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 Galpha_i/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 Galpha_i/o subunits of heterotrimeric G proteins. Inactivation of lin-5, gpr-1/gpr-2, or the Galpha_i/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 Galpha_i/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 Galpha_i/o interactions appear to be required for cell division and chromosome segregation, whereas no such role has been shown for Drosophila Galpha_i 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 G_alpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although G_alpha 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, G_alpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the G_alpha/GPR-1/2 signaling pathway, providing an explanation for how G_alpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, G_alpha 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 G_alpha/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 G_alpha/GPR signaling are consistent with this model. Loss of LET-99 causes gain of G_alpha/GPR-1/2-like phenotypes, hyperactive nuclear and spindle movements. These hyperactive movements are completely suppressed in G_alpha(RNAi); let-99 or gpr-1/2(RNAi); let-99 mutant embryos, suggesting that LET-99 opposes G_alpha/GPR-1/2 signaling. The antagonistic role of let-99 to G_alpha/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 G_alpha/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 G_alpha/GPR-1/2 signaling, thus downregulating cortical forces asymmetrically during both rotation and anaphase spindle elongation (Tsou, 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, 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).

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).

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 G_alphai. 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, 2003).

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, 2003).