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J. Biol. Chem., Vol. 277, Issue 18, 15897-15903, May 3, 2002

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Expression Analysis and Subcellular Distribution of the Two G-protein Regulators AGS3 and LGN Indicate Distinct Functionality

LOCALIZATION OF LGN TO THE MIDBODY DURING CYTOKINESIS^*

Joe B. Blumer, L. Judson Chandler, and Stephen M. Lanier^§

From the Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112 and the  Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, December 20, 2001, and in revised form, January 31, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activator of G-protein signaling 3 (AGS3) and LGN have a similar domain structure and contain four G-protein regulatory motifs that serve as anchors for the binding of the GDP-bound conformation of specific G-protein  subunits. As an initial approach to define further the different functional roles of AGS3 and LGN, we determined their expression profile and subcellular distribution. AGS3- and LGN-specific antisera indicated a widespread tissue distribution of LGN, whereas AGS3 is primarily enriched in brain. Brain punch biopsies of 13 discrete brain regions indicated that both AGS3 and LGN are expressed in all areas tested but are differentially regulated during development. LGN is expressed in neuronal, astroglial, and microglial cultures, whereas AGS3 expression is restricted to neurons. In primary neuronal cultures as well as in dividing cultures of PC12 cells, immunocytochemistry indicated distinct subcellular locations of AGS3 and LGN. The subcellular locations of the two proteins were differentially regulated by external stimuli and the cell cycle. In PC12 and COS7 cells, LGN moves from the nucleus to the midbody structure separating daughter cells during the later stages of mitosis, suggesting a role for G-proteins in cytokinesis. Thus, although AGS3 and LGN share a similar overall motif structure and both bind G-proteins, nature has endowed these proteins with different regulatory elements that allow functional diversity by virtue of tissue-specific expression and subcellular positioning.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heterotrimeric G-proteins transduce extracellular stimuli sensed by cell surface receptors into integrated cellular responses. An activated cell surface receptor initiates a change in the conformation of G subunits such that GDP is exchanged for GTP and G appears to dissociate from G. Both G and G then regulate the activity of various effectors with signal termination involving hydrolysis of bound GTP and apparent reassociation of GGDP and G. Recent data indicate that additional proteins influence the G-protein activation cycle with unexpected modes of regulation (1-17). Such proteins have broad implications for signal processing.

The family of RGS proteins acts to accelerate the rate of GTP hydrolysis by specific G subunits (12). RGS proteins apparently play important roles in terminating responses initiated by cell surface receptors and maintaining the dynamics of receptor-regulated events by promoting efficient cycling of activated G-proteins (13). A second mode of regulation is represented by proteins such as activators of G-protein signaling (AGS)¹ (1-10), the NG108-15 G-protein activator (14-15), phosphatidylethanolamine-binding protein (16), and neuromodulin (17), which regulate the activation state of G-protein independent of a G-protein-coupled receptor. Interestingly, the latter group of proteins may either act in a similar manner as a receptor, promoting nucleotide exchange on G subunits (i.e. AGS1, the NG108-15 G-protein activator, phosphatidylethanolamine-binding protein), or by influencing subunit interactions independent of nucleotide exchange (i.e. AGS2, AGS3, LGN, and PINS). The interactions among these proteins and their roles in integrated cellular responses are of keen interest.

In this study we focused on AGS3 and LGN, which possess a shared domain structure and exhibit 59% sequence identity. AGS3 was discovered in a functional screen for receptor-independent activators of G-proteins (1-2), whereas LGN was identified as a G_i-binding protein in a yeast two-hybrid screen (18). Both AGS3 (650 amino acids) and LGN (677 amino acids) consist of a three-module structure. The first third of the protein contains seven tetratricopeptide repeats (TPR) followed by a linker sequence that connects the TPR domain to a series of four G-protein regulatory (GPR) motifs (2, 4) also known as the GoLoco motif (19). The linker regions in the two proteins do not share sequence homology. The GPR domain is also found in other proteins including RGS12, RGS14, Rap1GAP, Pcp2, and G181b. Each of the four GPR motifs in AGS3 is capable of binding G_i, and a GPR peptide stabilizes the GDP-bound conformation of G_i (2-9) effectively acting as guanine nucleotide dissociation inhibitors for G_i subunits. A recombinant AGS3 protein consisting of four GPR repeats binds more than one G_i subunit simultaneously (4), suggesting a possible scaffolding function for AGS3 and LGN within a larger signal transduction complex.

