Loss of G Protein gamma 7 Alters Behavior and Reduces Striatal alpha olf Level and cAMP Production*

William F. SchwindingerDagger , Kelly S. BetzDagger , Kathryn E. GigerDagger , Angela SabolDagger , Sarah K. Bronson§, and Janet D. RobishawDagger

From the Dagger  Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 and the § Department of Cellular and Molecular Physiology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033-0850

Received for publication, October 30, 2002, and in revised form, December 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The G protein beta gamma -dimer is required for receptor interaction and effector regulation. However, previous approaches have not identified the physiologic roles of individual subtypes in these processes. We used a gene knockout approach to demonstrate a unique role for the G protein gamma 7-subunit in mice. Notably, deletion of Gng7 caused behavioral changes that were associated with reductions in the alpha olf-subunit content and adenylyl cyclase activity of the striatum. These data demonstrate that an individual gamma -subunit contributes to the specificity of a given signaling pathway and controls the formation or stability of a particular G protein heterotrimer.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The heterotrimeric G proteins control diverse biological processes by conveying signals from cell-surface receptors to intracellular effectors. Although function was originally ascribed to the GTP-bound alpha -subunit, it is now well established that the beta gamma -dimer plays active roles in the signaling process through upstream recognition of receptors and downstream regulation of effectors (1). Molecular cloning has identified at least 5 beta - and 12 gamma -subunit genes in the mouse and human genomes. Structurally, gamma -subunits are the most diverse, with four subgroups that show less than 50% identity to each other (2). Moreover, gamma -subunits exhibit very different temporal (3, 4) and spatial (5) patterns of expression. These characteristics suggest that gamma -subunits have heterogeneous functions. However, comparison of their biochemical properties has revealed only modest differences (6-8), perhaps because of the inherent limitations of transfection and reconstitution approaches. Gene ablation in mice has proven to be a powerful approach to identifying the functional roles of several G protein alpha -subunits (9). We report the first use of a gene targeting strategy to identify a unique function for a member of the gamma -subunit family.

The G protein gamma 7-subunit (Ggamma 7) was originally cloned from bovine brain (10). In situ hybridization of rat brain sections revealed that mRNA for Ggamma 7 is most highly expressed in the striatum (5), where it is found in 40-50% of medium sized neurons in the caudate putamen (11). The regional expression of mRNA for Ggamma 7 in the brain mirrors that of the striatum-enriched D1 dopamine receptor (D1R),1 Galpha olf, and adenylyl cyclase Type V (12), suggesting involvement of Ggamma 7 in the Galpha olf-mediated stimulation of adenylyl cyclase by dopamine. Single cell RT-PCR analysis confirms that D1R and Ggamma 7 are expressed in the same subset of rat neurons (13). Ribozyme suppression studies support a role for Ggamma 7 in the endogenous beta -adrenergic receptor pathway (14) and the heterologously expressed D1R pathway in human embryonic kidney cells (13).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Animal Care and Approval-- Mice were segregated by sex and group-housed in plastic microisolator cages in ventilated racks (Thoren Caging Systems, Inc., Hazelton, PA). Mice were given ad libitum access to water and Mouse Diet 9F (Purina Mills, St. Louis, MO). Environmental factors included temperature and humidity control and a 12-h light/dark cycle. The animal facility is maintained as virus antibody-free and parasite-free. Animal research protocols were approved by the Geisinger Clinic institutional animal care and use committee.

Genotyping-- Southern blot analysis was performed on genomic tail DNA cut with KpnI. The probe used for this analysis was an 0.8-kb fragment 5' of the modified Gng7 allele. Alternatively, PCR analysis was performed using primers (Invitrogen) shown in Table I. Briefly, primers flanking the 3' loxP site (JR385 and JR387) were used competitively to amplify the wild type and Gng7fl alleles, and a third primer upstream of the 5' loxP site (JR413) was included to amplify simultaneously the wild type and deleted Gng7 alleles. Amplification of the bacteriophage P1 Cre transgene was conducted with JR353 and JR354.

