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J Biol Chem, Vol. 274, Issue 38, 26931-26938, September 17, 1999

A Candidate Target for G Protein Action in Brain^*

Linda T. Chen, Alfred G. Gilman, and Tohru Kozasa

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An effector candidate for G protein action, GRIN1, was identified by screening a cDNA expression library with phosphorylated GTPS-G_z as a probe. GRIN1 is a novel protein without substantial homology to known protein domains. It is expressed largely in brain and binds specifically to activated G_z, G_o, and G_i through its carboxyl-terminal region. The protein KIAA0514 (GRIN2) is homologous to GRIN1 at its carboxyl terminus and also binds to activated G_o. Both GRIN1 and G_o are membrane-bound proteins that are enriched in the growth cones of neurites. Coexpression of GRIN1 or GRIN2 with activated G_o causes formation of a network of fine processes in Neuro2a cells, suggesting that these pathways may function downstream of G_o to control growth of neurites.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is likely that several targets for the action of G protein¹  subunits remain to be identified. Members of the G_i subfamily of these proteins, particularly G_o and G_i1, constitute roughly 1% of brain membrane protein, yet the only known effectors for G_o, the three G_i proteins, and G_z are certain isoforms of the enzyme adenylyl cyclase (1, 2). We have thus attempted to detect novel effectors that lie downstream of selected G protein  subunits, initially by utilizing ³²P-labeled  subunits activated (essentially irreversibly) with GTPS to probe cDNA expression libraries. We have taken advantage of the fact that G_z can be phosphorylated by protein kinase C at a site near its amino terminus that does not interfere with interaction between G_z and adenylyl cyclase (3). We have also appended a site for phosphorylation by cyclic AMP-dependent protein kinase to the carboxyl terminus of G_o; this region of G protein  subunits is also not involved in interactions with known effectors. This strategy led to isolation of a novel cDNA, initially designated Z-16, and detection of a homolog, KIAA0514. The protein products of these cDNAs interact selectively with GTPS- or GDP-AlF₄-bound forms of G_i subfamily members in vitro, and they cause extension of neurites in Neuro2a cells when coexpressed with activated forms of G_o. We thus tentatively refer to these two proteins as (GRIN1) (Z-16) and GRIN2 (KIAA0514) (G protein-regulated inducer of neurite outgrowth).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Phosphorylated G_z and G_o-- G_z and protein kinase C (PKC) were purified using a recombinant baculovirus-Sf9 cell expression system as described previously (2, 3). The recombinant catalytic subunit of cAMP-dependent protein kinase (protein kinase A) was expressed and purified from Escherichia coli (4). A tag encoding a phosphorylation site for protein kinase A (LRRASLG) followed by six histidine residues was added to the carboxyl terminus of G_o by polymerase chain reaction. The protein was then coexpressed with protein N-myristoyl transferase in E. coli and purified as described (5). Other recombinant G protein  subunits were prepared as described previously (2, 6).

G_z (250 µg) was incubated with GTPS at 30 °C for 60 min in the presence of 5 mM EDTA and 2 mM MgSO₄. Activated G_z was then phosphorylated with PKC (4 µg) in buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM MgSO₄, 0.125 mM CaCl₂, 1 mM DTT, 3 µM [-³²P]ATP (40,000 cpm/pmol), and 20 µg/ml phosphatidylserine-diolein (Sigma) for 30 min at 30 °C. Tagged G_o (600 µg) was similarly activated with GTPS and phosphorylated with the catalytic subunit of protein kinase A (3 µg) at 30 °C for 30 min as described by Baude et al. (4). After phosphorylation, free [-³²P]ATP was removed by gel filtration (PD-10 column; Amersham Pharmacia Biotech) in Buffer A (20 mM NaHepes (pH 7.4), 100 mM NaCl, 3 mM MgCl₂, 1 mM EDTA, 0.05% C₁₂E₁₀ (polyoxyethlene 10-lauryl ether), 1 mM DTT, 10 mM -glycerophosphate), and the proteins were used as probes for screening cDNA expression libraries.

