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

JCS005611 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Characterization and specificity of sG_i2 antibodies. (A) In western blots, incubation with anti-sG_i2 recognized a polypeptide of 39 kDa (arrow) in whole rat brain as well as in cortex and striatum of monkey brain. However, this protein band was not observed when preimmune serum or peptide preabsorbed antibody was used. (B) Immunoblots of BHK cells expressing sG_i2 protein showed reactivity to a protein band of 41 kDa (arrow) and not to cells expressing G_i1, G_i2, G_i3, G_S or G_O. Incubation with preimmune serum and preabsorbed antibody also did not show any reaction in cells with sG_i2. (C) Second antibody from different epitope of sG_i2 protein (Ab 332-342) showed similar reactivity on western blot as first antibody (Ab 343-354) in rat brain. Sizes of standard proteins are indicated in far left. Together these results suggest that antibody (Ab 343-354) is specific and subtype selective to sG_i2 protein. Antibody (Ab 343-354) was used throughout the studies of this manuscript except in this figure where the use of other antibody (Ab 332-342) is clearly indicated. Antibodies to D_2S and D_2L has been characterized and published previously (Khan et al., 1998, 2001).

• Supplemental Figure S2 -

  Fig. S2. sG_i2 reduces the efficiency of blockade of D₂-agonist-mediated Ca²⁺ influx in NG 108-15 cells. In contrast to antagonist, 10 μM of quinpirole, an agonist of dopamine D₂ receptor, inhibits Ca²⁺ influx when D₂ receptor alone was expressed, however, co-expression of sG_i2 significantly reduced the efficiency of this inhibition. Expression of G_i2 in place of sG_i2 did not show this change. *P<0.05, significant change from control. We have shown in main manuscript that D₂ antagonist treatment increases voltage-gated intracellular Ca²⁺ flow (Chronwall et al., 1995; Pauwels et al.,2001) in cells expressing dopamine D₂ receptor, here we show that activation by agonist blocks its flow (Lledo et al., 1990; Wolfe and Morris, 1999) in the same cells. However, when sG_i2 is co-expressed, these cells show lower blockade activity due to reduced numbers of D₂ receptor at plasma membrane.

• Supplemental Figure S3 -

  Fig. S3. Intracellular Ca²⁺ stores did not contribute to D₂ drug-mediated Ca²⁺ changes. Addition of IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (APB), in reaction did not show any effect in Ca²⁺ influx. However, experiments with Ca²⁺-free medium or medium with 5 mM EGTA and 8 mM CoCl₂, both blockers of membrane Ca²⁺ channels, showed no Ca²⁺ response. These results demonstrate that Ca²⁺ change observed in cells was due to activity in membrane-bound Ca²⁺ channels.

• Supplemental Figure S4 -

  Fig. S4. sG_i2 protein is localized in dendrites (A,B), in spines (C,D) and in axons (D). (A) Immunolabeling in dendrite (d; indicated by hollow arrows). (B) Immunolabeled dendrite is making both symmetric (arrows) and asymmetric (arrowhead) synaptic contacts with unlabeled axons (an). (C) Labeling was often seen in or near neck of spines (∼70%), which were originating from dendrites (hollow arrowheads). (D) shows the labeled axons (a) making asymmetric contacts (arrowheads) with labeled as well as unlabeled spines (sn).Bars, 500 nm (A,C), 200 nm (B,D). In addition to neurons, strong immunolabeling was also observed in neuronal elements, such as dendrites, axons and spines, where information traffic is very high. In contrast to the report of Wedegaertner (2002), our findings of sG_i2 protein localization in far distant sites from neuronal cell body where it is synthesized, in axons, dendrites and spines suggest that this protein is very stable. Furthermore, our findings are in agreement with other reports (Montmayeur and Borrelli, 1994; Picetti and Borrelli, 2000). For methodological description on electron microscopy, see Khan et al., 1998; Khan et al., 2001.

• Supplemental Figure S5 -

  Fig. S5. Protein expression analysis of deletion constructs of sG_i2 in BHK cells. The protein expression of a-f constructs (as shown in Fig. 6 of the manuscript) were tested in cells by immunoblotting. The antibody against sG_i2 protein residues, 674-687 (GenBank AY677118), common in all constructs, was prepared as described in Experimental Procedures of the manuscript. This antibody (Ab 674-687) recognized single polypeptide bands of 41, 40, 37, 27, 32 and 28 kDa size in cells expressing sG_i2-WT (a), Gα_i2-WT (b), sG_i2-N1 (c), sG_i2-N2 (d), sG_i2-C1 (e), and sG_i2-C2 (f), respectively. These results confirm the expression of correct size sG_i2 deletion constructs in experiments discussed in Fig. 6.