FIGURE 3.

The effect of calcium on the MPEG reactivity of cysteine substitution mutants in the C-terminal tail. A, schematic of the C-terminal tail of the IP₃R with the location of three endogenous cysteines and four cysteine substitution mutants indicated by open circles. The location of the transmembrane domain 6 (S6) and the coiled-coil domain (CC) as well as several sites for interaction with key proteins are indicated (for additional details, see text). GIT; G-protein-coupled receptor kinase-interacting protein, PP1-α; protein phosphatase 1-α, 4.1N; neuron-specific isoform of erythrocyte protein band 4.1; Cyt c, cytochrome c; PKC, protein kinase C. B, microsomal membranes were prepared from COS cells transfected with wild-type IP₃R (WT) or the indicated cysteine substitution mutants and then reacted with 0.5 mm MPEG-5 for 5 min in the presence or absence of a free [Ca²⁺] of 2.2 μm. The samples were then digested with trypsin. Cleaved fragments from the C-terminal domain were detected on immunoblots with affinity-purified IL-3 Ab. The open arrow indicates the position of the 95-kDa fragment V, and the closed arrows indicate its shift to higher molecular weights. The asterisk identifies a nonspecific IL3-reactive band that is not shifted by MPEG-5. C, experiments with MPEG-20 were performed with membranes expressing the A2749C mutant as described in B.