Isolation of Z-16 cDNA-- Candidate effector proteins were sought in a EXlox library from 16-day mouse embryo cDNA (Novagen) with BL21/DE3 as the host E. coli strain. Approximately 1 × 10⁶ clones were screened using phosphorylated G_z as a probe. Phage were plated at a density of 3-5 × 10⁴ per 150-mm plate and incubated at 37 °C for 7 h. The plates were overlaid with Hybond-C filters (Amersham Pharmacia Biotech) that had been saturated with 10 mM isopropyl--D-thiogalactopyranoside. They were further incubated at 37 °C for 3.5 h to induce expression of proteins encoded by cDNAs. The filters were then rinsed with Buffer A at room temperature and blocked with Buffer A containing 5% dry milk at 4 °C overnight. The filters were probed with 50 nM phosphorylated GTPS-bound G_z in Buffer A with 5% dry milk for 4 h at 4 °C. The filters were washed three times with Buffer A for 5 min at 4 °C, air dried, and exposed to film at 70 °C for 2 days. Secondary and tertiary screens were carried out under identical conditions. Two clones, Z-13 and Z-16, were isolated and cDNA inserts were sequenced. Z-13 had a 0.8-kb insert encoding mouse nucleobindin (7). Z-16 had a 1.8-kb insert encoding 273 amino acid residues of previously undescribed sequence.

To obtain a full-length Z-16 cDNA, a mouse brain gt11 library (generously provided by Dr. Melvin Simon, California Institute of Technology) was screened by plaque hybridization using a fragment from the 5' end of the initial Z-16 clone as a probe. An additional 0.8 kb of coding sequence was isolated; the reading frame was open throughout. Two rounds of 5'-rapid amplification of cDNA ends reactions were then performed with a mouse brain cDNA library (CLONTECH), using oligonucleotides based on sequences at the 5' end of the Z-16 cDNA. The final Z-16 cDNA contains an ATG codon that agrees well with Kozak's translation initiation criteria and an in-frame, upstream stop codon. A related cDNA clone, designated KIAA0514 (8), was identified in public data bases and was generously supplied by Dr. T. Nagase (Kazusa DNA Research Institute).

Northern Analysis-- Northern blots of RNA from various tissues or brain regions (CLONTECH) were probed with GRIN1 (Z-16) or GRIN2 (KIAA0514) cDNA fragments labeled with [-³²P]dATP (random primer labeling; Stratagene, Prime-it II). Blots were hybridized with probe (5 × 10⁶ cpm/ml) in ExpressHyb (CLONTECH) at 68 °C for 2 h. The blots were then washed in 2× SSC and 0.1% SDS for 45 min at room temperature, followed by a high stringency wash with 0.1× SSC containing 0.1% SDS for 30 min at 50 °C. Then, the blots were finally subjected to phosphorimaging analysis (TR2040S imaging plates and BAS1500 scanner, Fuji Medical System).

Antisera-- Two peptides were synthesized based on the amino acid sequence of GRIN1: P1 (⁵⁵⁰SSAQPQRDTRSIGSLPER⁵⁶⁷) and P2 (⁷⁴⁷EVEVLGMAIQKHLERQIE⁷⁶⁴); these peptides also included an additional cysteine residue at their amino termini and a tyrosine residue at their carboxyl termini. The cysteine residue was utilized to facilitate cross-linking of peptide to keyhole limpet hemocyanin (Sigma) with m-maleimidobenzoyl-N-hydroxysuccinimide ester. Antisera were produced in New Zealand White rabbits. Antisera T116 and T114 were generated against peptides P1 and P2, respectively. The specificities of the antisera were confirmed by immunoblotting. An antiserum specific for G_o (U1901) was generated in a rabbit against a synthetic peptide with the amino acid sequence of G_o (⁹⁴EYGDKERKADSKMVC¹⁰⁸) conjugated to keyhole limpet hemocyanin. Other rabbit polyclonal antisera against various G subunits have been described previously (2, 9-11), as has mouse monoclonal antibody (mAb2A) against G_o (12).

Purification of Recombinant GRIN1 and GRIN2 Proteins-- GRIN1 and GRIN2 were subcloned into the pFastBacHTb vector (Life Technologies, Inc.), and recombinant baculoviruses encoding His₆-GRIN1 or His₆-GRIN2 were generated according to the manufacturer's protocol. Membranes from Sf9 cells infected with baculoviruses encoding His₆-GRIN1 or His₆-GRIN2 were prepared and extracted with 1% C₁₂E₁₀. His₆-GRIN1 and His₆-GRIN2 were then purified from these extracts using Ni-NTA chromatography. The proteins were further purified by Mono Q and Superdex 200 column chromatography in 20 mM NaHepes (pH 8.0), 5 mM MgCl₂, 2 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.5% C₁₂E₁₀.

