Figure 1. GINIP, a Highly Conserved Protein, Specifically Interacts with GTP-Bound Forms of G_αi.

(A) Alignment of amino acid sequences of GINIP from various vertebrate species. Residues shaded in black are identical in >85% of the predicted proteins; similar residues are highlighted in gray. The predicted PHD zinc finger (dotted box), the two EF-hand domains (#1 and #2; black lines), and the core domain of each EF-hand (gray box) are indicated. The sequence corresponding to the spliced exon in mouse is indicated by a double line. Phosphorylated residues identified by large-scale proteomic studies are indicated by asterisks (http://www.phosphosite.org/). Schematic representation of GINIP architecture is drawn on the bottom.

(B) In situ hybridization of DRGs with gnai1, gnai2, and gnai3 antisense probes followed by immunostaining using rat anti-GINIP antibody. gnai1 is mainly expressed in large neurons and excluded from GINIP⁺ population, whereas gnai2 and gnai3 were mainly coexpressed with GINIP in smaller-diameter neurons. Scale bar, 100 μm.

(C) GST pull-down experiments: GST-GINIP (lanes 3 and 6) or GST alone (lanes 2 and 5) was incubated with lysate from HEK293 cells transfected with WT (lanes 1–3) or constitutively active forms (lanes 4–6) of G_αi1, G_αi2, G_αi3, Gαo, Gαolf, Gαq, and Gαs. All forms are fused with Venus protein. One-twentieth of the incubated volume of lysate was loaded in lanes 1 and 4. Western blot performed with anti-GFP antibody shows a specific interaction of GINIP with the active forms of only the three G_αi variants.