FIGURE 4. Effect of transgenic expression of Rab4 in the myocardium on the density of β-AR in the plasma membrane.

A, immunoblot analysis of Rab expression levels in hearts from transgenic mice overexpressing Rab4 and NTG mice. Fifty μg of total homogenate prepared from ventricles of four Rab4 and four NTG mice was separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Rab4 expression levels were detected by Western blotting using anti-Rab4 and anti-FLAG antibodies and Rab1 and Rab5 expression by Rab-isoform specific antibodies. B, immunoblot analysis of the ER marker calregulin, the Golgi marker GM130, and the plasma membrane marker Na⁺-K⁺-ATPase in cytosolic and membrane fractions. Myocardial cytosolic and plasma membrane fractions were prepared by homogenization and centrifugation at 10,000 × g as described under “Experimental Procedures.” Twenty-five μg of protein from the cytosolic and membrane fractions was analyzed. The blots shown are representatives of three separate experiments. C, specific [¹²⁵I]iodocyanopindolol binding to membrane fractions prepared from Rab4 and NTG mouse ventricles. Twenty-five μg of membrane protein was incubated with the nonselective β-AR ligand [¹²⁵I]iodocyanopindolol (400 pM) in a total volume of 500 μl of binding buffer for 1 h. Specific binding was performed in duplicate and nonspecific binding determined in the presence of 20 μM alprenolol. Receptor density was expressed as fmol/mg membrane protein and presented as the means ± S.E. (n = 6). *, p < 0.05 versus NTG. D, Expression of G_s, G_i, Gβ, and GRK2 in four Rab4 and four NTG mouse hearts. Fifty μg of total homogenate prepared from mouse ventricles was analyzed.