RT-PCR-- RNA was isolated from olfactory bulb, frontal cortex, striatum, hypothalamus, midbrain, cerebellum, pons, or whole brain using TRIzol reagent (Invitrogen). First-strand cDNA, prepared from 2 µg of RNA using Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Promega Corp., Madison, WI), was used as a template to amplify the Ggamma 7 transcript with primers JR384 and JR386 or the elongation factor transcript with primers EFs and EFas, respectively (Table I).

                              
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Table I
Primers used in this study

Immunoblotting-- Powdered brain tissues were homogenized in HME with proteinase inhibitors (20 mM Hepes, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 1 mM benzamidine, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µM leupeptin, 1.4 µM pepstatin, 27 µM 1-chloro-3-tosylamido-7-amino-2-heptanone, 28 µM L-1-tosylamido-2-phenylethyl chloromethyl ketone). Membranes were obtained by centrifugation onto a sucrose cushion (see below), and membrane-associated proteins were extracted with 1% cholate at 4 °C overnight. Protein concentrations were determined using an Amido Black assay (15). Equal amounts of proteins were loaded onto 12% Nu-Page gels (Invitrogen) and transferred to NitroPure nitrocellulose (Osmonics, Inc., Westborough, MA) using a high temperature transfer procedure (16). Immunoblotting was performed as described previously (14) with antisera specific for Ggamma 7 (17) at a 1:200 dilution, rat Na+/K+-ATPase beta -subunit (Research Diagnostics, Inc., Flanders, NJ) at a 1:1000 dilution, Galpha s (a generous gift of Dr. Catherine Berlot) used at a 1:500 dilution, Galpha olf (a generous gift of Dr. Denis Hervé) used at a 1:1000 dilution, Galpha o (16) used at 1:500, Galpha 13 (a generous gift of Dr. N. Dhanasekaran) used at 1:1000, Galpha q/11 (16) used at 1:200, and Ras (BD Biosciences) used at a 1:2000 dilution. Immunoblots were imaged with a PhosphorImager and analyzed with ImageQuant software (Amersham Biosciences).

Adenylyl Cyclase Assay-- Brain tissues were homogenized in Buffer A (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.3 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 30 µM leupeptin, 1 µM pepstatin A) with 10% sucrose using a Brinkmann homogenizer (Brinkmann Instruments). Membranes were then isolated by centrifugation (65 min at 100,000 × g) onto a cushion of Buffer A with 44.5% (w/v) sucrose. The membranes at the interface were transferred to a new tube, washed twice with Buffer A, and collected by centrifugation (30 min at 100,000 × g). Protein concentrations were determined with Coomassie Plus (Pierce). Adenylyl cyclase activity (18) was determined by incubating membrane protein (20 µg) at 30 °C for 10 min in 0.1 ml of buffer containing 50 mM Hepes, pH 7.4, 0.2 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, 0.5 mM ATP, 1 mM isobutylmethylxanthine, 5 mM creatine phosphate, 50 units/ml creatine phosphokinase, and various agonists as indicated in Fig. 3. For stimulation of striatal membranes with dopamine agonists, more consistent results were obtained using buffer containing 10 mM imidazole, pH 7.3, 0.2 mM EGTA, 0.5 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM ATP, and 0.5 mM isobutylmethylxanthine (19). Reactions were terminated by the addition of 1 ml of 0.1 N HCl, 1 mM EDTA. The cAMP concentrations were determined by automated radioimmunoassay using a Gamma-Flo instrument (Atto Instruments, Inc., Potomac, MD) as described previously (20).

Open Field Study-- A functional observational battery was used to assess the behavior of mice outside of their home cages. On 5 successive days, mice were placed in the center of a translucent polypropylene box (45 × 30 × 60 cm). The behavior of each mouse was observed for 2 min by a researcher who was unaware of the genotypes of the mice. The latency to first step in seconds was recorded. The number of times the mouse reared was counted. The fraction of the box floor explored by the mouse was estimated. The startle response of the mouse to the sound of a latex glove being snapped was graded. After the mouse was removed from the box, the fecal boli and urine pools left in the box were counted, and the box was wiped clean with a contact disinfectant.