Binding of Purified GRIN1 or GRIN2 to G Protein  Subunits-- Hexa-histidine-tagged GRIN1 or GRIN2 purified from SF9 cells (1.5 µg) was mixed with 2 µg (500 nM) of recombinant G_o or G_z bound with GDP, GTPS, or GDP-AlF₄, as indicated, in 100 µl of Buffer B (50 mM NaHepes (pH 8.0), 5 mM MgCl₂, 10 mM -mercaptoethanol, 0.1% C₁₂E₁₀) and incubated on ice for 30 min. NaF (10 mM) and AlCl₃ (30 µM) were included in the buffer to prepare G-GDP-AlF₄. Ni-NTA resin (Qiagen) was equilibrated with Buffer B, and 25 µl was added to the mixture of proteins, followed by further incubation on ice for 5 min. The resin was collected by brief centrifugation, and the supernatant was saved as the flow-through fraction. The resin was washed twice with 150 µl of Buffer B containing 500 mM NaCl and 10 mM imidazole. NaF and AlCl₃ were included in the wash buffer when they were present initially. The resin was finally eluted twice with 50 µl of Buffer B containing 200 mM imidazole. Fractions were analyzed by immunoblotting after SDS-PAGE.

Expression Vectors-- An expression vector for GRIN1 was constructed by adding a FLAG tag at the amino terminus of GRIN1 by polymerase chain reaction and subcloning the resultant GRIN1 cDNA (KpnI-HindIII fragment, 3.6 kb) into the pCMV5 vector. For GRIN2, a FLAG tag or a hexahistidine tag was added at the amino terminus, and the resultant cDNA (EcoRI-HindIII fragment, 1.4 kb) was subcloned into pCMV5.

Cell Culture and Transfection-- Simian kidney COS-M6 cells, simian renal epithelium MA104 cells, and murine neuroblastoma Neuro2a cells were cultured in Dulbecco's modified Eagle's medium supplemented with 25 mM glucose and 10% heat-inactivated fetal bovine serum at 37 °C in an atmosphere of 10% CO₂. Cells were transiently transfected with appropriate expression plasmids using LipofectAMINE (Life Technologies, Inc.). Cell lysates were fractionated into particulate and soluble fractions as described (13, 14).

Immunoprecipitation-- A crude membrane fraction of COS cells expressing FLAG-tagged GRIN1 was extracted with immunoprecipitation buffer (50 mM sodium phosphate (pH 7.2), 100 mM NaCl, 1 mM DTT, and 0.5% C₁₂E₁₀ supplemented with fresh protease inhibitors). The extracts were centrifuged at 100,000 × g for 20 min at 4 °C. The resulting supernatants were incubated with 200 nM G protein  subunits either in the presence or absence of 10 mM NaF and 30 µM AlCl₃ for 10 min on ice, mixed (50 µg of protein) with 5 µl of anti-FLAG monoclonal antibody M2 (3 mg/ml; Eastman Kodak Co.) in a total volume of 50 µl, and incubated at 4 °C overnight. Fixed Staphylococcus aureus bacteria (5 µl of a 10% suspension; Pansorbin, Calbiochem) were added and incubated on ice for an additional 1 h. The extracts were then centrifuged at 13,000 × g for 1 min, and the precipitates were suspended in 100 µl of immunoprecipitation buffer. The suspensions were layered over 0.9 ml of immunoprecipitation buffer containing 20% sucrose (w/v) and centrifuged for 6 min. The pellets were washed with 0.5 ml of phosphate-buffered saline, suspended in SDS-PAGE sample buffer, resolved by SDS-PAGE, and analyzed by Western blotting.

Subcellular Fractionation of Mouse Brain-- Subcellular fractionation of adult mouse brain was performed based on the procedure described in Ref. 15. Mouse brain was homogenized in Buffer C (10 mM Tris-HCl (pH 8.0), 1 mM DTT, 11% sucrose with protease inhibitors) and centrifuged at 600 × g for 10 min. The supernatants were collected and centrifuged at 11,700 × g for 20 min. The pellets were resuspended in Buffer C and loaded onto a discontinuous sucrose gradient (10-40%) and centrifuged at 150,000 × g for 2 h. Fractions at the interfaces of the gradient were analyzed by immunoblotting. Plasma membrane is concentrated at the 25-30% sucrose interface. Subcellular fractions of embryonic mouse brain were obtained as described (16, 17).

Immunocytochemistry-- To detect endogenous GRIN1, Neuro2a cells were plated on laminin-coated coverslips at 50% confluency and grown for 2 days to generate neurites. For transient transfection, Neuro2a cells were plated on nontreated coverslips at 60-70% confluency and allowed to grow for 1 day. Cells were then transfected with expression plasmids and incubated for 48 h.