The modular domain structure of AGS3 and LGN defines an evolutionarily conserved family of proteins with a single gene product identified in Drosophila melanogaster (Partner of Inscuteable, PINS) (20-22) and Caenorhabditis elegans (F32A6.4) (4). Recent studies (20, 23, 24) in Drosophila indicate that PINS is required for asymmetric division of neuroblasts and for regulating planar polarity of developing sensory organ precursor cells. The functional role for the C. elegans protein is not known. Of particular note is that the role of PINS in asymmetric cell division apparently involves heterotrimeric G-proteins (24, 25). A broader role for heterotrimeric G-proteins in cell polarity and cell division is indicated by studies in both C. elegans and higher organisms (24-27). Thus, nature evolved two related proteins, AGS3 and LGN, but the roles of these two proteins in orthologous functions observed in lower organisms have yet to be determined.

As an initial approach to understand the relationship between the mammalian proteins AGS3 and LGN, we defined the tissue distribution and subcellular distribution of each protein. LGN is expressed in all rat tissues and cell lines tested, whereas AGS3 is primarily enriched in the brain, testes, and heart (6). In primary neuronal cultures as well as in dividing cultures of PC12 cells, immunocytochemistry indicated distinct subcellular locations of AGS3 and LGN. Of particular note, the subcellular distributions of AGS3 and LGN are differentially regulated by external stimuli and the cell cycle. During the late phases of mitosis in PC12 and COS7 cells, LGN moves from the nucleus to the midbody structure separating the daughter cells, suggesting a role for G-proteins in cytokinesis. Such migration of LGN within the cell during the cell cycle is reminiscent of the behavior of the AGS3/LGN Drosophila ortholog PINS during cell division and development. Thus, although AGS3 and LGN share a three-module structure and both bind G-proteins, nature has endowed these proteins with different regulatory elements that allow functional diversity by virtue of tissue-specific expression and subcellular positioning.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Polyvinyl difluoride membranes were obtained from Pall Gelman Sciences (Ann Arbor, MI). Poly-L-lysine, N-methyl-D-aspartate (NMDA), plasma-derived horse serum (PDHS), and DNase were purchased from Sigma. VectaShield mounting medium was purchased from Vector Laboratories (Burlingame, CA). Normal donkey serum was purchased from Jackson ImmunoResearch (West Grove, PA). Ionomycin was obtained from Calbiochem. Tetrodotoxin and (+)-5-methyl-10,11-dihydro-5H-dibenzo[,]cyclohepten-5,10-imine hydrogen maleate (MK801) were obtained from Alexis (San Diego, CA). Fluorescently labeled goat anti-rabbit secondary antibodies, phalloidin 594, and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Molecular Probes (Eugene, OR). An adult rat multiple tissue immunoblot (WB48) was obtained from Oncogene Research Products (La Jolla, CA). Restriction endonucleases were obtained from New England Biolabs (Beverly, MA). Fetal bovine serum, B27 supplements, Dulbecco's modified Eagle's medium (DMEM), and neurobasal media were obtained from Invitrogen. Trypsin was obtained from Worthington Biochemicals (Lakewood, NJ). FuGENE 6 reagent was obtained from Roche Molecular Biochemicals.

Plasmid Constructs-- LGN cDNA was obtained from the I.M.A.G.E. Consortium and was used as template for a polymerase chain reaction using Pfu (Stratagene, La Jolla, CA) as the polymerase with forward oligonucleotide 5'-CTTGGTACCGATGAGAGAAGACCATTCT-3' with a 5' KpnI site and reverse oligonucleotide 5'-ATGCTCGAGCTAATGGTCTGCCGATTTTTTCCC-3'with a 5' XhoI site and cloned into the mammalian expression vector pcDNA3. The construct was sequenced before use to confirm the fidelity of the PCR.