Locomotor Activity-- Locomotor activities were quantified in CLAMS cages (Columbus Instruments, Columbus, OH). The cages were clear plastic boxes (20 × 10 × 12.5 cm) fitted with three rows of eight photoelectric sensors (x, y and z directions). The mice were placed in the CLAMS cages at 11 a.m. and remained in the cages for 3 h. During this time the mice had ad libitum access to water. Every minute the number of total and consecutive photobeam breaks for each of the three sensor arrays and the number of contacts with the sipper tube were recorded.

Acoustic Startle Reflex-- Acoustic startle was measured using an SR-Lab startle reflex system (San Diego Instruments, San Diego, CA). A speaker delivered a continuous background noise of 65 db. During each trial a 40-ms pulse of broadband noise at 110 db was delivered. In prepulse trials, 20 ms of broadband noise at 70, 80, or 90 db was administered at 100 ms before the pulse. Each mouse received six consecutive pulse trials followed by six of each prepulse, pulse, or no stimulation trial, in random order. The interval between trials varied from 12 to 30 s. Data were collected at 1-ms intervals starting 50 ms before the first stimulus. The startle magnitude was calculated by summing the voltage from the startle chamber (minus the base-line voltage) over the 100 ms after the onset of the pulse. A single chamber was used for all mice.

Statistical Methods-- Data are presented as the mean ± S.E. Data were analyzed by t test, chi square, or repeated measures MANOVA using JMP (SAS Institute Inc., Cary, NC).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To investigate the physiologic function of Ggamma 7, we targeted Gng7, the murine gene encoding Ggamma 7, using a Cre/Lox strategy to provide the potential for conditional inactivation of the gene in future studies (Fig. 1A). The complete coding region of Gng7 was contained within an ~10-kb mouse genomic clone (21). This included the two coding exons split by an ~1.1-kb intron. A targeting vector was designed to flank the coding region of Gng7 with loxP sites. Targeted embryonic stem cells, chimeric mice, and F1 heterozygotes with the floxed Gng7 allele (Gng7fl/+) were produced at Lexicon Genetics, Inc., The Woodlands, TX. Gng7fl/+ mice were mated with mice carrying the Cre recombinase driven by the CMV promoter, BALB/c-TgN(CMV-Cre)#Cgn (Jackson Laboratories, Bar Harbor, Maine). This resulted in deletion of the coding region of Gng7 following Cre recombinase-mediated excision (Fig. 1B). Heterozygous mice with the deleted Gng7 allele (Gng7+/-) were back-crossed with C57BL/6J mice (Jackson Laboratories) for up to five generations, to eliminate the CMV-Cre transgene and to obtain a more homogeneous genetic background, and then were intercrossed to obtain the mice used in the following experiments (Fig. 1C). To confirm the effectiveness of the gene knockout, expression of Gng7 was examined in brain, where the gene is most highly expressed (10). RT-PCR analysis demonstrated that the Ggamma 7 mRNA was absent in brains from Gng7-/- mice (Fig. 1D). Likewise, immunoblot analysis with Ggamma 7 antisera (17) showed that the Ggamma 7 protein was reduced by 50 ± 6% in cholate-solubilized membrane extracts of brains from Gng7+/- mice and was not detectable in brains from Gng7-/- mice (Fig. 1E).