Transfected MA104 or Neuro2a cells were rinsed and incubated with Dulbecco's modified Eagle's medium without serum for 6 h, fixed with 4% paraformaldehyde in phosphate-buffered saline at 4 °C for 20 min, and washed with Dulbecco's modified Eagle's medium containing 5 mM glycine on ice for 5 min. Cells were permealized in buffer (10 mM sodium phosphate (pH 7.4), 150 mM NaCl, 2 mM MgCl₂, and 0.1% Triton X-100) or cold 100% methanol for 10 min. Cells were then incubated in 5% goat serum in buffer containing 10 mM sodium phosphate (pH 8.0), 150 mM NaCl, and 2 mM MgCl₂ for 1 h, followed by incubation at 4 °C overnight with G_o monoclonal antibody (mAb2A, 1:50 dilution), anti-GRIN1 antibody (T116, 1:300 dilution), or GAP43 antibody (Zymed Laboratories Inc., 1:100 dilution). After washing, coverslips were incubated with fluorescent, conjugated secondary antibodies (10 µg/ml; Oregon Green-conjugated goat anti-rabbit IgG or Texas Red-conjugated goat anti-mouse IgG) for 20 min at room temperature. The coverslips were washed twice with 1% Triton X-100 in phosphate-buffered saline, twice with phosphate-buffered saline, and twice with water, and then air-dried for 30 min before mounting with Fluoromount G.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of Z-16 cDNA-- Phosphorylated GTPS-G_z was utilized to screen a mouse embryo EXlox expression library. Two clones, designated Z-13 and Z-16, were eventually isolated based on clearly positive signals in several successive screens. The product of the Z-13 cDNA bound GDP-G_z and GTPS-G_z equally well, whereas the product of the Z-16 cDNA bound GTPS-G_z selectively (Fig. 1). Sequencing and data base searches revealed that Z-13 encoded the protein nucleobindin. Interactions of G_i2 with nucleobindin have been detected previously (18). Nucleobindin is described as a secreted protein that interacts with DNA (7). We did not pursue nucleobindin further because of these characteristics and its failure to recognize GTPS-bound G_z selectively. The protein product of the Z-16 cDNA also bound to phosphorylated protein kinase A-tagged, GTPS-bound G_o.

View larger version (119K): [in this window] [in a new window]   Fig. 1.   Binding of [32P]Gz to Z-13 and Z-16. The interactions of [32P]Gz with purified phage encoding Z-13 (A and B) or Z-16 (C and D) are shown. Phosphorylated Gz was tested in either its GDP-bound (A and C) or GTPS-bound (B and D) forms.

Sequencing of the 1800-base pair insert designated Z-16 revealed that it had an open reading frame encoding 273 amino acid residues fused to the T7 gene 10 product, as anticipated. The sequence was incomplete at the 5'-end. An apparently full-length cDNA was obtained by screening a mouse brain cDNA library and by 5'-rapid amplification of cDNA ends, using a 5'-stretch of randomly primed brain cDNA library (Fig. 2A). The deduced amino acid sequence specifies a protein containing 827 residues, with M_r = 84,700. Nearly 45% of the residues have small side chains: Ala (11%), Gly (9.8%), Pro (10.5%), and Ser (13.4%). Data base searches revealed that KIAA0514, a cDNA clone isolated from a human brain cDNA library, and Z-16 encode homologous ~100-150 amino acid residue domains near their carboxyl termini (Fig. 2B). No other matches or homologies were detected, except for a few previously unidentified Z-16 expressed sequence tags (AI427420, W54141, and AI413422). In addition, related genes were not detected by Southern blot analysis of mouse genomic DNA (data not shown). Because of the functional properties of the proteins encoded by the Z-16 and KIAA0514 cDNAs (described below), we refer to these two proteins as GRIN1 and GRIN2, respectively.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   A, the nucleotide and deduced amino acid sequence of GRIN1 (Z-16). B, alignment of the amino acid sequences of GRIN1 and GRIN2 (KIAA0514) using NIH Blast 2 (27).

Northern and Western Analysis of GRIN1-- Northern blot analysis of GRIN1 was performed using several regions of the cDNA as probes. Messenger RNA for GRIN1 (about 4 kb) was detected in brain but not in heart, liver, spleen, lung, skeletal muscle, kidney, or testis (Fig. 3A). GRIN1 mRNA is widely distributed in the central nervous system; the highest concentration was detected in the spinal cord (Fig. 3B). Messenger RNA for GRIN2, which is about 8 kb, is also specifically expressed in brain. In contrast to GRIN1, expression of GRIN2 was only detected in cerebellum (Fig. 3B).