Antibody Generation and Immunoblotting-- AGS3-specific antisera were generated as described previously (4, 6). For LGN antisera, a peptide from human LGN (Ser⁴²⁰-Lys⁴⁴⁹) was synthesized and conjugated for generation of rabbit polyclonal antisera using the Peptide Synthesis and Antibody Production Facility at the Medical University of South Carolina. The antisera were affinity-purified prior to use for immunoblotting and immunocytochemistry as described previously (4, 6). Denaturing gel electrophoresis, immunoblotting, and reprobing of membrane transfers were performed as described previously (4).

Cell Culture, Transfection, and Tissue Fractionation-- PC12 and COS7 cells were cultured as described previously (28). Subconfluent 100-mm dishes of cells were transiently transfected with FuGENE 6 reagent according to the manufacturer's directions, and cells were allowed to grow 2 days prior to use. Cerebral cortical cultures (high density) were prepared from newborn rat pups as described previously (29). Low density hippocampal cultures used for immunocytochemistry were prepared as follows. Cells were plated onto poly-L-lysine precoated culture dishes at a density of ~250,000 cells/35-mm dish and incubated at 37 °C in a humidified incubator with 5% CO₂, 95% air for ~3 h to allow attachment. The DMEM containing 10% PDHS was then replaced with 2 ml of astroglial conditioned neurobasal media with B27 supplements. The neurobasal medium without B27 was conditioned by prior incubation with astroglia for 24 h. After 3 days in culture, 5 µM -cytosine arabinoside (ARC) was added to the dish, and a 1/4 media change was performed once/week. Cultures were typically used for experiments after 2-3 weeks of growth. Astroglia were prepared by plating cell suspensions used for preparation of cortical cultures at low density without ARC in 150-mm flasks. After 2 days, the DMEM/PDHS media were replaced with DMEM containing 10% fetal bovine serum and were allowed to grow to confluency before use in conditioned neurobasal media. Microglia were isolated from astroglia. 150-mm flasks of astroglial cultures were shaken on a rotating shaker for 6 h. The medium containing floating microglia was removed and spun at 600 × g for 10 min to obtain the microglial cell pellet.

Primary cultures of cortical neurons were harvested by washing twice with phosphate-buffered saline (4 °C) and then lysed in 2% SDS by sonication. Tissue lysates were generated by homogenization in 5 mM Tris-HCl, pH 7.4, 5 mM EDTA, 5 mM EGTA, 1% Nonidet P-40 at 4 °C. Brain punch biopsies were obtained from adult rat brain by gross dissection on ice followed by punches of discrete brain areas from the dissected areas. Brain sections were snap-frozen on dry ice and stored at 80 °C. Frozen tissue was prepared for immunoblot analysis by placing in 2% SDS followed by probe sonication. Samples were boiled for 5 min and then centrifuged for 30 min at 15,000 × g to remove insoluble material. Protein concentrations were determined by a Bio-Rad DC protein assay.

