Article Figures & Data
Figures
- Figure 3.
Schematic representation (N to C terminus) of the RGS9 constructs. The numbers in parentheses indicate the position of each indicated domain in the original wild-type protein. The numbers at either end of the open boxes represent the span of the wild-type protein used in each construct. A, Schematic representation and domain composition of full-length wild-type RGS9-2. B, The full-length RGS9-2-EGFP fusion construct. The black bar between the RGS9-2 and EGFP represents a flexible spacer/linker with the following sequence: GSGGGSG. C, The DEPless RGS9-2-EGFP fusion construct in which the N-terminal region of RGS9 containing the DEP domain was deleted but was otherwise identical to the full-length RGS9-2-EGFP fusion. D, The RGS9 DEP domain fusion construct. The linker represented by the black bar has the following sequence: GDPPVAT. GGL, G-protein γ-subunit-like domain.
- Figure 1.
Behavioral effects of dopamine agonists in reserpinized or haloperidol-treated mice and distribution of D2DR and RGS9-2 in wild-type striatal neurons. Mice were injected once per day for 3 d with reserpine or haloperidol (see Materials and Methods). Three days after recovery, them ice were injected with dopaminergic agonists. Open bars represent wild-type mice, and filled bars represent RGS9-deficient mice. A, Quantification of abnormal involuntary movements in reserpinized mice after apomorphine (apo; n = 13 and 18 in wild-type and knock-out, respectively), quinpirole (quin; n = 10, 14), or SKF 38393 (SKF; n = 9, 8) injection (# and * indicate significant differences between genotypes; p < 0.01; Wilcoxon rank test). B, Locomotion measured (for 15 min) in the reserpinized mice (from A) immediately before (baseline) and after apo, quin, or SKF injection (*p < 0.01; t test). C, Quantification of AIM in haloperidol-treated mice after quin or SKF injection (n = 10; *p < 0.01; Wilcoxon rank test). D, D2 DR distribution (red; left), RGS9-2 distribution (green; middle), and their overlay (right) in a striatal section. The left and middle panels represent sequential confocal scans of the same field. E, D1DR distribution (red; left), RGS9-2 distribution (green; middle), and their overlay (right) in a striatal section. The left and middle panels represent sequential confocal scans of the same field. Scale bar: (in D) D, E, 10 μm. Error bars represent SEM.
- Figure 2.
Effects of the D2-like DR agonist quinpirole (quin) on excitatory currents elicited from medium spiny striatal neurons. A, Representative traces of currents evoked from the same voltage-clamped (-70 mV) striatal medium spiny neuron in response to glutamate (glut; 100 μm; black trace) or glutamate and quinpirole (glut, 100 μm plus quin, 100 μm; gray trace). Left, Wild-type neuron; right, RGS9-deficient neuron. Black bars above the traces represent drug application. B, Glut- and quin-elicited current expressed as a percentage of the current evoked by glut alone (100 μm) from wild-type (open bars) and RGS9-deficient (filled black bars) neurons. The mean ± SEM peak current amplitudes in picoamperes for wild-type cells were: glut alone, 607 ± 81 (n = 9); glut plus 1 μm quin, 488 ± 63 (n = 9); glut plus 10 μm quin, 574 ± 85 (n = 9); glut plus 100 μm quin, 602 ± 108 (n = 9); and for RGS9-deficient cells: glut alone, 316 ± 71 (n = 9); glut plus 1 μm quin, 257 ± 54 (n = 8); glut plus 10 μm quin, 250 ± 52 (n = 6); glut plus 100 μm quin, 155 ± 52 (n = 8). C, Current evoked by NMDA (100 μm; n = 7) or AMPA (10 μm; n = 5) in the presence of quin (100 μm) expressed as a percentage of the current evoked, respectively, by NMDA or AMPA alone from RGS9-deficient neurons. The mean ± SEM peak current amplitudes in picoamperes were as follows: NMDA alone, 442 ± 106; NMDA plus quin, 230 ± 87; AMPA alone, 266 ± 66; AMPA plus quin, 247 ± 28. The # symbol indicates that the quinpirole-sensitive fraction of the current differs significantly from zero. p values using a paired t test are 0.03, 0.04, and 0.008 at 1, 10, and 100 μm quinpirole, respectively. The corresponding p values for the wild-type animals were 0.07, 0.8, and 0.9, respectively. The asterisk indicates that the quinpirole-sensitive fraction of the current differs significantly from the corresponding wild-type sample. p < 0.01; ANOVA. Error bars represent SEM.
- Figure 4.
