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

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [(35)S]GTP gamma S revealed a class of binding sites with an apparent K(d) value of 100 nM and a B(max) of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX decreased B(max) by 50%. The mRNA levels for G alpha(i-3), G alpha(0), G alpha(s), and G alpha(q) 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 [alpha-(32)P]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 G(i) proteins and stimulates the expression of a specific GTP-binding protein of M(r) 59.5 kDa.