Immunocytochemistry-- PC12 cells were seeded onto sterile 25-mm polylysine-coated coverslips. Primary cultures of hippocampal neurons were grown as described and plated onto 35-mm polylysine-coated plastic dishes (Falcon, BD PharMingen). For experiments using NMDA or ionomycin, primary hippocampal neuronal cultures growing for 12-24 days were washed once in HEPES buffer (HB: 25 mM HEPES, pH 7.4, 25 mM NaHCO₃, 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 150 mM glucose) and incubated in the presence of 1 µM tetrodotoxin for 1 h at 37 °C prior to any treatment. Conditions of cell treatment are as indicated in the figure legend, and all incubations were at 37 °C. Cells were washed twice with ice-cold PBS and then fixed with 4% paraformaldehyde, 4% sucrose in PBS for 15 min. After washing 3 times for 5 min with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, followed by three 5-min PBS washes. 5% normal donkey serum was used as a blocking agent for 1 h, followed by a 2-h incubation with primary antibodies diluted 1:50 in PBS and a 1-h incubation with secondary antibody (goat anti-rabbit AlexaFluor488 or AlexaFluor594, highly cross-adsorbed, Molecular Probes) diluted 1:2000 in PBS. All antibody dilutions were centrifuged at 10,000 × g for 10 min prior to use. In some cases, 1 µg/ml DAPI was added to the diluted secondary antibody. In some cases, cells were incubated with 5 units/ml AlexaFluor 594 phalloidin in PBS for 5 min after incubation with secondary antibodies. Slides were then mounted with glass coverslips and analyzed by confocal or deconvolution microscopy. Images were obtained either from an Olympus confocal fluorescence microscope (FV5-PSU laser, BX50, 60× oil immersion objective) or a Leica DMRXA deconvolution fluorescence microscope using a 40× oil immersion objective. Images were deconvoluted using SlideBook deconvolution software (Intelligent Imaging Innovations, Denver, CO) with a "no neighbors" deconvolution algorithm. All images were obtained from approximately the middle plane of the cells.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genes Encoding AGS3 and LGN-- AGS3 (AF107723) and LGN (U54999) possess the same motif architecture and exhibit 59% amino acid sequence identity (Fig. 1). AGS3 cDNA was initially isolated from a rat brain cDNA library, and searches of human genome and EST data bases reveal multiple AGS3 ESTs encoding the human counterpart.² The genes for rat or mouse AGS3 and LGN are not yet defined, and only the final three exons of the AGS3 gene (NT 024000.7) are identified in the Human Genome Resources data base (chromosomal location, 9q34.2-34.3). The human LGN gene (NT 029860.2) is located on chromosome 1p13.1, spans 44,690 bp, and contains 14 exons (Fig. 1). AGS3 and LGN possess identical exon/intron junctions and size for the region of AGS3 currently available in the genome (Fig. 1). The gene for the AGS3/LGN ortholog in C. elegans (F32A6.4, GenBank^TM/EBI Data Bank accession number U40409.1) also contains 14 exons and exhibits an overall structure similar to the human LGN and AGS3 genes (Fig. 1). In contrast, the gene encoding the Drosophila AGS3/LGN ortholog PINS (chromosome 3R98A3-4) contains only 2 exons. Thus, the C. elegans F32A6.4 gene structure is clearly more closely related to the human LGN and AGS3 gene structure than is the Drosophila pins gene.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the exon structure of AGS3-related genes. TPR and GPR domains in the proteins are represented by black and striped boxes, respectively. Predicted exon structure is indicated by the narrow boxes placed below the protein schematic. Exon predictions were obtained from the Human Genome Resources data base (human AGS3 and LGN), GadFly (PINS), and WormBase (F32A6.4). The complete gene for human AGS3 is not yet defined, and only the final three exons are annotated. Human LGN exon translated amino acid lengths are as follows: 12, 75, 41, 49, 42, 39, 52, 36, 46, 24, 61, 53, 73, 79; human AGS3 exon translated amino acid lengths are as follows: 54, 71, 68; F32A6.4 exon translated amino acid lengths are as follows: 24, 49, 45, 48, 41, 38, 34, 53, 31, 35, 58, 40, 39, 36; and PINS exon translated amino acid lengths are 513 and 147.

The individual motifs (TPR and GPR) within the two proteins are generally encoded by distinct exons. Exons 2-8 each encode most, if not all, of a single TPR domain, suggesting that each of the TPR domains incorporated into the gene structure as a separate unit. GPRI is encoded by a single exon (LGN exon 12, AGS3 exon 1), whereas GPRII and a portion of GPRIII are encoded by another exon (LGN exon 13, AGS3 exon 2). The remainder of GPRIII and all of GPRIV are contained within the final exon (LGN exon 14 and AGS3 exon 3).

