QUANTITATIVE STOICHIOMETRY OF THE PROTEINS OF THE STIMULATORY ARM OF THE ADENYLYL-CYCLASE CASCADE IN NEUROBLASTOMA-X-GLIOMA HYBRID, NG108-15 CELLS

To understand the details of regulation of guanine-nucleotide-binding- protein-linked transmembrane cellular-signalling cascades, it is impor tant to know the absolute levels of each polypeptide component and the stoichiometry of their interactions. Amounts of the IP prostanoid rec eptor, the stimulatory G protein of the adenylyl cyclase cascade (G(s) alpha) and the functional complex of G(s) alpha with adenylyl cyclase , which acts as the cyclic AMP generator, were measured in membranes o f neuroblastoma x glioma hybrid, NG108-15, cells. As measured by the s pecific binding of [H-3]prostaglandin E1, the IP prostanoid receptor w as present in some 100000 copies/cell. G(s) alpha assessed by quantita tive immunoblotting with recombinantly expressed protein, was present in considerably higher levels (1250000 copies/cell). However, the maxi mal formation of a complex of G(s) alpha and adenylyl cyclase represen ted only some 17500 copies/cell. The previously established 8:1 stoich iometry of concurrent downregulation of G(s) alpha and the IP prostano id receptor in these cells [Adie, E. J., Mullaney, I., McKenzie, F.R. & Milligan, G. (1992) Biochem. J. 285, 529-536] indicates that full-ag onist occupation of the receptor should be able to activate some 65% o f the expressed G(s), Despite the potential 70-fold excess of G(s) alp ha over the G(s) alpha/adenylyl cyclase complex, IP prostanoid-recepto r-agonist-mediated reduction of G(s) alpha levels by some 35% resulted in a 25% reduction in the maximal formation of the G(s) alpha/adenyly l cyclase complex. Such results demonstrate that adenylyl cyclase is q uantitatively the least highly expressed component of this signalling cascade and suggests that much of the cellular G(s) alpha may not have access to adenylyl cyclase.