Distribution of D2DR and RGS9-2 in transfected CHO and PC12 cells. D2DR distribution is depicted in red, whereas RGS9-2 constructs and EGFP are shown in green. A, D2DR distribution in CHO cells that were transfected with cDNA for D2DR alone. B, RGS9-2 distribution in CHO cells that were transfected with cDNA for the RGS9-2 alone. C, D2DR distribution (red; left) and RGS9-2 distribution (green; right) in CHO cells transfected with cDNA for both D2DR and the RGS9-2. D, EGFP distribution in CHO cells transfected with cDNA for EGFP alone. E, D2DR distribution (red; left) and EGFP distribution (green; right) in CHO cells transfected with cDNA for both D2DR and EGFP. F, DEPless RGS9-2 distribution in CHO cells that were transfected with cDNA for DEPless RGS9-2. G, D2DR distribution (red; left) and DEPless RGS9-2 distribution (green; right) in CHO cells transfected with cDNA for both D2 DR and DEPless RGS9-2. H, RGS9 DEP domain distribution in CHO cells that were transfected with cDNA for the RGS9 DEP domain alone. I, D2DR distribution (red; left) and RGS9 DEP domain distribution (green; right) in CHO cells transfected with cDNA for both D2DR and RGS9 DEP domain. J, D2DR distribution in PC12 cells that were transfected with cDNA for D2DR alone. K, RGS9-2 distribution in PC12 cells that were transfected with cDNA for the RGS9-2 fusion protein alone. L, D2DR distribution (red; left) and RGS9-2 distribution (green; right) in PC12 cells transfected with cDNA for both D2DR and the RGS9-2 fusion construct. M, DEPless RGS9-2 distribution in PC12 cells that were transfected with cDNA for DEPless RGS9-2. N, D2DR distribution (red; left) and DEPless RGS9-2 distribution (green; right) in PC12 cells transfected with cDNA for both D2DR and DEPless RGS9-2. The left and right pictures, respectively, in panels C, E, G, I, L, and N represent confocal scans of the same field. Outlines of cells in which the specific fluorescent signal does not extend to the cell boundary are marked out with a yellow dashed line. Each CHO cell image is representative of the distribution pattern in >90% of cells in at least five observed fields, each containing >50 cells transfected with the relevant constructs. For PC12 cells, each image is representative of the distribution pattern observed in >90% of cells from at least 20 observed fields, each containing at least one cell transfected with the relevant constructs. Scale bar: (in A) A-N, 5 μm.
- Figure 5.
Distribution of m1-mAChR, D1DR, m2-mAChR, and RGS9-2 in transfected CHO cells and effect of RGS9-2 and DEPless RGS9-2 on the deactivation kinetics of D2DR- or m2-mAChR-elicited GIRK currents in Xenopus oocytes. A, m1-mAChR (labeled m1-mR) distribution in cells that were transfected with cDNA for D2DR alone. B, m1-mR distribution (red; left) and RGS9-2 distribution (green; right) in cells transfected with cDNA for both m1-mR and RGS9-2. C, D1DR distribution in cells that were transfected with cDNA for D1DR alone. D, D1DR distribution (red; left) and RGS9-2 distribution (green; right) in cells transfected with cDNA for both D1DR and the RGS9-2. E, m2-mAChR (labeled m2-mR) distribution in cells that were transfected with cDNA for D2DR alone. F, m2-mR distribution (red; left) and RGS9-2 distribution (green; right) in cells transfected with cDNA for both D2DR and RGS9-2. The left and right pictures, respectively, in B, D, and F represent confocal scans of the same field. Outlines of cells in which the fluorescence does not extend to the cell boundary are marked out with a yellow dashed line. Each image is representative of the distribution pattern in >90% of cells in at least five observed fields, each containing >50 cells transfected with the relevant constructs. Scale bar: (in A) A-F, 5 μm. In the Xenopus oocyte experiments, control oocytes were injected with cRNA for m2-mR, GIRK1, and GIRK2 or with cRNA for D2DR, GIRK1, and GIRK2. Some oocytes in each of the control groups were also injected with cRNA for either RGS9-2 or for the DEPless RGS9-2. G, H, Representative normalized traces of deactivation waveforms for dopamine-evoked (dop; D2DR agonist; 100 nm; red trace) and acetylcholine-evoked (m2-mR agonist; 100 nm; black trace) GIRK currents recorded from the oocyte group expressing the appropriate receptor and either RGS9-2 (G) or DEPless RGS9-2 (H). The horizontal bar above the traces represents either dopamine or acetylcholine application. I, Deactivation rate constants, koff (1/τdeactivation), were from exponential fits of the deactivation phase of the GIRK currents. For each receptor, acceleration of deactivation by RGS9-2 was calculated as follows: (individual values for koff in the oocytes injected with RGS9-2 cRNA)/(mean koff in the corresponding control group lacking RGS9-2). Acceleration of deactivation by DEPless RGS9-2 was calculated similarly. All bars are means ± SEM from the same oocyte donor (red open bar, D2DR response; black open bar, m2-mR response; * indicates significant difference; p < 0.05; Student's t test). In the experiment measuring RGS9-2-mediated acceleration of the deactivation kinetics, the mean ± SEM koff values in seconds-1 for the different oocyte groups were as follows: D2DR, 0.057 ± 0.006 (n = 4); D2DR plus RGS9-2, 0.102 ± 0.007 (n = 5); m2-mR, 0.055 ± 0.003 (n = 5); m2-mR plus RGS9-2, 0.078 ± 0.004 (n = 5), and the mean ± SEM steady-state current amplitudes in nanoamperes were: D2DR, 1094 ± 284; D2DR plus RGS9-2, 1125 ± 128; m2-mR, 1460 ± 153; m2-mR plus RGS9-2, 1416 ± 102. In the experiment measuring DEPless RGS9-2-mediated acceleration, the mean ± SEM koff values in seconds-1 were as follows: D2DR, 0.131 ± 0.021 (n = 5); D2DR plus DEPless RGS9-2, 0.211 ± 0.014 (n = 8); m2-mR, 0.123 ± 0.008 (n = 12); m2-mR plus DEPless RGS9-2, 0.225 ± 0.007 (n = 12), and the mean ± SEM steady-state current amplitudes in nanoamperes were: D2DR, 618 ± 43; D2DR plus DEPless RGS9-2, 943 ± 158; m2-mR, 964 ± 90; m2-mR plus DEPless RGS9-2, 953 ± 92. Error bars represent SEM.
