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First published online 5 June 2007
doi: 10.1242/jcs.005611

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Role of a G_i2 protein splice variant in the formation of an intracellular dopamine D₂ receptor pool

Manuel F. López-Aranda^1,*, Maria J. Acevedo^1,*, Antonia Gutierrez², Peter Koulen³ and Zafar U. Khan^1,

¹ Neurobiology Laboratory, CIMES, Faculty of Medicine, University of Malaga, Campus Teatinos s/n, 29071-Malaga, Spain
² Department of Cell Biology, Faculty of Science, University of Malaga, Campus Teatinos s/n, 29071-Malaga, Spain
³ Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA

View larger version (21K): [in this window] [in a new window]   Fig. 1. Expression of sGi2 reduces the dopamine D2 receptor density at the plasma membrane. (A) Infection of BHK cells with 30 µl pseudovirions of D2L (long isoform of D2 receptor) and 0-15 µl of sGi2 () or Gi2 (Gi2, ) protein showed a gradual decrease in the number of plasma-membrane-bound D2 receptors as the expression of sGi2 protein was increased. Gi2 had no effect. (B) Immunoblots of cells from A show that, as the addition of sGi2 pseudovirion was increased, higher expression of this protein. D2L expression remained the same. Non-transfected cells are shown in lane 4. (C) In JEG-3 cells that lack endogenous Gi2 protein, co-expression of sGi2 with either D2L or D2S (short isoform of D2 receptor) receptors also yielded a 32-35% reduction in cell surface D2 receptors, similar to that as seen in BHK cells in A. *P<0.05, significant change from control. Values are representative of six to eight different experiments.

View larger version (52K): [in this window] [in a new window]   Fig. 2. (A-D) Co-accumulation of D2 receptor and sGi2 protein complex in cytoplasmic space. (A) D2S receptors were expressed alone in BHK cells and immunodetected by antibodies to D2S receptor. In these cells, most of the D2S proteins were localized at the cell surface (arrowheads). However, when D2S receptors were co-expressed with sGi2 proteins (B and C) in these cells, cell surface localization of receptors was reduced markedly (arrowheads indicate protein immunolabeling). They co-accumulated in cytoplasm space with sGi2 protein (arrowheads in D). To detect the co-labeling of both proteins, specific antibodies against D2S (B) and sGi2 (C) were used in double labeling immunofluorescence experiment. Bar, 12 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Reduced D2 receptor-mediated plasma membrane Ca2+ channel activity after co-expression with sGi2 demonstrates the reduced D2 receptor density at cell surface. (A,B) Typical intracellular Ca2+ transient in a single BHK cell expressing (A) D2S and (B) D2L receptor, after application of 15 µM raclopride, a D2 antagonist. This activity was significantly reduced in cells when sGi2 was co-expressed. (C) Summary of the results from experiments using NG108-15 cells. Co-expression of sGi2 with either D2L or D2S produced a 30-35% decrease in Ca2+ transients, similar to receptor binding results shown in Fig. 1. This activity was associated with D2 and not to D1 receptors. In addition, expression of Gi2 instead of sGi2 did not show any change. F/F0 represents the change in fluorescence intensity over baseline (see Materials and Methods). *P<0.05, significant change from control. Values are representative of six different experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Co-immunoelution of sGi2 protein with D2S and D2L receptors. (A) BHK cell extracts were passed through immunoaffinity columns and resultant proteins were identified on immunoblots with a mixture of antibodies against D2L and sGi2 (lanes 1, 2) and antibodies to D2S (lanes 3, 6), sGi2 (lanes 4, 5), D2L (lane 7) and D1 (lane 8). sGi2 protein (41 kDa) co-eluted with both D2L (57 kDa) and D2S (49 kDa) antibodies and vice-versa. D1 receptor was not co-eluted with sGi2. (B) sGi2 antibodies co-immunoprecipitated D2 (3H-raclopride) but not D1 (3H-SCH 23390) receptor binding sites from extracts of BHK cells expressing sGi2 and D2S or D1. Values are representative of four different experiments.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Co-immunoelution of sGi2 protein with D2S and D2L receptor from the extract of monkey brain. Similar to Fig. 4, extract from the substantia nigra was passed through immunoaffinity columns as indicated. Eluted proteins were then identified in immunoblots with antibodies raised against D2S (lanes 1, 4), sGi2 (lanes 2, 3), D2L (lane 5), D1 (lane 6). sGi2 antibody co-eluted D2S and D2L receptors but not D1 receptor.

View larger version (12K): [in this window] [in a new window]   Fig. 6. The extreme C-terminal end of sGi2 is essential for the intracellular interaction. (A) For deletion experiments, sGi2 constructs were prepared by eliminating the part of the complete cDNA sequence with the use of specific PCR primers and then cloned. Each bar reflects the approximate size and location of each construct included in the study. Numbers on the right indicate start and end of the cDNA sequence. sGi2 (WT) and Gi2 (WT) are complete genes and rest (c-f) are the constructs derived from sGi2 (WT). Shaded areas show the sequences that differ between sGi2 and Gi2. Gi2 was included in the study as control. (B) Each of the constructs or WT (a-f) from (A) was co-expressed with D2S receptor in BHK cells and the receptor density at cell surface was assayed in intact cells. The protein expression of these constructs was confirmed by western blots (see supplementary material Fig. S5).The 108 base pairs on the extreme 3'-end were necessary for the intracellular interaction and retention of the receptor. Values are representative of five different experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Treatment with dopamine agonists increases the D2 receptor density at plasma membrane and decreases the D2 level from sGi2-D2 complex. (A) Incubation of BHK cells expressing D2S and sGi2 protein with 10 µM dopamine (D) or 5 µM quinpirole (Q) for 30 minutes showed a significant increase (18%) in D2 receptor localization at plasma membrane. By contrast, however, treatment with antagonist [raclopride (R), 5 µM] produced no effect. Controls are from C1 to C5 (C1, cells expressing D2S receptors alone and treated with dopamine; C2, cells treated with quinpirole; C3, cells expressing Gi2 as well as D2S receptor and treated with dopamine; C4, cells treated with quinpirole; C5, cells expressing sGi2 and D2S receptor but left untreated). (B) Extracts of same cells as used in A were passed through sGi2 immunoaffinity columns and eluted to determine sGi2-bound D2S receptor in above conditions. Immunoblot assays show that agonist treatment resulted in loss of 75% (equals to 22% of the binding results) D2S component (49 kDa) from sGi2-D2S complex, whereas the level of sGi2 was the same in all conditions. (C) Average OD change in immunoblot experiments from B. Results show that only 28.6±2.48% and 25±2.78% of the D2S receptor remained bound in the sGi2-protein complex eluted from dopamine (D)-treated and quinpirole (Q)-treated cells, respectively. Values are representative of five different experiments.