View larger version (71K): [in this window] [in a new window]   Fig. 3.   Northern and Western analysis. A, Northern analysis of mouse tissues for GRIN1 expression. One µg of poly(A)+ mRNA from mouse heart (lane 1), brain (lane 2), liver (lane 3), spleen (lane 4), lung (lane 5), skeletal muscle (lane 6), kidney (lane 7), or testis (lane 8) was resolved electrophoretically and hybridized with a 32P-labeled GRIN1 probe (1416-1662) (top panel) or a human -actin probe (bottom panel) as described under "Experimental Procedures." The migration positions of markers (size in kb) are shown to the left. B, Northern analysis of human brain regions for GRIN1 and GRIN2 expression. One µg of poly (A)+ mRNA from human amygdala (lane 1), caudate nucleus (lane 2), corpus callosum (lane 3), hippocampus (lane 4), whole brain (lane 5), substantia nigra (lane 6), subthalamic nucleus (lane 7), thalamus (lane 8), cerebellum (lane 9), cerebral cortex (lane 10), medulla (lane 11), spinal cord (lane 12), occipital pole (lane 13), frontal lobe (lane 14), temporal lobe (lane 15), or putamen (lane 16) was resolved electrophoretically and hybridized with a 32P-labeled GRIN1 probe (1416-1662) (top panel), a 32P-labeled full-length GRIN2 probe (middle panel), or a 32P-labeled human -actin probe (bottom panel). C, Western immunoblot analysis of mouse tissues for GRIN1 expression. Homogenate (10 µg of protein) from mouse heart (lane 1), brain (lane 2), liver (lane 3), spleen (lane 4), lung (lane 5), skeletal muscle (lane 6), kidney (lane 7), or placenta (lane 8) was resolved electrophoretically (SDS-PAGE; 9.5% gels). Immunoblots generated with a GRIN1-specific antiserum (T116) and a Go-specific antiserum (U1901) are shown.

Two antibodies specific for GRIN1 (designated T114 and T116) were prepared using GRIN1 peptides as immunogens. In lysates of COS cells transfected with GRIN1 cDNA, both antibodies detected an immunoreactive band with an apparent molecular weight of 110,000. Consistent with the results of Northern analysis, this 110-kDa band was also detected in mouse brain homogenates but not in homogenates of other tissues (Fig. 3C). The appearance of GRIN1 as a doublet band in brain lysate may reflect proteolysis during sample preparation, or it may indicate the existence of splice variants of the protein. This immunoreactive band was not detected if antisera were first incubated with peptides used as immunogens or with expressed GRIN1 protein (data not shown). We thus conclude that this band represents GRIN1 protein and that the protein migrates anomalously during SDS-polyacrylamide gel electrophoresis. Among several cell lines tested, endogenous GRIN1 was detected in mouse neuroblastoma Neuro2a (see Fig. 5B) and rat pheochromocytoma PC12 cells as a doublet similar to its appearance in brain lysate (data not shown).

The peptide used to generate antibody T114 shares 13 amino acid residues (of 18) with GRIN2. This antibody, but not T116, recognized the GRIN2 product expressed in COS or Sf9 cells (data not shown). GRIN2 (461 amino acid residues) has a calculated molecular weight of 47,600 but migrates with an apparent molecular weight of 65,000 during SDS-PAGE.

