FIGURE 3.

The α5 subunit alters the relative subunit association among α4+β2+β4. Increasing amounts of α5 were co-transfected with fixed amounts of α4, β2, and β4 cDNA (0.25 μg of each). Crude membranes were solubilized in detergent and subjected to progressive immunoprecipitation with either anti-β2 followed by anti-β4 or reciprocally anti-β4 followed by anti-β2 as described (see “Experimental Procedures”). A, representative results from cells transfected as described above. A Western blot reveals the relative amount of R3b epitope-tagged α4 or α5 as indicated in association with β2 or β4 after removal of all β2 complexes. Quantitation of these blots is shown where all of the values are normalized to samples with no α5 co-transfected. The error bars reflect plus or minus the standard error of the mean as calculated from three independent experiments done in the same manner. B, average percentages of the relative amounts of mixed subunit complexes that were either directly measured or derived from measuring the relative amount of α4 remaining after successive anti-β subunit immunoprecipitations as in A. The averages are from three independent experiments. C, ligand binding ([³H]EB) to total membranes decreases as α5 input is increased. The loss of ∼56% of the total radioligand binding relative to non-α5 transfection mixtures corresponds well with the increase of α4α5β2β4 complexes (B).