Tissue Distribution of AGS3 and LGN-- To define the tissue and subcellular distribution of LGN and AGS3, we generated AGS3- and LGN-specific antisera in rabbits using peptides derived from the insert region for the two proteins (Fig. 2A) (4, 6). These peptides did not exhibit any sequence homology and are completely conserved in the human, rat, and mouse proteins. LGN antisera did not recognize AGS3, and AGS3 antisera did not recognize LGN following transient transfection of the two proteins in COS7 cells (Fig. 2B).³ Subsequent immunoblots indicated that LGN and AGS3 exhibited clear differences in their tissue distribution. LGN is widely expressed in the rat where it is found in liver, brain, kidney, lung, spleen, ovary, testes, heart, and pancreas (Fig. 2C). In addition to the expected M_r ~74,000 peptide, the LGN antibody also recognized peptides of lower apparent molecular weight (Fig. 2, B and C) suggesting the existence of splice variants or alternative promoters as reported previously for AGS3 (6).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Specificity of AGS3 and LGN antisera and analysis of tissue distribution. A, diagram indicating the sequence and location of peptides used to generate AGS3- and LGN-specific antisera. BLAST searches indicate that these peptide sequences are unique to each protein. B, lysates from COS7 cells (35 µg per lane) transiently transfected with plasmids overexpressing AGS3 or LGN cDNA were immunoblotted with AGS3- and LGN-specific antisera. The lines to the left of the blot indicate the migration of size standards (Bio-Rad low molecular weight) × 103. The immunoblots shown are representative of three experiments. C, an adult rat multiple tissue immunoblot was probed with AGS3 and LGN antisera as described under "Experimental Procedures." The blot was first probed with AGS3 antisera then stripped and reprobed with LGN antisera. The lines to the left of the blot indicate the migration of size standards (Bio-Rad low molecular weight) × 103, and the arrows to the right indicate the major immunoreactive species.

In contrast to the wide distribution of LGN, AGS3 was primarily expressed in brain and testes. A truncated, carboxyl-terminal portion of AGS3 (AGS3-SHORT) is enriched in heart compared with the M_r ~74,000 AGS3 protein (6). However, AGS3-SHORT is not recognized by the AGS3 antisera used in this study (6), and the M_r ~28,000 species observed in heart in Fig. 2C suggests the existence of additional forms of the protein. Immunoblots of lysates from a panel of cell lines indicated that LGN is expressed in all cell lines tested, whereas AGS3 is primarily expressed in cell lines of neuronal origin.⁴ Thus, LGN has a much wider tissue distribution than does AGS3 indicative of distinct functionality.

Within the central nervous system, AGS3 and LGN are expressed in all brain regions tested including entorhinal cortex, posterior cortex, pons, and spinal cord (Fig. 3).⁴ However, analysis of postnatal expression patterns over time indicated differential regulation or cell type-specific expression of the two proteins. Although LGN expression increased with time of development, AGS3 expression is maximal at day 7 and is significantly decreased at day 22 (Fig. 4A). Lysates from primary cultures of cortical neurons, astroglia, and microglia indicated expression of LGN in all three cell types, whereas AGS3 is expressed primarily in neuronal cultures, with very slight expression in astroglia (Fig. 4B). The expression of AGS3 and LGN in neuronal cultures was fairly constant over time (Fig. 4C) suggesting that the expression of these proteins is regulated by factors or mechanisms present only in intact tissue. Alternatively, the increased expression of LGN with time of development may reflect the relative increase in the proportion of glial cells versus neuronal cells, the former of which express LGN but little if any AGS3.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Distribution of AGS3 and LGN within brain. Rat brain regions were dissected on ice and subjected to immunoblotting with AGS3 and LGN specific antisera as described under "Experimental Procedures." Each lane contains 35 µg of protein. The blot was first probed with AGS3 antisera and then stripped and reprobed with LGN antisera.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Developmental regulation and cell type-specific expression of AGS3 and LGN in brain. A, brain regions (brainstem, cerebellum, cerebral cortex, hippocampus, and midbrain) from postnatal day 1, 7, and 22 rats were dissected on ice and subjected to immunoblotting with AGS3- and LGN-specific antisera. Each lane contains 35 µg of protein. B, lysates from primary cultures of cortical neurons, astroglia, and microglia were immunoblotted with AGS3- and LGN-specific antisera. Each lane contains 35 µg of protein. Immunoblots shown are representative of two experiments. C, lysates from cortical neuronal cultures prepared from newborn rats were isolated 0, 1, 3, 5, 8, 12, and 16 days after plating and immunoblotted with AGS3- and LGN-specific antisera. Day 0 samples were harvested before plating. Each lane contains 35 µg of protein. Immunoblots shown are representative of two experiments.

