COMMUNICATION
Interaction of Heterotrimeric G Protein Galpha o with Purkinje Cell Protein-2
EVIDENCE FOR A NOVEL NUCLEOTIDE EXCHANGE FACTOR*

Yuan LuoDagger and Bradley M. Denker§

From the Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The heterotrimeric G protein Galpha o 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 Galpha o, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galpha o in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galpha o is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63-76). Galpha o and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galpha o. In addition, when Galpha o and Pcp2 were cotransfected into COS cells, Galpha o was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galpha o function, kinetic constants kcat and koff of bovine brain Galpha o were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galpha o 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 Galpha o is important to Purkinje cell function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cell surface receptors coupled to G proteins1 enable cells to respond to a wide range of extracellular signals. G proteins, composed of Galpha and Gbeta gamma subunits, associate at the plasma membrane in a complex with a serpentine transmembrane receptor. Agonist-liganded receptors activate Galpha by inducing a change in conformation that leads to GDP release and GTP-binding. GTP-liganded Galpha dissociates from Gbeta gamma , and both subunits can interact with a variety of intracellular effectors. Galpha and Gbeta gamma remain activated until the intrinsic GTPase activity of Galpha hydrolyzes GTP to GDP. The mechanisms utilized by cells to respond to specific signals in a precise manner is not well understood. In reconstituted systems there is ample evidence that multiple G proteins can couple to the same sets of receptors and effectors (reviewed in Ref. 1), and in transfected cells a single Galpha subunit can couple to at least three different effector pathways (2). One important mechanism that contributes to regulation of signaling pathways is the existence of proteins that modulate particular points in the pathway. For example, G protein receptor kinases desensitize receptors (such as beta -adrenergic receptor kinase, (reviewed in Ref. 3) and RGS (regulators of G protein signaling) proteins turn off effector responses by accelerating the GTPase activity of Galpha subunits (reviewed in Ref. 4). Since some G protein family members are predominantly expressed in specific tissues, cell type-specific modulators of G protein signaling are likely to exist.

Galpha o is a member of the pertussis toxin family of Galpha subunits and is predominantly expressed in the central nervous system and heart. Although Galpha o comprises 0.2-0.5% of brain particulate protein (5), many of its functions are yet to be defined. Galpha o couples to several well characterized receptors in the brain and can regulate both N-type Ca+2 channels as well as some K+ channels (see Ref. 6 and references therein). In addition, Galpha o can be regulated by neuromodulin (GAP43 (growth cone-associated protein)) in developing neurites (7). Knockout of neuromodulin in mice causes significant abnormalities in neuronal pathfinding (8), but Galpha o knockout mice have anatomically normal brains. Despite the normal central nervous system anatomy in the Galpha o knockout mice, they develop a spectrum of neurologic abnormalities (tremor and impairments of motor control and behavior) and shortened survival (6, 9). We utilized the yeast two-hybrid interaction system to search for unique modulators of Galpha o that are expressed in the central nervous system. An interaction between Galpha o and Pcp2 (Purkinje cell protein-2), a protein of unknown function expressed in Purkinje cells and retinal bipolar neurons was identified. Although Galpha o and Pcp2 have not yet been definitively colocalized in cerebellar Purkinje cells, this interaction was confirmed in vitro and by coimmunoprecipitation from transfected cells. Furthermore, Pcp2 can function as a nucleotide exchange factor by stimulating GDP release from Galpha o.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Yeast Two-hybrid Screening-- Galpha o cDNA in pBS (previously described in Ref. 10) was cloned into EcoRI/SalI sites of PAS2-1 (CLONTECH), a vector that encodes GAL4 DNA-binding domain. Galpha o-PAS2-1 was used as a "bait" to screen a mouse brain library in pACT (CLONTECH). Both Galpha o-PAS2-1 and the mouse brain cDNA libraries were cotransformed into Y190, a yeast lacZ/HIS3 reporter strain, using standard methods. The transformed mix was screened for growth on plates containing selective medium (synthetic complete medium lacking tryptophan, leucine, and histidine in the presence of 25 mM 3-aminotriazole) and incubated at 30 °C for 8-12 days. His+ colonies were screened for beta -galactosidase activity using a filter lift assay, and positive "blue" colonies (beta -galactosidase-positive) were further confirmed by a yeast mating assay. Individual plasmids were transformed into Escherichia coli by electroporation and plasmids analyzed by restriction analysis and dideoxynucleotide sequencing. GenBankTM data bases were screened with each sequence using BLAST analysis.