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Fig. 1.   A, targeting strategy. Exons 2 and 3 (black boxes) and the 5'-probe (gray box) are shown for the wild type allele (Gng7). The ~10-kb genomic clone is between sites S1 and S3. The targeted allele (Gng7fl) with LoxP-neoR cassette in intron 1 and LoxP cassette in the 3'-untranslated sequence (gray boxes with arrows or "neo") are shown. The targeting fragment is between sites S2 and S3. The deleted allele (Gng7-) shows the remaining LoxP site following Cre-mediated deletion of the coding region of Gng7. PCR primers are located at arrows a, b, and c. Expected sizes of KpnI (K) fragments are illustrated. B, Southern blot of tail biopsy DNA digested with KpnI and detected with the 5'-probe. Lane 1, molecular weight ladder; lanes 2-7, progeny of a Gng7+/fl × TgN(CMV-Cre) cross. All lanes show the 14-kb wild type Gng7 allele, and lanes 4, 5, and 7 also show the 12-kb Gng7- allele. C, PCR products obtained with primers a, b, and c of tail biopsy DNA from progeny of a Gng7+/- × Gng7+/- cross. Lane 1, phi X HaeIII marker; lanes 2, 4, 5, and 8, wild type mice with a single 452-bp band derived from primers a and b; lanes 3 and 6, Gng7-/- mice with a single 574-bp band derived from primers b and c; lanes 7 and 9, heterozygous Gng7+/- mice with both bands. D, top, RT-PCR products obtained after 25 cycles of amplification with primers for Gng7 from cDNA prepared from whole brains of three wild type, three Gng7+/-, and three Gng7-/- mice or without added cDNA (W). Bottom, RT-PCR products obtained after 25 cycles of amplification with primers for elongation factor 1alpha 2 (Eef1a2) from identical aliquots of the cDNAs. E, Western blot analysis of proteins prepared from brains of three wild type, three Gng7+/-, and three Gng7-/- mice or SF9 cells expressing Ggamma 7 (Std). The lower panel shows Ggamma 7 is reduced in membranes from Gng7+/- mice and absent in membranes from Gng7-/- mice. The upper panel shows that rat sodium/potassium ATPase beta -subunit (ATPase) is equal in all lanes.

Genotype analysis of offspring of heterozygous (Gng7+/-) intercrosses revealed the expected numbers of wild type, heterozygous, and homozygous mice, indicating that disruption of Gng7 did not affect survival to weaning. There were no significant differences in the weights of Gng7-/- mice and their wild type littermates. Moreover, no increased mortality was seen in Gng7-/- mice over a 6-month median observation time. Finally, in two homozygous crosses, Gng7-/- mice were fertile and weaned litters of apparently normal size. These observations are in stark contrast to mice with a deficiency of other components of the D1R signal transduction pathway. Mice with a homozygous deletion of Gnas, the gene encoding Galpha s, die in utero before implantation (22). Most mice with a homozygous deletion of Gnal (23), the gene encoding Galpha olf, fail to thrive and die in the neonatal period. Moreover, although Gnal-/- mice are fertile, dams lack fostering skills and none of their litters survive. Similar failure to thrive is observed in mice with a homozygous deletion of Drd1a (24, 25), the gene encoding the D1R. These feeding and fostering deficits may be related to loss of the sense of smell in Gnal-/- mice (23) and impaired motivated behavior in Drd1a-/- mice (26), respectively. Thus, Gng7-/- mice display a more circumscribed phenotype than mice with a deficiency of Gnas, Gnal, or Drd1a; and offer a more robust animal model for behavioral studies.

Behavior reflects the underlying function of the brain, making it a sensitive indicator of alterations induced by genetic manipulation (27). Anecdotal reports of increased handling reactivity from the animal care technicians provided the first evidence for a behavioral phenotype. More systematic screens of their behavior were carried out using a functional observational battery (28). For this purpose, mice were observed outside of their home cages; measurements of neuromuscular function (gait abnormalities), sensorimotor defects (auditory startle), autonomic responses (fecal boli and urine pools), and activity levels (latency to first step, rears, and exploration) were scored on predefined rating scales by a trained observer who was unaware of the genotypes of the animals. Using this test battery, the most striking observation was that the Gng7-/- mice exhibited an increased startle response as compared with their wild type littermates. To quantify this effect, a Startle Reflex System (San Diego Instruments) was used to measure the startle reactivity of Gng7-/- mice and their wild type littermates. Notably, the Gng7-/- mice displayed a greater startle amplitude than their wild type littermates for each stimulus tested (Fig. 2A). Prepulse inhibition refers to the reduction in startle response that occurs when the startling stimulus is preceded by a stimulus of lower intensity and is often used as a measure of sensorimotor gating. Despite the enhanced startle response, the Gng7-/- mice showed a similar degree of prepulse inhibition compared with their wild type littermates for each stimulus tested (Fig. 2A, inset).


