exemplified by the identification of AGS3 as a gatekeeper for the behavioral manifestation of drug dependence. Whereas AGS3 expression has no detectable direct effect on GPCR-mediated activation or inhibition of adenylyl cyclase, AGS3 is required for the paradoxical activation of protein kinase A (PKA) by opiates acting on Gi-coupled receptors. Indeed, AGS3 at the nucleus accumbens core plays an instrumental role in the relapse of heroin-seeking behavior (Wiser, 2006 and references therein).
The functional role of LGN in neurons is an interesting open question. In proliferating cells, LGN regulates mitotic spindle organization, analogous to the function of its Drosophila and C. elegans counterparts. This function involves interaction of its TPR domain with its GPR domain as well as the Nuclear Mitotic Apparatus (NuMA) protein, and the ability of its GPR domain to bind Gα-GDP, apparently without the involvement of any GPCR. Whether LGN contributes to GPCR signaling has not been explored, notwithstanding the broad LGN expression in postmitotic neurons, the interesting redistribution of LGN upon activation of NMDA receptors in hippocampal neurons, and its likely involvement in NMDA receptor trafficking due to its ability to bind the MAGUK protein SAP102. How might GDIs like LGN regulate GPCR signaling? Do they reduce GPCR coupling to G proteins and hence diminish GPCR signaling? Or do they enhance activation of Gβγ effectors by stabilizing and sequestering Gα-GDP so as to prolong the action of Gβγ? Could LGN regulate Go-coupled receptors (Wiser, 2006 and references therein)?
This study characterizes LGN modulation of the Gβγ effector, the GIRK (Kir3) channels that mediate slow synaptic inhibition in mammalian brain. LGN was found to increase basal GIRK current but reduce GPCR-induced GIRK current in expression systems such as Xenopus oocytes and HEK293 cells; these functions have been verified by RNAi-mediated knockdown of LGN endogenous to HEK293 cells. This study demonstrates LGN modulated signaling of Go-coupled receptors as well as Gi-coupled receptors, but not Gs-coupled receptors. Moreover, reducing LGN endogenous to hippocampal neurons abolished basal GIRK current and increased excitability. Whereas elevating or reducing LGN protein level had opposite effects on basal GIRK channel activity, the bidirectional effects on neuronal excitability likely involve other proteins under LGN modulation as well. Finally, expression of the TPR-containing protein interaction domains of LGN in mammalian HEK293 cells or hippocampal neurons mimics the effects of LGN and GPR, indicating that LGN may be modulated not only by self interaction but also by sequestration in the postmitotic neurons (Wiser, 2006).
Chronic cocaine administration reduces G protein signaling efficacy. The expression of AGS3, which binds to GialphaGDP and inhibits GDP dissociation, is upregulated in the prefrontal cortex (PFC) during late withdrawal from repeated cocaine administration. Increased AGS3 is mimicked in the PFC of drug-naive rats by microinjecting a peptide containing the Giα binding domain (GPR) of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of Tat-GPR mimicked the phenotype of chronic cocaine-treated rats by manifesting sensitized locomotor behavior and drug seeking and by increasing glutamate transmission in nucleus accumbens. By preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Giα was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. It is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC (Bowers, 2004).
Neurons in the developing mammalian brain are generated from progenitor cells in the proliferative ventricular zone, and control of progenitor division is essential to produce the correct number of neurons during neurogenesis. This study establishes that GΔgamma subunits of heterotrimeric G proteins are required for proper mitotic-spindle orientation of neural progenitors in the developing neocortex. Interfering with Gbetagamma function in progenitors causes a shift in spindle orientation from apical-basal divisions to planar divisions. This results in hyperdifferentiation of progenitors into neurons as a consequence of both daughter cells adopting a neural fate instead of the normal asymmetric cell fates. Silencing AGS3, a nonreceptor activator of Gbetagamma, results in defects similar to the impairment of Gbetagamma, providing evidence that AGS3-Gbetagamma signaling in progenitors regulates apical-basal division and asymmetric cell-fate decisions. Furthermore, the observations indicate that the cell-fate decision of daughter cells is coupled to mitotic-spindle orientation in progenitors (Sanada, 2005).
The spatio-temporal regulation of symmetrical as opposed to asymmetric cell divisions directs the fate and location of cells in the developing CNS. In invertebrates, G-protein regulators control spindle orientation in asymmetric divisions, which generate progeny with different identities. This study investigated the role of the G-protein regulator LGN (also called Gpsm2) in spindle orientation and cell-fate determination in the spinal cord neuroepithelium of the developing chick embryo. LGN was shown to be located at the cell cortex and spindle poles of neural progenitors, and it regulates spindle movements and orientation. LGN promotes planar divisions in the early spinal cord. Interfering with LGN function randomizes the plane of division. Notably, this does not affect cell fate, but frequently leads one daughter of proliferative symmetric divisions to exit the neuroepithelium prematurely and to proliferate aberrantly in the mantle zone. Hence, tight control of planar spindle orientation maintains neural progenitors in the neuroepithelium, and regulates the proper development of the nervous system (Morin, 2007).
During mammalian development, neuroepithelial cells function as mitotic progenitors, which self-renew and generate neurons. Although spindle orientation is important for such polarized cells to undergo symmetric or asymmetric divisions, its role in mammalian neurogenesis remains unclear. This study shows that control of spindle orientation is essential in maintaining the population of neuroepithelial cells, but dispensable for the decision to either proliferate or differentiate. Knocking out LGN, (the G protein regulator), randomizes the orientation of normally planar neuroepithelial divisions. The resultant loss of the apical membrane from daughter cells frequently converts them into abnormally localized progenitors without affecting neuronal production rate. Furthermore, overexpression of Inscuteable to induce vertical neuroepithelial divisions shifts the fate of daughter cells. These results suggest that planar mitosis ensures the self-renewal of neuroepithelial progenitors by one daughter inheriting both apical and basal compartments during neurogenesis (Konno, 2008).
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