In Vitro Binding Assay-- Pcp2 was excised from pACT2 using EcoRI/XhoI and cloned into pGEX4-2 (Amersham Pharmacia Biotech). The resulting Pcp2-pgex cDNA and pGEX4-2 without insert (GST alone) were transformed into E. coli, and the expressed proteins were purified from bacterial pellets after induction with isopropyl-1-thio-beta -D-galactopyranoside. Bacterial lysates were incubated with glutathione-agarose beads (2 ml) and rotated at 4 °C for 30 min. After centrifugation, GST-Pcp2 and GST alone were eluted from beads using glutathione elution buffer for 10 min at room temperature (Amersham Pharmacia Biotech). Samples were recentrifuged, and the resulting supernatant containing the fusion proteins was analyzed according to the Bradford method to determine protein concentration. Galpha o (subtype 1) and Galpha i2 in pBS (Galpha i2 plasmid construction described in Ref. 11) and Galpha s in pcDNAI (from ATCC, Manassas, VA) were used for in vitro translation. Labeled Galpha subunits were made using 1 µg of cDNA, appropriate RNA polymerase in a coupled rabbit reticulocyte translation system (TNT system, Promega, Madison WI) plus [35S]methionine (NEN Life Science Products; 20 µCi/reaction) as described previously (11). [35S]Methionine-labeled Galpha subunits were analyzed by SDS-PAGE and autoradiography, and the amounts were normalized by densitometric analysis of translated products (NIH Image 1.61/fat, Wayne Rasband, NIH, Bethesda, MD). Equivalent amounts of fusion proteins (GST or GST- Pcp2) were incubated with glutathione-agarose beads for 30 min at room temperature, followed by incubation with equivalent amounts of in vitro translated [35S]methionine-labeled Galpha o, Galpha s, or Galpha i2 in PBS with 0.05% Triton X-100 overnight at 4 °C with rocking. Beads were centrifuged, washed with PBS with 0.05% Triton X-100 three times, eluted with SDS sample buffer, and analyzed by SDS-PAGE and autoradiography.

Coimmunoprecipitation from Transiently Transfected COS Cells-- To preserve the HA epitope located at the N terminus of Pcp2 in pACT2, the plasmid was cut with BglII and filled with Klenow to generate a blunt end. Following XhoI digest, the fragment was cloned into the EcoRV/XhoI sites of pcDNA3 (Invitrogen). Galpha o in pcDNA3 (12) was transfected into COS-7 cells alone or in combination with Pcp2 using LipofectAMINETM (Life Technologies, Inc.) according to the manufacturer's protocol. At 72 h after the transfection, cells were washed with ice-cold PBS and lysed for 30 min in lysis buffer (50 mM Hepes, pH 7.5, 6 mM MgCl2, 1 mM EDTA, 75 mM sucrose, 2.5 mM benzamidine, 1 mM dithiothreitol, and 1% Triton X-100). Lysates were cleared by low speed centrifugation, and the supernatant was incubated with the HA-specific monoclonal antibody 12CA5 (1:100) overnight at 4 °C. Protein A-Sepharose (Sigma) was added for 1 h, and the samples were rocked at 4 °C. Samples were then centrifuged, and the pellets were washed three times with PBS with 0.05% Triton X-100. The precipitated proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE and Western blotting using a rabbit polyclonal anti-Galpha o antibody (5), and bands were visualized by chemiluminescence (Pierce).