Subcellular Distribution of AGS3 and LGN-- As studies in Drosophila indicated that the AGS3/LGN ortholog PINS is a key player in several aspects of cell polarity, we compared the subcellular distribution of AGS3 and LGN. We used both primary neuronal cultures, which do not undergo any cell division, as well as cell lines actively undergoing mitosis. In primary neuronal cultures, both LGN and AGS3 are non-homogeneously distributed in the cell body and neuronal processes (Fig. 5). In contrast to AGS3, LGN is also found in the nucleus. The two proteins also differed in their response to activation of NMDA receptors and elevation of intracellular calcium. NMDA or the calcium ionophore ionomycin did not alter the expression pattern of AGS3, but both agents redistributed LGN into punctate structures in both the cell body and neuronal processes (Fig. 5). The redistribution observed with NMDA was blocked by the NMDA receptor antagonist MK801 (Fig. 5). Thus, AGS3 and LGN exhibit different modes of regulation and/or are found in distinct subcellular compartments.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Subcellular distribution of AGS3 and LGN in primary cultures of hippocampal neurons. Cells were cultured and processed for immunocytochemistry as described under "Experimental Procedures." Images were captured using confocal microscopy, and the images presented were taken from approximately the middle plane of the cells. Primary hippocampal neurons were washed and incubated for 1 h with 1 µM tetrodotoxin at 37 °C to inhibit spontaneous synaptic activity prior to addition of drugs. Addition of tetrodotoxin did not alter the subcellular distribution of either AGS3 or LGN when compared with untreated cells, and no specific staining was observed in cells incubated only with secondary antibody (data not shown). Cells were then treated with either 1 µM ionomycin 15 min or 50 µM NMDA for 60 min. Images are representative of three separate experiments performed in duplicate. The bar corresponds to 50 µm.