Interaction of GRIN1 and GRIN2 with Various G Protein  Subunits-- Baculoviruses encoding full-length GRIN1 or GRIN2 (with hexahistidine tags at the amino terminus) were generated, and recombinant GRIN1 or GRIN2 protein was purified from Sf9 cells using Ni-NTA, Mono-Q, and Superdex 200 column chromatography. Purified GRIN1 or GRIN2 was mixed with 500 nM G_o or G_z bound with either GDP, GDP-AlF₄, or GTPS, and interactions were analyzed by co-elution from Ni-NTA columns (Fig. 4A). Both GRIN1 and GRIN2 interacted with the GTPS- or GDP-AlF₄-bound form of G_o and G_z; interactions with the GDP-bound forms of these two proteins were not detected.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Interactions of GRIN1 and GRIN2 with G protein  subunits. A, purified recombinant (Sf9 cell-derived) hexahistidine-tagged GRIN1 (left) or hexahistidine-tagged GRIN2 (right) (1.5 µg of each) was mixed with 2 µg of recombinant Go or Gz bound with GDP, GTPS, or GDP-AlF4, as indicated. Ni-NTA resin was added to the mixture and processed as described under "Experimental Procedures." Fractions were resolved by SDS-PAGE, and immunoblots were developed with antibodies reactive with GRIN1 or GRIN2 (top panels, left and right, respectively) or with Go or Gz, as appropriate. The applied proteins are shown in lane 1 of each panel; lane 2, flow through; lanes 3 and 4, sequential washes with 500 mM NaCl and 10 mM imidazole; lanes 5 and 6, sequential elutions with 200 mM imidazole-HCl. B, immunoprecipitation of G protein  subunits associated with Flag-tagged GRIN1. Flag-tagged GRIN1 was expressed in COS cells and extracted as described under "Experimental Procedures." Extracts were supplemented with G protein  subunits in various nucleotide-bound states and then subjected to immunoprecipitation as described using an anti-Flag monoclonal antibody. Electrophoretically resolved immunoprecipitates were immunoblotted using antibodies appropriate for the protein listed to the left of each panel. Top two panels: lane 1, GTPS-Go was mixed with extract from COS cells not expressing Flag-tagged GRIN1; lanes 2-4, GDP-Go, GDP-AlF4-Go, and GTPS-Go, respectively, were mixed with extracts containing Flag-tagged GRIN1. Lower six panels, lane 1, the indicated G protein  subunit (GTPS-bound) was mixed with extract from COS cells not expressing Flag-tagged GRIN1. Lanes 2 and 3, the indicated GDP- or GDP-AlF4-bound G protein  subunit, respectively, was mixed with extract from COS cells expressing Flag-tagged GRIN1.

Interactions between GRIN1 and various G proteins were also examined using full-length Flag-tagged GRIN1 expressed in COS cells. Detergent extracts containing GRIN1 were then mixed with G protein  subunits, and complexes were precipitated with an anti-Flag monoclonal antibody (Fig. 4B). Specific interactions were detected between GRIN1 and members of the G_i subfamily of G subunits, including G_o, G_z, and G_i1. A weak interaction was detected with G₁₂, but none was observed with G_q or G_s. Again, GRIN1 interacted preferentially with the GTPS or the GDP-AlF₄-bound forms of the G proteins compared with the GDP-bound proteins.

Subcellular Distribution of GRIN1; GRIN1 Is Found in Growth Cones with G_o-- The subcellular distribution of GRIN1 was first examined in transfected COS cells. Exogenously expressed GRIN1 and G_o were largely confined to the particulate fractions, and only small amounts appeared in the cytosol (Fig. 5A). Similarly, endogenous GRIN1 and G_o in Neuro2a cells and brain are predominantly membrane-bound (Fig. 5, B and C). The amino acid sequence of GRIN1 contains no obvious hydrophobic domain sufficient to explain membrane localization. There are also no consensus sequences for covalent modification by myristoylation or prenylation.

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Subcellular distribution of GRIN1 and Go. A-C, subcellular fractions (10 µg of protein in A and B; 20 µg of protein in C) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. A, transfected COS cells expressing GRIN1 and Go. Lane 1, 1000 × g supernatant from untransfected COS cells; lane 2, 1000 × g supernatant from transfected COS cells; lane 3, 1000 × g pellet from transfected cells; lane 4, 100,000 × g supernatant from transfected cells; lane 5, 100,000 × g pellet from transfected cells. B, Neuro2a cells. Lane 1, cell lysate; lane 2, 1000 × g pellet; lane 3, 1000 × g supernatant; lane 4, 100,000 × g supernatant; lane 5, 100,000 × g pellet. C, mouse brain. Lane 1, whole homogenate; lane 2, discontinuous sucrose gradient 10-15% interface; lane 3, 15-20% interface; lane 4, 20-25% interface; lane 5, 25-30% interface; lane 6, recombinant GRIN1 standard. D and E, immunofluorescence of GRIN1 and Go in differentiated Neuro2a cells. Differentiated Neuro2a cells were stained with rabbit anti-GRIN1 antibody and Oregon Green-conjugated goat anti-rabbit IgG (D) or with mouse monoclonal anti-Go antibody and Texas Red-conjugated goat anti-mouse IgG (E). F shows the overlapped images of D and E. The calibration bar corresponds to 17 µm.