Determination of kcat and koff-- Bovine brain Galpha o was kindly provided by E. Neer (Harvard Medical School) and used for kinetic analysis in the presence and absence of Pcp2. Pcp2 was prepared from GST fusion proteins by cleavage with thrombin at 2.5 units/mg protein for 1 h at room temperature and separated from GST by incubation with glutathione-agarose beads. Samples were concentrated and protein concentration determined (Bradford). Single turnover GTP hydrolysis (kcat) was determined by incubating Galpha o (50 nM) with 1 µM [gamma -32P]GTP (5500 cpm/pmol) for 20 min in the presence (1:1 molar ratio) or absence of Pcp2 in buffer A (50 mM Tris, pH 7.6, 5 mM EDTA, 1 mM dithiothreitol, and 0.1% Triton X-100). The hydrolysis reaction was started by addition of MgCl2 (final concentration, 10 mM) and 100 µM GTP. Aliquots were diluted into 1 ml of 5% (w/v) trichloracetic acid in 5% charcoal and counted as described previously (13). The amount of gamma -32P released at each time point was fit to an exponential function using GraphPad Prism. For determining koff, Galpha o (2 pmol) in the presence and absence of Pcp2 or GST (1:1 ratio with Galpha o) was incubated in buffer A with 10 mM MgCl2, 1 µM GTP [alpha -32P]GTP (specific activity 1.1 × 105 cpm/pmol), and 10 µg/ml bovine serum albumin for 20 min at room temperature. The stoichiometry of binding was approximately 60%, and the amount bound prior to initiating nucleotide exchange was set at 100%. GDP dissociation from Galpha o was initiated by the addition of 1 mM GDP, and aliquots were filtered onto nitrocellulose, washed, and counted. The data were fit to a one phase exponential decay using GraphPad Prism (San Diego, CA).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The yeast two-hybrid interaction system detects low affinity protein interactions and has been successfully used for finding novel proteins that interact with G protein alpha  subunits (14). The initial screen yielded 34 beta -galactosidase-positive clones, and this number was reduced to six positive clones after yeast mating controls. These six clones were sequenced and searched in GenBankTM (BLAST) data bases. Three of these cDNAs had no sequence homology to known genes, and one was identified as lactate dehydrogenase. One of the remaining genes was a previously identified human mosaic protein, LGN, that was discovered in a yeast two-hybrid screen using Galpha i2 as bait (15). The other remaining gene was a full-length clone of PCD5 (now called Pcp2) (16) that had been initially identified as a candidate gene important for Purkinje cell development. In pcd mice, Purkinje cells develop normally but then begin to degenerate at 15-18 days after birth leading to the development of ataxia (17).

To determine whether Galpha o could interact with Pcp2 in an independent assay, GST pull-down experiments were performed. Galpha subunits were translated and [35S]methionine-labeled in vitro and then incubated with equivalent amounts of GST or GST-Pcp2 fusion proteins. Fig. 1 shows that in vitro translated Galpha o binds to GST-Pcp2 (lane 2), whereas no significant interaction was seen with the GST control protein (black arrow, lane 1). We consistently detected approximately 10% of in vitro translated Galpha o coprecipitating with GST-Pcp2. Nonspecific interactions of Galpha o with GST were less than 1%. We next asked whether the conformation of Galpha o affected the interaction with GST-Pcp2. Galpha o was preincubated with GTPgamma S (nonhydrolyzable GTP analogue) or GDP prior to binding. As shown in Fig. 1, the amount of Galpha o that associates with Pcp2 is similar irrespective of the nucleotide bound to Galpha o. To address the issue of which Galpha subunits could interact with Pcp2, two other Galpha subunits expressed in the central nervous system were studied. Galpha i2 (pertussis toxin family member; ~70% amino acid identity to Galpha o) and Galpha s (cholera toxin family member; ~40% amino acid identity to Galpha o) were characterized in pull-down experiments with GST-Pcp2. Equal amounts of 35S-labeled Galpha i2 and Galpha s were translated in vitro and used for binding to GST proteins, and the amount of nonspecific binding between Galpha i2 or Galpha s with GST was similar to that seen with Galpha o (not shown). Fig. 1 shows that in vitro translated Galpha i2 interacts with GST-Pcp2, but Galpha s is barely detectable in GST-Pcp2 precipitates (lane 6, 52 kDa, open arrow). Taken together, these results suggest that the related pertussis toxin family members (Galpha o and Galpha i2) interact in vitro with Pcp2 but that Galpha s subunits do not.