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Fig. 2.   Behavior of Gng7-/- mice and wild type littermates. Data are presented as the mean ± S.E. of 32 mice in each group. A, startle response of wild type (black bars) and Gng7-/- (gray bars) mice quantified by testing with the SR-Lab startle reflex system. The startle response is shown as the area-under-the-curve (voltage from startle chamber versus time) for each stimulus. p110a represents an average of 6 successive startle pulses of 110 db. The remaining bars represent an average of 6 stimuli presented in pseudo-random order at varying intervals and consisting of pulse alone (p110), pulse with prepulse of 70 db (pp70), 80 db (pp80), 90 db (pp90), or no pulse (none). A repeated measures MANOVA showed that stimulus (F4,59 = 63.7, p < 0.0001) and genotype (F1,62 = 35.6, p < 0.0001) were significant factors and that there was a significant interaction between stimulus and genotype (F4,59 = 9.1, p < 0.0001). The inset shows percent prepulse inhibition (Percent PPI) of the startle response, calculated as 100 × (p110 - ppX)/p110. A repeated measures MANOVA shows that stimulus (F4,59 = 51.8, p < 0.0001) was a factor, but there was no significant effect of genotype. B, locomotor activity of wild type (black bars) and Gng7-/- (gray bars) mice studied in CLAMS cages for a 3-h period on each of 2 consecutive days. Ambulatory activity calculated as adjacent photobeam breaks per minute, in the x and y axis directions, averaged over 1-h periods. A repeated measures MANOVA of horizontal activity showed that day (F1,124 = 64.4, p < 0.0001) and hour (F2,123 = 279.1, p < 0.0001) were significant factors, but genotype (F1,124 = 3.1, p = 0.08) was not a statistically significant factor; the only significant interaction was between hour and day (F2,123 = 50.2, p < 0.0001).

The finding of increased startle response but normal prepulse inhibition of the startle response is a phenotype that has been observed previously in mice with mutations in the glycine binding site of the N1-subunit of the NMDA receptor, Grin1D481N/D481N (29) and Grin1D481N/K483Q (30). This is intriguing because earlier studies had shown that dopamine acting through cAMP to stimulate protein kinase A, and through DARPP-32 (32-kDa dopamine- and cAMP-responsive phosphoprotein) to inhibit protein phosphatase 1, increases the phosphorylation state of the N1-subunit of the N-methyl-D-aspartic acid (NMDA) receptor and potentiates NMDA responses (31). This suggests that Ggamma 7 may be involved in the signal transduction pathway that regulates the response of striatal gamma -aminobutyric acid-producing (GABAergic) neurons to glutamate, a pathway that has been implicated in the pathogenesis of schizophrenia (32). Alternate mechanisms by which a deficiency of Ggamma 7 may increase the startle response are suggested by other mouse models. Mice with a deficiency of adrenergic alpha 2c-receptors demonstrate an increased startle response; however, these mice have diminished prepulse inhibition (33). Transgenic mice expressing a dominant mutant of the human inhibitory glycine receptor alpha 1-subunit, TgN(GLRA1R271Q), as well as mice with the recessive mutation spasmodic (Glraspd/spd), display a complex neuromuscular phenotype that includes increased startle response and mimics the human disease of hyperekplexia (34).

The abundant expression of Gng7 in the striatum suggests a possible role in control of locomotor activity. As a quantitative test of this activity, Gng7-/- mice and their wild type littermates were placed in CLAMS cages (Columbus Instruments), which are equipped with photobeams to measure movement in the x, y, and z directions. Both wild type and Gng7-/- mice exhibited elevated horizontal and vertical locomotor activity when introduced into this new environment. Gng7-/- mice showed less horizontal (Fig. 2B) and vertical activity (not shown) than their wild type littermates at all time points, but these differences were not statistically significant. Habituation refers to the tendency for the increased locomotor activity to decline upon repeated or sustained exposure to a new environment, which is often used as a measure of a learned response. Importantly, both groups of mice displayed comparable levels of habituation between and within sessions.

