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ight:700;'>RGS9-2 in native brain tissue and transfected cells. Immunocytochemical and immunoblot experiments revealed an unexpectedly high proportion of RGS9-2 within the nuclei of forebrain neurons. A similar intracellular distribution was seen in transfected COS-7 cells. The RGS9 binding partner G(beta5) further enhanced the nuclear localization of RGS9-2, but did not affect the strongly cytoplasmic localization of RGS9-1, the retinal form of RGS9. Deletion construct analysis revealed that the unique polyproline-rich C-terminus of brain-specific RGS9-2 contains sequences necessary and sufficient to target RGS9 to the nucleus of COS-7 cells, as well as cultured striatal neurons. Furthermore, RGS9-2 transfection increased the transcriptional activity of a neuronal gene construct normally expressed in RGS9-positive neurons, suggesting that nuclear RGS9 directly or indirectly regulates transcription in vivo. The nuclear localization of RGS9-2 suggests a heretofore-unanticipated role for this brain-specific protein in transducing signals to the nuclei of forebrain neurons.

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protein containing an 118 amino acid RGS domain, which corresponds to an evolutionarily conserved domain that is present in all members of the RGS family of proteins. Outside of the homology domain, RGS9 shows more extended similarity to human RGS6 and RGS7, rat RGS12, and the C. elegans protein EGL-10. During embryonic and early postnatal stages of development, two RGS9 transcripts of approximately 1.4 Kb and 1.8 Kb were detected in whole brain. After postnatal day 10, accumulation of the larger transcript increased progressively until adulthood at the expense of the smaller transcript, which was undetectable in the adult. In adult rat brain, the 1.8-Kb RGS9 transcript was detected in the striatum but not in other brain regions or peripheral tissues. In situ hybridization in rat and mouse demonstrates that RGS9 mRNA is expressed predominantly in medium-sized, spiny neurons of the neostriatum and in neurons of the nucleus accumbens and olfactory tubercle. Relatively strong signals were also detected in some hypothalamic nuclei. Its selective expression suggests that RGS9 may play an important role in modulation of the complex signalling pathways of the basal ganglia.

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so has the unique ability to interact with the G protein beta subunit Gbeta(5). Here, we examined the behavior of Gbeta(5), RGS7, RGS9, and Galpha in tissue extracts using immunoprecipitation and conventional chromatography. Native Gbeta(5) and RGS7 from brain, as well as photoreceptor-specific Gbeta(5)L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gbeta(5) nor Gbeta(5)-free RGS could be detected. Co-expression in COS-7 cells of Gbeta(5) dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gbeta(5):RGS stoichiometry in a manner similar to Gbetagamma complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gbeta(5)-RGS and their coupled expression argue that in vivo, Gbeta(5) and Ggamma-like domain-containing RGSs only exist as heterodimers. Native Gbeta(5)-RGS7 did not co-precipitate or co-purify with Galpha(o) or Galpha(q); nor did Gbeta(5)L-RGS9 with Galpha(t). However, in transfected cells, RGS7 and Gbeta(5)-RGS7 inhibited Galpha(q)-mediated Ca(2+) response to muscarinic M3 receptor activation. Thus, Gbeta(5)-RGS dimers differ from other RGS proteins in that they do not bind to Galpha with high affinity, but they can still inhibit G protein signaling.

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br>DESIGN: Retrospective case series.
PARTICIPANTS: Eight affected individuals from 7 families.
METHODS: Ophthalmologic examination, color vision testing, fundus photography, and detailed electrophysiologic assessment were undertaken. Blood samples were taken for DNA extraction from affected subjects and, where possible, unaffected relatives. Mutation screening of RGS9 and R9AP was performed.
MAIN OUTCOME MEASURES: Detailed clinical, electrophysiologic, and molecular genetic findings.
RESULTS: All 8 patients had normal ocular examination results, with visual acuity ranging from 6/12 to 6/18. Four subjects were found to harbor mutations in RGS9 or R9AP, with 3 of the identified sequence variants being novel. Three subjects, 2 Pakistani sisters and an Afghani female, had mutations in R9AP. A novel homozygous nonsense mutation, p.G205fs, was identified in the simplex case, and a second novel homozygous in-frame deletion, p.D32_Q34del, was found in the 2 sisters. The remaining patient, a British male, had a compound heterozygous mutation in RGS9 (p.R128X/p.W299R). The mutation p.R128X represents the first nonsense mutation reported in RGS9. The 4 mutation-positive subjects had concordant characteristic previously described electrophysiologic findings that were not present in the 4 individuals in whom mutations were not identified. Novel findings associated with these mutation-positive patients included that they all showed electroretinogram evidence of severe cone system dysfunction under photopic conditions but normal cone function to a red flash under scotopic conditions. Such findings seem unique for the disorder.
CONCLUSIONS: This is the first report describing a nonsense mutation in RGS9. We have established novel electrophysiologic observations associated with RGS9 and R9AP mutations, including those relating to dark-adapted cone function and S-cone function. Patients with either RGS9/R9AP mutations (bradyopsia) or oligocone trichromacy have very similar clinical phenotypes, characterized by stationary cone dysfunction, mild photophobia, normal color vision, lack of nystagmus, and normal fundi. The distinctive electrophysiologic features associated with RGS9 and R9AP mutations enable directed genetic screening.
FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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proteins. Herein, we assess the functional role of RGS9-2 on LID. In monkeys, Western blot analysis of striatal extracts shows that RGS9-2 levels are not altered by MPTP-induced DA denervation and/or chronic L-dopa administration. In MPTP monkeys with LID, striatal RGS9-2 overexpression--achieved by viral vector injection into the striatum--diminishes the involuntary movement intensity without lessening the anti-parkinsonian effects of the D1/D2 receptor agonist L-dopa. In contrasts, in these animals, striatal RGS9-2 overexpression diminishes both the involuntary movement intensity and the anti-parkinsonian effects of the D2/D3 receptor agonist ropinirole. In unilaterally 6-OHDA-lesioned rats with LID, we show that the time course of viral vector-mediated striatal RGS9-2 overexpression parallels the time course of improvement of L-dopa-induced involuntary movements. We also find that unilateral 6-OHDA-lesioned RGS9-/- mice are more susceptible to L-dopa-induced involuntary movements than unilateral 6-OHDA-lesioned RGS9+/+ mice, albeit the rotational behavior--taken as an index of the anti-parkinsonian response--is similar between the two groups of mice. Together, these findings suggest that RGS9-2 plays a pivotal role in LID pathophysiology. However, the findings also suggest that increasing RGS9-2 expression and/or function in PD patients may only be a suitable therapeutic strategy to control involuntary movements induced by nonselective DA agonist such as L-dopa.