Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C.

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.     

Somatic genetic analysis of cyclic AMP action: selection of unresponsive mutants.

Dibutyryl cyclic AMP and theophylline kill S49.1 mouse lymphoma tissue culture cells. When cells are grown in soft agar with these drugs, the few clones that survive are resistant to cytolysis. The rate of mutation to resistance is 1-3 times 10-7/cell/generation in both diploid and tetraploid cells. The incidence of mutants is increased by treatment with a chemical mutagen, ICR 191. The mutation is consistently associated with greatly reduced or absent cytoplasmic cyclic AMP binding protein. These results suggest that a somatic mutation leads to a defect of the protein kinase regulatory subunit and that activity of this kinase is required for induction of cell death by cyclic AMP.