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Fig. 1.   Interaction of in vitro translated Galpha subunits with GST fusion proteins. Equivalent amounts of GST or the fusion protein GST-Pcp2 were incubated with glutathione-agarose for 30 min and then overnight with equal amounts of [35S]methionine-labeled Galpha o, Galpha i2, or Galpha s translated in vitro. Samples were washed and eluted with SDS sample buffer and analyzed by SDS-PAGE followed by autoradiography. Nonspecific binding of in vitro translated Galpha o with GST is shown in lane 1 and was similar for Galpha i2 and Galpha s (not shown). A representative interaction of Galpha o with GST-Pcp2 is shown in lane 2 and was consistently seen in four independent experiments performed in duplicate. Preincubating Galpha o with GTPgamma S (100 µM, lane 3) or GDP (100 µM, lane 4) for 10 min at 30 °C did not affect binding. The binding of GST-Pcp2 to Galpha i2 is seen in lane 5, and there was minimal interaction of GST-Pcp2 with Galpha s (lane 6). The closed arrow is the size of Galpha o (39 kDa), and the open arrow is the expected size of Galpha s (52 kDa). The exposure time was 16 h.

To look for evidence that Galpha o and Pcp2 could interact in cells, cotransfection studies were performed in COS cells. COS cells were transfected with Galpha o, Pcp2, empty expression vector pcDNA3 (PC) alone or in combination. Cell lysates were immunoprecipitated using the 12CA5 antibody directed toward the hemagglutinin epitope on the N terminus of Pcp2 and then analyzed by Western with anti-Galpha o antibody (Fig. 2). COS cells do not normally express Galpha o, and in cells transfected with vector (PC), Pcp2, or Galpha o and then immunoprecipitated with 12CA5 there is no detectable Galpha o immunoreactivity (Fig. 2, arrow). However, when both Galpha o and Pcp2 are cotransfected into the same cells, a fraction of Galpha o is found in association with Pcp2 (Fig. 2, lane 4). We were unable to detect Pcp2 in immunoprecipitates of Galpha o, presumably due to disruption of the association with Galpha o by the stringent detergent conditions necessary to immunoprecipitate Galpha o (18). Attempts to detect Galpha i2 in Pcp2 immunoprecipitates were unsuccessful, although the level of Galpha i2 expression was much lower than for Galpha o. The observation that Galpha o and Pcp2 interact in an intact cell raises the possibility that Pcp2 could be a modulator of Galpha o.


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Fig. 2.   Coimmunoprecipitation of Galpha o and PCP2 from transfected COS cells. COS cells were transfected with pcDNA 3 (PC), Galpha o, Pcp2, or Galpha o plus Pcp2 and immunoprecipitated (IP) with HA-specific monoclonal antibody 12CA5 directed toward the N terminus of Pcp2. 72 h after the transfection, cellular lysates were immunoprecipitated and eluted with SDS sample buffer followed by SDS-PAGE and Western blotting with anti-Galpha o antibody. The migration of Galpha o in cell lysates of COS cells transfected with Galpha o is shown in lane 1. The arrow denotes the size of Galpha o at 39 kDa. Lanes 1-3 are from a different gel (and divided by white line) than lanes 4-6 but represent a single experiment. Similar results were seen in three independent experiments. Immunoprecipitation of Pcp2 was confirmed in independent experiments (not shown). In cells cotransfected with both Galpha o and Pcp2, a fraction of Galpha o can be detected in association with precipitated Pcp2 (lane 4, arrow). In cells transfected with vector alone (PC), Galpha o alone, or Pcp2 alone (lanes 2, 3, 5, and 6) and analyzed in parallel with Galpha o + Pcp2, no Galpha o (39 kDa, arrow) is detectable.

There are several possible mechanisms for regulation of Galpha subunits by accessory proteins, including effects on nucleotide binding or GTP hydrolysis. To address the possibility that Pcp2 modulates the enzymatic properties of Galpha o, we measured kcat and koff of bovine brain purified Galpha o (5) in the presence and absence of Pcp2. Fig. 3 shows a representative experiment, and the results are summarized in Table I. The kcat and koff values obtained for Galpha o are similar to literature values (19), and there was no significant difference in kcat of Galpha o in presence of Pcp2 (Fig. 3a and Table I). However, as seen in Fig. 3b and Table I, there is significant stimulation of koff in the presence of Pcp2. In experiments simultaneously comparing koff of Galpha o in the presence and absence of Pcp2 or GST, there was 5.2 ± 0.5-fold stimulation of koff in the presence of Pcp2 (n = 7). Incubating Galpha o with GST had no effect on koff (Fig. 3b and Table I), and including bovine serum albumin in the binding buffer also had no effect. The increase in koff of Galpha o in the presence of Pcp2 is similar to the observed increases seen with Galpha subunits reconstituted with receptors plus agonists (4-6-fold increases in koff) (20). This finding suggests that Pcp2 could mimic a receptor by stimulating GDP release. A family of proteins that promote nucleotide exchange have been described for monomeric G proteins such as Ras (21), but only neuromodulin has been shown to affect nucleotide binding to Galpha o. This mechanism of activation is likely to be distinct from Pcp2 because neuromodulin stimulates GTPgamma S binding through its N-terminal domain, which is homologous to the cytoplasmic tail of G protein-coupled receptors (7).