Co-localization of GRIN1 and G_o in Neuro2a cells was also demonstrated by immunofluorescence. Neuro2a cells were induced to extend neurites by culture on laminin-coated dishes. Permeabilized cells were then incubated with both rabbit polyclonal anti-GRIN1 antibodies (T116) and a mouse monoclonal antibody specific for G_o (mAb2A). Oregon Green-conjugated goat anti-rabbit IgG or Texas Red-conjugated goat anti-mouse IgG were used as secondary antibodies to detect GRIN1 and G_o, respectively. The specificity of observed fluorescence was confirmed by competition with the immunogenic peptide or GRIN1 protein (data not shown). The pattern of immunofluorescence for GRIN1 and G_o in differentiated Neuro2a cells is similar, and the two proteins are predominantly found at the plasma membrane (Fig. 5, D-F).

G_o and GAP43 are enriched in growth cone membranes (19). Growth cone membrane fractions were prepared as described by Pfenninger et al. (16) and Simkowitz et al. (17) and analyzed by immunoblotting using GRIN1 antisera. Fig. 6A shows that GRIN1 is highly enriched in the growth cone membrane fraction (lane 4), as are GAP43 and G_o. By contrast, G_z and the low-affinity NGF receptor are found in all membrane fractions. Furthermore, immunofluorescence of differentiated Neuro2a cells shows that GRIN1 and GAP43 are concentrated in putative growth cone membranes (Fig. 6, B-D, arrows).

View larger version (62K): [in this window] [in a new window]   Fig. 6.   Go and GRIN1 are enriched in growth cone membranes. A, homogenate from embryonic mouse brain (day 17) was fractionated according to the method of Pfenninger et al. (16) to prepare growth cone membranes. Fractions (10 µg of protein) were then resolved electrophoretically and immunoblotted with antibodies that react with the indicated proteins. Lane 1, homogenate; lane 2, 1660 × g supernatant; lane 3, 1660 × g pellet; lane 4, discontinuous sucrose gradient 10-26% interface; lane 5, 26-34% interface; lane 6,: 34-75% interface. B-D, immunofluorescence of GRIN1 and GAP43 in differentiated Neuro2a cells. Differentiated Neuro2a cells were stained with rabbit anti GRIN1 antibody (Oregon Green) (B) and mouse anti GAP43 (Texas Red) (C). D shows the overlapped images of B and C. The arrowheads indicate growth cones. The calibration bar corresponds to 4 µm.

GRIN1 and GRIN2 Cause Morphological Changes in MA104 and Neuro2a Cells-- GRIN1 or GRIN2 was cotransfected into MA104 (simian renal epithelium) cells with either wild type G_o or a constitutively active (GTPase-deficient) mutant of the protein (G_o Q205L), and the transfected cells were examined by immunofluorescence microscopy. As shown in Fig. 7A, MA104 cells transfected with only wild type G_o, G_oQ205L, or GRIN1 had a relatively unperturbed morphology. However, cells spread irregularly on coverslips and generated many fine, neurite-like processes if they were cotransfected with GRIN1 and G_oQ205L. This effect was much less apparent if wild type G_o was expressed with GRIN1. Expression of GRIN2 alone caused extension of processes in some cells, and co-transfection of G_o or G_oQ205L increased both the frequency and extent of these changes. Similar experiments were performed with Neuro2a cells (Fig. 7, B and C). Again, co-transfection of GRIN1 or GRIN2 with G_oQ205L caused long neurites to appear. In addition, these neurites displayed many hair-like processes. Co-transfection of GRIN1 or GRIN2 with wild type G_o caused less extensive changes. In contrast to MA104 cells, expression of G_oQ205L alone induced formation of neurites, albeit with lower efficiency. This effect is perhaps explained by the endogenous content of GRIN1 in Neuro2a cells. These results indicate that G_o and GRIN1 interact (directly or indirectly) in vivo and that this causes formation and extension of neurite-like processes. Similar effects of G_oQ205L have been described in PC12 cells (20).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Morphological changes of cells transfected with GRIN1 and GRIN2. MA104 cells (A) or nondifferentiated Neuro2a cells (B) were transfected with control vector (a), wild type Go (b), Go Q205L (c), GRIN1 (d), GRIN1 and wild type Go (e), GRIN1 and Go Q205L (f), GRIN2 (g), GRIN2 and wild type Go (h), or GRIN2 and GoQ205L (i). GRIN1 or GRIN2 was stained with Oregon Green, and wild type Go or Go Q205L was stained with Texas Red. C, nondifferentiated Neuro2a cells were transfected with GRIN1 and Go Q205L. After transfection (48 h), cells were further incubated with serum-free medium for 24 h to extend neurites. GRIN1 and Go Q205L were stained with Oregon Green and Texas Red, respectively. The overlapped images of one transfected cell with long neurites are combined. The calibration bar in each panel corresponds to 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We screened a mouse embryo cDNA expression library to search for proteins capable of interacting with phosphorylated GTPS-G_z. Two clones were isolated, designated Z-13 and Z-16. Z-13 encoded nucleobindin, the interactions of which with G_i2 were detected previously using a yeast two-hybrid screen (18). We did not pursue this lead further because of the apparently similar affinity of GDP-G_z and GTPS-G_z for nucleobindin and the presence of a signal sequence in the protein. By contrast, the protein now designated GRIN1 interacts preferentially with activated forms of G protein subunits in the G_i subfamily (G_i, G_o, G_z). Furthermore, GRIN1 is specifically expressed in brain and shares with G_o substantial enrichment in membranes from neuronal growth cones. We thus hypothesize that GRIN1 may function as a downstream effector for G_o.

