Tissue-specific variations in the expression and regulation of the small GTP-binding protein Rho

Rho proteins are involved in the regulation of the assembly of the microfilamental cellular network and are known to be specific substrates for the ADP-ribosyltransferase C3 from Clostridium botulinum. Here, we studied the distribution of Rho and Rho-regulating proteins in extracts from various rabbit tissues. The highest amounts of [32P]ADP-ribosylated proteins were detected in cell extracts from lung and kidney. Compared to these tissues, 50-95% reduced labeling of Rho proteins was observed in extracts from liver, spleen, brain, heart and muscle. The level of the C3-mediated [32P]ADP-ribosylation of Rho did not correlate with the amount of RhoA proteins detected by Western analysis. The relative amounts of [32P]ADP-ribosylated proteins located in cytosolic or membrane fractions, respectively, depended on the type of tissue investigated, indicating a tissue-specific variation in the subcellular distribution of Rho proteins. The same was true for the complexation of Rho with other factors and the expression of diverse Rho species. In respect to Rho-regulating proteins, extracts from lung and brain contained the highest amounts of guanine nucleotide dissociation-inhibitor proteins (Rho-GDI). The association of Rho with Rho-GDI however showed tissue specificity and did not correlate with Rho-GDI amounts. The highest Rho-GAP (GAP = GTPase-activating protein) activities were observed in extracts from lung, kidney and spleen, the lowest ones in extracts from muscle and heart. In total, our data demonstrate tissue-specific differences in the expression of RhoA, [32P]ADP-ribosylated proteins and Rho-regulating factors, indicating a tissue-specific variation in the activity and regulation of Rho proteins.