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Fig. 3.   Determination of kcat and koff for Galpha o in the presence and absence of Pcp2. a, single turnover GTPase activity for Galpha o was determined in the presence () and absence (open circle ) of PCP2 (1:1 molar ratio). The fraction of GTP hydrolyzed was measured for bovine brain purified Galpha o in the presence of [gamma -32P]GTP, and kcat was determined by fitting the data to a single exponential association function (GraphPad Prism). The results are summarized in Table I. b, the rate constant for GDP release (koff) from Galpha o was determined alone (open circle ) or in the presence of PCP2 () or an equivalent amount of GST (×). The percentage of GDP remaining bound to Galpha o was determined by equilibrating 1 µM [alpha -32P]GTP for 20 min and initiating nucleotide exchange with 1 mM GDP. The fraction of GDP bound to Galpha o was measured over time. The data were fit to a single exponential dissociation, and the results are summarized in Table I. The fold increase in koff for this experiment was 5.9 (the mean of all experiments (n = 7) was 5.2 ± 0.5).

                              
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Table I
Summary of Galpha o rate constants
Results are expressed as the means of individual experiments ± S.E. (n) where n = the number of experiments. There is no significant difference in any values except in koff for Galpha o + Pcp2 versus Galpha o alone or Galpha o + GST (p < 0.006).

The gene for Pcp2 is located on mouse chromosome 8 and encodes a cytosolic 99-amino acid protein without significant homology to other proteins (including G protein-coupled receptors). There is some amino acid sequence similarity to the c-sis/PDGF2 gene, but the implications of this are unknown (22). The expression profile of Pcp2 is consistent with a role in Purkinje cell development (23), but in mice without Pcp2 expression, Purkinje cells develop normally (presumably through other compensatory mechanisms) (24, 25). The function of Pcp2 and the mechanism(s) for increased Purkinje cell apoptosis in pcd mice remains unknown. Several genes in pcd mice have altered expression patterns including up-regulation of the early response genes fos and jun and down-regulation of the anti-apoptosis gene bcl-2 (26). Several G protein alpha  subunits trigger apoptosis (27), and Galpha o may be involved in apoptosis triggered by a mutated amyloid precursor protein in Alzheimer's disease (28). The results described in these studies suggest that Pcp2 may be involved in a signal transduction pathway involving Galpha o, but it remains to be determined whether this pathway relates to apoptosis. In addition, our results do not exclude important interactions of Pcp2 with other Galpha subunits, particularly Galpha i1, which is highly expressed in the brain. Increasingly, novel functions (and locations) for G proteins are being identified (such as in the Golgi, in the endoplasmic reticulum, and on intracellular vesicles) where they regulate protein processing and vesicular targeting (29-31). Because G protein-coupled transmembrane receptors have not been identified in many of these locations, Galpha subunits may be activated by nucleotide exchange factors. Pcp2 may be such a factor for Galpha o in cerebellar Purkinje cells, and future studies will define the functional consequences of this interaction.

    FOOTNOTES

* This work was supported by the National Institutes of Health and by a pilot award from the Harvard Digestive Disease Center (to B. M. D.).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.

Dagger Present address: Dept. of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS 39406.

§ To whom correspondence should be addressed: Harvard Inst. of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5809; Fax: 617-525-5830; E-mail: bdenker{at}rics.bwh.harvard.edu.

    ABBREVIATIONS

The abbreviations used are: G proteins, guanine nucleotide-binding proteins; GTPgamma S, guanosine 5'-(gamma -thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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