An even more startling difference in the subcellular distribution of the two proteins is observed in actively dividing cells. Subcellular distribution of LGN appeared to be dynamic and influenced by the cell cycle. Although primarily found in the nucleus, LGN was also found in the cytoplasm in some populations of cells, whereas AGS3 exhibited a non-homogenous distribution in the cytoplasm and was generally excluded from the nucleus (Fig. 6).⁵ Within the nucleus, the distribution of LGN is not homogeneous consistent with a specific and regulated localization. LGN underwent a dramatic relocalization during the cell cycle that resulted in its movement from the nucleus to the midbody at the site of daughter cell separation during cytokinesis (Fig. 7, arrows in merged panels).^5,6 At this late stage of the cell cycle, LGN is colocalized with F-actin (Fig. 7), which plays a key role in the constricture and abscission process at the site of cell separation (30). In some instances, comets of LGN immunoreactivity extended from points of cell contact (Fig. 7, arrowheads). These comets were not colocalized with F-actin as in the midbody (Fig. 7). Analysis of multiple fields of PC12 cells revealed no apparent differences of AGS3 expression in dividing cells again indicating that the two proteins differ in their cellular function. Thus, LGN, as observed with the Drosophila AGS3/LGN ortholog PINS, exhibited dramatic differences in its localization at specific stages of the cell cycle.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   AGS3 and LGN exhibit distinct differences in subcellular distribution in PC12 cells. Immunolocalization of AGS3 and LGN in untransfected PC12 cells. Cells were fixed and stained with AGS3 and LGN-specific antisera as described under "Experimental Procedures." Images were obtained from approximately the middle plane of the cells. Images are representative of four experiments performed in duplicate. Bar corresponds to 20 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   LGN localizes to the midbody during cytokinesis. Untransfected PC12 cells were fixed and stained for DNA (DAPI, blue), LGN (green), and filamentous actin (phalloidin, red) as described under "Experimental Procedures." Arrows and arrowheads point to areas in the cell where LGN colocalizes or does not colocalize with phalloidin, respectively. Images were captured using deconvolution microscopy. Images are representative of four experiments performed in duplicate. Bar corresponds to 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The diversity of recently discovered proteins that regulate or bind to components of the signal transduction system involving heterotrimeric G-proteins provide many opportunities for fine-tuning the signaling system and maintaining signaling specificity. Although we do not yet fully understand how these regulators function within the cell, their existence suggests novel concepts for G-protein signaling. First, such proteins can function as alternative binding partners for G or G. Second, such regulatory proteins allow signal input to G-protein signaling systems from a source other than a G-protein-coupled receptor. Both LGN and AGS3 are particularly interesting examples of such regulatory proteins. Although the TPR motifs of the two proteins likely play a role in subcellular location of the proteins (6), the GPR motif stabilizes the G_i-GDP complex and inhibits GDP dissociation. Thus, such proteins may replace G as a G-GDP-binding partner.

As AGS3 and LGN both possess four GPR motifs, these proteins would provide a scaffold for tethering GDP-bound G subunits in a defined spatial orientation (6). It is not clear whether such complexes target G to specific subcellular domains/cellular compartments or await an activating signal. Such a signal might promote nucleotide exchange on G-GPR in much the same way as if the signal was received by a G heterotrimer. Such a signal may come from within the cell or be initiated by extracellular stimuli. Alternatively, AGS3 or LGN that is not complexed with G-proteins may be activated by an as yet undefined mechanism and then transfer this signal to G complexes in microdomains of the cell, displacing G from G and selectively activating signals involving G-sensitive effectors.

Whereas only one such AGS3/LGN protein is found in C. elegans and Drosophila, higher organisms evolved two such proteins. The expressions of AGS3 and LGN are differentially regulated, and they have likely assumed distinct roles in cellular function based upon differences in their subcellular distribution. Whereas LGN is found in all cells and tissues examined, full-length, M_r ~74,000 AGS3 is primarily expressed in brain and testes. Within the brain, both proteins are widely expressed. AGS3 is restricted to neurons, whereas LGN is found in both neuronal and glial cell types. Thus, nature may have evolved a primordial AGS3 gene to serve specialized functions in the neuron.

A second striking difference between LGN and AGS3 is defined by their subcellular distribution. Both LGN and AGS3 are found throughout the cell body and neuronal processes in primary cultures of hippocampal neurons, but they are in different microdomains of the cell, and the two proteins respond differently to extracellular stimuli. More striking differences in the subcellular distribution and regulation of the two proteins are apparent in cell lines capable of undergoing cell division. In both PC12 and COS7 cells, LGN, but not AGS3, is found in the cell nucleus. Additional G-protein regulators, but typically not G-proteins themselves, are also present in the nucleus (31-36). Neither AGS3 nor LGN have any apparent hydrophobic domains or consensus sequences for acylation that would mediate a membrane association. Both AGS3 and LGN possess a loosely defined nuclear localization signal at ³⁷⁹RPKR³⁸², ⁴³⁸RRPR⁴⁴¹, ⁴²³PMRSRKY⁴²⁹ for AGS3 (6), and ⁴⁴⁶KKYK⁴⁴⁹ for LGN.⁷ Thus, there are no obvious "targeting" motifs that would explain the differences in subcellular location of the two proteins. Exon 1 of LGN encodes an amino-terminal 12-amino acid sequence that is not shared by AGS3, and this sequence is not found in all ESTs for LGN. However, it is not clear if this sequence influences cellular location of the protein. The differences in the subcellular location of the AGS3 and LGN likely reflect distinct binding partners that interact with the two proteins in the TPR or linker regions.⁸

