Figure 2.

Calnuc specifically interacts with both activated and inactivated Gαi3 in vivo, and the interaction is Ca²⁺/Mg²⁺-dependent. (A) Pull-down assays. In vitro translated [³⁵S]Gαi3 was incubated with calnuc-Sepharose 4B at 4°C for 1 h in the presence of 15 mM Mg²⁺ (lane 1), 2 mM Ca²⁺ (lane 2), 15 mM Mg²⁺ + 2 mM Ca²⁺ (lanes 4 and 5), or 5 mM EDTA (lane 3), followed by extensive washing, SDS/PAGE separation, and autoradiographic analysis. Calnuc interacts with [³⁵S]Gαi3 in the presence of Mg²⁺, Ca²⁺, or both, but weak interaction is observed in the presence of Ca²⁺ alone. Binding is abolished by 5 mM EDTA as well as excess unlabeled Gαi3 (lane 5). (B) coIP of Gαi3 and calnuc. NIH 3T3 cells overexpressing Gαi3(Q204L) (activated) and Gαi3(G203A) (inactivated) were lysed with 0.5% TX-100 at 4°C for 30 min in the presence of 2 mM Ca²⁺/15 mM Mg²⁺ or 5 mM EDTA. coIP was performed on the cell lysates with anti-calnuc IgG in the presence of 1 M NDSB-195 and protein A beads, as described in Materials and Methods, followed by immunoblotting with anti-Gαi3 IgG. Calnuc interacts with both activated and inactivated Gαi3 in the presence (+) but not in the absence (−) of 2 mM Ca²⁺/15 mM Mg²⁺, suggesting that the interaction between calnuc and Gαi3 is Ca²⁺/Mg²⁺-dependent but does not depend on the state of activation of Gαi3. (C) Crosslinking of calnuc and Gαi3. NIH 3T3 cells overexpressing Gαi3(Q204L) and Gαi3(G203A) cells were treated with 10 μM ionomycin plus either 2 mM Ca²⁺/15 mM Mg²⁺ or 5 mM EDTA. Crosslinking was carried out by using the membrane permeant crosslinker dithiobis (succinimidylpropionate) (2 mM), followed by lysis with 1% TX-100 and immunoprecipitation with anti-calnuc IgG as above. Similar results to those in B were obtained. Both coIP and crosslinking experiments were repeated four times.