A homolog of GRIN1-GRIN2 (KIAA0514), was isolated and sequenced previously as a newly identified brain cDNA by Nagase et al. (8). The regions of GRIN1 and GRIN2 that are similar are at the carboxyl termini of both proteins, and we had shown independently that this is the G_o-binding domain of GRIN1 (data not shown). Significantly, GRIN2 also interacts preferentially with activated members of the G_i subfamily of G protein  subunits. Although GRIN1 is widely distributed throughout the central nervous system, GRIN2 is apparently restricted to the cerebellum.

G_o is the most abundant G protein  subunit in mammals. It is expressed predominantly in brain and is enriched in neural growth cones (19). Despite these interesting properties, physiological roles for G_o have not been identified, other than its interactions with G protein subunits and appropriate receptors. The heterotrimeric G_o protein is responsible for receptor-mediated inhibition of voltage-sensitive N-type or P/Q-type Ca²⁺ channels in presynaptic nerve terminals, but this effect appears to be mediated by the G protein subunit complex (21). G_o is a weak inhibitor of some isoforms of adenylyl cyclase (1), but the physiological significance of this is difficult to evaluate. G_o has also been hypothesized to regulate neurite extension. Binding of GTPS to G_o is stimulated by GAP43 (neuromodulin), an abundant growth cone protein that is important for neural pathfinding (19). The expression of both GAP43 and G_o starts in brain regions when differentiated neurons begin to extend neurites (22). Furthermore, expression of constitutively activated mutant forms of G_o stimulates neurite outgrowth in neuronal cell lines (20). The molecular mechanism for this phenomenon has not been defined.

We have shown herein that either GRIN1 or GRIN2 induces extensive outgrowth of neurites from Neuro2a cells when coexpressed with activated forms of G_o. This result implies interaction between proteins in vivo, although not necessarily a direct one. However, the fact that G_o does interact directly with both GRIN1 and GRIN2 in vitro suggests that these latter proteins may function physiologically as downstream targets for G_o and/or other members of the G_i subfamily to regulate neurite outgrowth. The amino acid sequences of GRIN1 and GRIN2 show no significant homology with known kinases, enzymes that generate second messengers, or other identified effectors for G protein action. Although GRIN1 has a proline-rich domain (residues 590-710), we detected no other provocative signatures. Recent evidence suggests that Rho family GTPases are important components of signaling pathways that control axonal growth and guidance (23). Rho itself is involved in collapse of growth cones and retraction of neurites. By contrast, Cdc42 and Rac1 stimulate the formation and advance of growth cones through formation of filopodia and lamellipodia (24). It is thus possible that Cdc42 or Rac1 are downstream components of signaling pathways that include G_o and GRIN1 or GRIN2. Perhaps relevant is the fact that the G proteins G₁₃ and G₁₂ have recently been shown to control the activity of a guanine nucleotide exchange factor, p115, that activates Rho (25, 26).

	ACKNOWLEDGEMENTS

We thank Jeffrey Laidlaw and Linda Hannigan for excellent technical assistance, Dr. Takahiro Nagase for KIAA0514 cDNA, and members of our laboratory for valuable discussions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM34497 and the Raymond and Ellen Willie Distinguished Chair of Molecular Neuropharmacology (to A. G. G.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF146569.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-2370; Fax: 214-648-8812; E-mail: alfred.gilman@email.swmed.edu.

	ABBREVIATIONS

The abbreviations used are: G protein, guanine nucleotide binding regulatory protein; GTPS, guanosine 5'-3-O-(thio)triphosphate; C₁₂E₁₀, polyoxyethlene 10-lauryl ether; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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