In cells undergoing cytokinesis, LGN moves to a constricture between dividing cells where it colocalizes with F-actin. This defined structure is termed the midbody, and it serves as a focal point for the localization of proteins that eventually lead to the abscission of the dividing cells (30). The migration of LGN during the process of mitosis and cell division is remarkably similar to the protein survivin (37) and so-called passenger proteins T60, INCENPs, and AIM-1 (38). The function of these proteins within the midbody is not fully defined, but they are implicated as playing key roles in the latter stages of cytokinesis (37, 39-42). Loss of function at this stage of the cell cycle leads to multinucleated cells that underwent mitosis but failed to complete cell division (43, 44). The cell cycle-dependent location of LGN in the midbody structure is of particular interest due to the role of AGS3 orthologs and/or G-proteins in cell polarity and division (23-27). It is not clear at this point whether it is full-length LGN or a smaller version of the proteins that actually moves to the midbody. Our data suggest that at some point during the cell cycle, LGN receives a signal from inside the dividing cell, which initiates the movement of LGN out of the nucleus with eventual concentration at the midbody. As LGN moves toward its destination, it may interact with G-proteins and release G for functions required as part of the final stages of cell division.

	ACKNOWLEDGEMENTS

We appreciate the technical assistance provided by Jane Jordan and Maureen Fallon. We thank Ezekiel Carpenter-Hyland and Dr. Peter Kalivas (Department of Physiology and Neuroscience, Medical University of South Carolina) for preparation of neuronal cultures and dissection of brain regions, respectively. We thank Luis Marrero (Morphology and Imaging Core Facility, Gene Therapy Consortium, Louisiana State University Health Sciences Center, New Orleans, LA) for assistance with deconvolution microscopy and Dr. Mark Alliegro (Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, LA) for discussions on the midbody location of LGN.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants MH90531 and NS24821 (to S. M. L.) and AA10983 (to L. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence be addressed: Dept. of Pharmacology, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. E-mail: slanie@lsuhsc.edu.

Published, JBC Papers in Press, February 6, 2002, DOI 10.1074/jbc.M112185200

² Alignment of the genomic sequence of AGS3 with that of express sequence-tagged cDNAs for human AGS3 suggests there may have been a sequencing error in the DKFZP727I051 cDNA sequence resulting in a shift in the reading frame, giving rise to an additional 336 amino acids in the translated sequence.

³ The M_r ~76,000 species observed in untransfected COS7 cells is likely endogenous LGN. The endogenous LGN in COS7 cells, which are derived from simian monkey kidney, was slightly larger than the M_r ~74,000 peptide observed in COS7 cells transfected with human pcDNA3::LGN suggesting that COS7 LGN is either a slightly larger protein or that there are differences in the splicing pattern of endogenous LGN in COS7 cells.

⁴ J. B. Blumer and S. M. Lanier, unpublished observations.

⁵ J. B. Blumer and S. M. Lanier, unpublished observations. Similar results were observed in COS7 and Chinese hamster ovary cells.

⁶ J. B. Blumer and S. M. Lanier, unpublished observations. Similar results were obtained using an LGN-specific antibody derived against the carboxyl terminus of the protein.

⁷ N. Pizzinat, S. M. Lanier, and J. B. Blumer, unpublished observations.

⁸ Du et al. (45) recently reported the interaction of LGN with the nuclear mitotic apparatus protein and a role for LGN in mitotic spindle organization.

	ABBREVIATIONS

The abbreviations used are: AGS, activator of G-protein signaling; PINS, Partner of Inscuteable; NMDA, N-methyl-D-aspartate; PDHS, plasma-derived horse serum; DAPI, 4',6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GPR, G-protein regulatory; TPR, tetratricopeptide repeats.

	REFERENCES

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