On the basis of pharmacologic studies showing D1 dopamine agonists produce a strong stimulatory effect on locomotor activity (35), one might have expected a more marked decrease in locomotor activity in Gng7-/- mice. In this regard, however, other gene knockout studies have not provided strong support for the pharmacologic studies. For example, Drd1a-/- mice have been variously reported as being either hypoactive (24, 26), hyperactive (25), or neither but showing an altered pattern of activity (36). Moreover, Gnal-/- mice have increased locomotor activity in an open field (12), which is the opposite of the result expected based on pharmacologic studies. This variability may reflect the complex nature of locomotor activity (37), the confounding effects of olfactory deficits in Gnal-/- mice (23), or unidentified compensatory changes that may occur in knockout mice (38). Further study will be needed to clarify this issue, including more a restricted disruption of Gng7 within specific regions of the brain that is made possible by our floxed mouse model.

Finally, the expression of Gng7 along with Drd1a in a subset of neurons within the striatum suggests a possible role in regulation of adenylyl cyclase activity (13). To evaluate this possibility, adenylyl cyclase activity was compared between those regions of the brain that normally express Gng7, such as striatum, and other regions that do not normally express Gng7, such as cerebellum (Fig. 3A). Intriguingly, dopamine and the D1-specific agonist 6-chloro-PB had stimulatory effects on adenylyl cyclase activity in the striatum from wild type mice, but the responses were virtually abolished in the striatum from Gng7-/- mice (Fig. 3B). By contrast, dopamine had no effect on adenylyl cyclase activity in the cerebellum, but the response to isoproterenol was comparable in both wild type and Gng7-/- mice (Fig. 3C). These results establish a functional link between the expression of Ggamma 7 and adenylyl cyclase activity. Forskolin is a potent activator of adenylyl cyclase activity. Remarkably, forskolin had a potent stimulatory effect on adenylyl cyclase activity in the striatum from wild type mice, but the response was reduced by 50% in the striatum from Gng7-/- mice (Fig. 3D). By contrast, response to forskolin was comparable in the cerebellum from both groups of mice (Fig. 3E). Taken together, these data demonstrate that Ggamma 7 has an important but regionalized role in the regulation of adenylyl cyclase activity in the brain.


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Fig. 3.   A, regional expression of Gng7 in brain. RT-PCR products were amplified from RNA prepared from various regions of the mouse brain: pons (Pon), cerebellum (Cer), midbrain (Mid), hippocampus (Hip), striatum (Str), cortex (Cor), and olfactory bulbs (Olf). Upper panel, the first lane is the phi X HaeIII molecular weight marker (Mkr). The remaining lanes show products after 25 cycles of amplification with primers Gng7. mRNA for Gng7 is present at highest levels in the striatum and at lowest levels in the cerebellum and olfactory bulb. Lower panel, products after 25 cycles of amplification with primers for eukaryotic translation elongation factor 1alpha 2 (Eef1a2) showing equal amounts of mRNA from all regions. B-D, adenylyl cyclase activity of membranes prepared from wild type (black bars) and Gng7-/- (gray bars) brain regions in pmol of cAMP/mg of membrane protein/min. Data are presented as the mean ± S.E. for n mice in each group. B, striatal membranes (n = 9) in response to 1 µM GTP alone or with 100 µM dopamine or 10 µM 6-chloro-PB, a D1-like agonist. In wild type membranes but not in Gng7-/- membranes, cyclase activity is increased significantly by dopamine (p < 0.01) and 6-chloro-PB (p < 0.05) as compared with GTP alone. C, cerebellar membranes (n = 3) in response to 1 µM GTP alone or with 100 µM dopamine or 1 µM isoproterenol. Adenylyl cyclase activity was not stimulated by dopamine but was significantly stimulated by isoproterenol in both wild type (p < 0.05) and Gng7-/- membranes (p < 0.05). D, striatal membranes (n = 9) in response to 100 µM forskolin. Adenylyl cyclase activity is significantly greater (p < 0.0001) in wild type than in Gng7-/- striatal membranes. E, cerebellar membranes (n = 3) in response to 100 µM forskolin. There is no significant difference between wild type and Gng7-/- cerebellar membranes.

