Figure 9. Calcium-dependent assembly of a centrin1/G-protein complex.

Figure 9

Calcium-dependent assembly of a centrin1/G-protein complex. A) Co-Immunoprecipitation of transducin with centrin from lysed retinal photoreceptor cell fragments.

Lanes 1: Western blot analysis with mAb anti-Gtα of an immunoprecipitation with mAb anti-centrin (clone 20H5) from photoreceptor cell fragments of bovine retina. (Upper and lower bands in lane 1 correspond to the heavy (HC) and light chains (LC) of mouse antibodies.) Lane 2: Western blot analysis with polyclonal anti-Gtβ of anti-centrin of an immunoprecipitation with mAb anti-centrin (clone 20H5) from photoreceptor cell fragments of bovine retina. Gtα and Gtβ co-immunoprecipitate with centrin. The upper and lower bands in lane 1 correspond to the heavy (HC) and light chains (LC) of the mouse antibodies.

B) Combined Western blot-overlay analysis identifies retinal centrin-interacting protein P37 as Gtβ subunit of transducin.

For specific determination of the centrin-binding protein, Western blotted lanes were cut in half and parallel processed for immunolabeling with subunit-specific antibodies against Gtβ (upper lane 1), and Gtα transducin (lower lane 1) and for overlays with recombinantly expressed MmCen1p (67 μg/ml) (OL). The 37 kDa centrin-binding protein (P37 in Fig. 7) is identified by centrin overlays had the exact mobility as the Gtβ subunit.

C) Calcium-dependent enhancement of kinetic light-scattering (KLS) Gt-binding signal in the presence of MmCen1p. Upper panel represents KLS binding signals (3 μM rhodopsin, 0.5 μM Gt) in the presence of calcium, and 0 (control, black curve), 0.6, 1.2, 2.5, 3.6, 5, 7.3, and 10 μM MmCen1p (gray curves), respectively. Lower panel represents KLS binding signals under the same conditions as in the upper panel, but with EGtα instead of calcium. Experimental conditions were 50 mM BTP, pH 7.5 containing 80 mM NaCl, 5 mM MgCl2 and either 100 μM CaCl2 or 1 mM EGtα at 20°C, sample volume of 300 μL, and cuvette path length of 1 cm; 32% rhodopsin was photolyzed per flash (500±20 nm).

D) Competition between Gtβγ-subunit and Gt for binding to MmCen1p. Calcium-dependent inhibition of the MmCen1p enhanced amplitude of flash-induced KLS Gt-binding signals by the βγ-subunit of Gt. The KLS assay was carried out as described in (A). Experiments were performed with the βγ-subunit of Gt. Data points represent the normalized amplitude of the MmCen1-dependent enhancement of the Gt-binding signal (AMmCen1) divided to the control Gt-binding signal without added MmCen1 (control). Filled and empty circles indicate the results obtained from experiments with calcium and with EGTA, respectively.

E) Calcium-dependent interaction of MmCen1p with Gt and its subunits analyzed by size-exclusion chromotography and SDS-PAGE.

Upper panels represent elution profiles of MmCen1p alone (...), Gt or its subunits alone (---), and the mixture of MmCen1p with Gt or its subunits (—) in the presence of calcium. The gray dotted lines are the calculated superpositions of the respective single component profiles (MmCen1p plus Gt or its subunits) yielding the predicted profiles for the mixture of the two noninteracting components. Arrows indicate the shift of the formed complexes. In the lower panel the SDS-PAGE analysis of the fractions of the size-exclusion chromatography is shown. Interaction of MmCen1p with the transducin holoprotein is shown in 1st panel with the Gtα-subunit in 2nd panel and with the Gtβγ-subunit in 3rd in the presence of calcium.

Experimental conditions: 10 μg of MmCen1p and 10 μg of Gtholo (or Gt subunits) were incubated in 50 mM BTP, pH 7.0 containing 80 mM NaCl, 1 mM MgCl2 and 100 μM CaCl2 for 5 min at room temperature, loaded on a Superose TM 12 column (using the Smart System, Pharmacia Biotech. Inc., flow rate, 40 μL/min) equilibrated with the same buffer, eluted by monitoring the absorbance at 280 nm and subsequent analyzed by SDS-PAGE. Note: Gt holoprotein elutes at an apparently lower MW, as compared to its subunits.73

From: Centrins, a Novel Group of Ca2+-Binding Proteins in Vertebrate Photoreceptor Cells

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