Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone

Abstract

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPγS revealed a class of binding sites with an apparent K d value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Gαi-3, Gα0, Gαs, and Gαq were not increased after aldosterone. The patterns of small M r G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [α-32P]GTP, in control membranes, showed either no labeling or a faint band of M r 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of M r 59.5 kDa.