Because the striatal adenylyl cyclase Type V isoform (39) is synergistically activated by a combination of forskolin and Galpha s (40), one mechanism that could account for defects in both receptor-mediated and forskolin-stimulated adenylyl cyclase activity is a reduced level of the stimulatory G protein alpha -subunit in the striatum of Gng7-/- mice. To test this possibility, the levels of Galpha s and Galpha olf, the two known activators of adenylyl cyclase activity, were determined. Notably, the levels of Galpha s were comparable in the striatum from both groups of mice level, but the amount of Galpha olf was reduced by 82 ± 3% in the striatum (n = 8) from Gng7-/- mice (Fig. 4A). To provide additional evidence for a specific reduction in Galpha olf, we examined the levels of representative members of other G protein alpha -subunit subunit families. Notably, the levels of Galpha o, Galpha 13, and Galpha q/11 were not significantly reduced in the striatum of Gng7-/- mice (Fig. 4B). These results provide the first evidence that loss of a gamma -subunit can result in loss of its alpha -subunit partner.


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Fig. 4.   Western blot analysis. A, expression of the two stimulatory G protein alpha -subunits, Galpha olf and Galpha s, in striatal membranes of four wild type and four Gng7-/- mice. Note that the level of Galpha olf is markedly reduced in Gng7-/- mice, whereas expression of the 45- and 52-kDa forms of Galpha s is essentially unchanged. B, expression of representative members of other G protein alpha -subunit families in striatal membranes. There is no significant decrease in the levels of these G protein alpha -subunits. Blotting with antisera to Ras demonstrates even loading in all lanes.

In summary, this paper provides several novel insights. First, Ggamma 7 plays a unique role in regulation of adenylyl cyclase signaling in certain regions of the brain. This is demonstrated by the finding that loss of Ggamma 7 produces both a behavioral and a biochemical phenotype, indicating that other types of gamma -subunit are not able to substitute for this function. Thus, members of the gamma -subunit family are not functionally interchangeable in the context of the whole animal. Second, Ggamma 7 plays a role in the stabilization or formation of a G protein heterotrimer (alpha olfbeta gamma 7) that is required for stimulation of adenylyl cyclase activity in the striatum. This is substantiated by the finding that loss of Ggamma 7 reduces the level of Galpha olf in the striatum in a specific and coordinate fashion. Further studies are needed to address the underlying mechanisms and to determine whether this process is applicable to other gamma -subunits. If widespread, these results will reveal an important new signaling paradigm, namely, the level of a specific gamma -subunit controls the stability or assembly of a particular G protein heterotrimer. This, in turn, provides a probable basis for the selectivity of the multitude of G protein-coupled receptor signaling pathways that are now known to exist. Finally, the production of mice lacking Ggamma 7 provides a unique mouse model for the study of numerous diseases in which dysfunction of the adenylyl cyclase signaling pathway in the striatum has been implicated, such as Parkinson's disease, Huntington's chorea, Tourette's syndrome, and schizophrenia.

    ACKNOWLEDGEMENTS

We thank the outstanding technicians in our animal care facility, Cynthia J. Rhone, Gail L. Gregory, and Shannon Wescott. We are grateful to Dr. Denis Hervé for providing the alpha olf antibody, to Dr. Catherine Berlot for supplying the alpha s antibody and providing helpful advice, and to Dr. N. Dhanasekaran for supplying the alpha 13 antibody. We are indebted to Drs. Michael C. Nehls and Jean-Pierre Revelli (Lexicon Genetics Inc.) for producing the Gng7+/fl mice.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM39867 (to J. D. R.).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 should be addressed: Weis Center for Research, Geisinger Clinic, 100 North Academy Ave., Danville, PA 17822. Tel.: 570-271-6684; Fax: 570-271-6701; E-mail: jrobishaw@geisinger.edu.

Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211132200

    ABBREVIATIONS

The abbreviations used are: D1R, dopamine D1 receptor; Ggamma 7, G protein gamma 7-subunit; MANOVA, multivariate analysis of variance; RT, reverse transcriptase; CLAMS, comprehensive laboratory animal monitoring system; CMV, cytomegalovirus; olf, olfactory; NMDA, N-methyl-D-aspartic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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