G protein betagamma11 complex translocation is induced by Gi, Gq and Gs coupling receptors and is regulated by the alpha subunit type.

G protein activation by Gi/Go coupling M2 muscarinic receptors, Gq coupling M3 receptors and Gs coupling beta2 adrenergic receptors causes rapid reversible translocation of the G protein gamma11 subunit from the plasma membrane to the Golgi complex. Co-translocation of the beta1 subunit suggests that gamma11 translocates as a betagamma complex. Pertussis toxin ADP ribosylation of the alphai subunit type or substitution of the C terminal domain of alphao with the corresponding region of alphas inhibits gamma11 translocation demonstrating that alpha subunit interaction with a receptor and its activation are requirements for the translocation. The rate of gamma11 translocation is sensitive to the rate of activation of the G protein alpha subunit. alpha subunit types that show high receptor activated rates of guanine nucleotide exchange in vitro support high rates of gamma11 translocation compared to alpha subunit types that have a relatively lower rate of guanine nucleotide exchange. The results suggest that the receptor induced translocation of gamma11 is controlled by the rate of cycling of the G protein through active and inactive forms. They also demonstrate that imaging of gamma11 translocation can be used as a non-invasive tool to measure the relative activities of wild type or mutant receptor and alpha subunit types in a live cell.

A fluorescence resonance energy transfer-based sensor indicates that receptor access to a G protein is unrestricted in a living mammalian cell.

Fluorescence recovery after photobleaching of muscarinic receptors and G protein subunits tagged with cyan or yellow fluorescent protein showed that receptors and G proteins were mobile and not immobilized on the cell membrane. The cyan fluorescent protein-tagged Galpha and yellow fluorescent protein-tagged Gbeta subunits were used to develop sensors that coupled selectively with the M2 and M3 muscarinic receptors. In living Chinese hamster ovary cells, imaging showed that sensors emitted a fluorescence resonance energy transfer signal that was abrogated on receptor activation. When sequentially activated with highly expressed muscarinic receptors and endogenous receptors expressed at low levels, sensor molecules were sensitive to the sequence of activation and the receptor numbers. The results distinguish between models proposing that receptor and G protein types interact freely with each other on the cell membrane or that they function as mutually exclusive multimolecular complexes by providing direct support for the former model in these cells.

Role of the G protein gamma subunit in beta gamma complex modulation of phospholipase Cbeta function.

The G protein betagamma complex regulates a wide range of effectors, including the phospholipase C isozymes (PLCbetas). Different domains on the beta subunit are known to contact phospholipase Cbeta and affect its regulation. In contrast, the role of the gamma subunit in Gbetagamma modulation of PLCbeta function is not known. Results here show that the gamma subunit C-terminal domain is involved in mediating Gbetagamma interactions with phospholipase Cbeta. Mutations were introduced to alter the position of the post-translational prenyl modification at the C terminus of the gamma subunit with reference to the beta subunit. These mutants were appropriately post-translationally modified with the geranylgeranyl moiety. A deletion that shortened the C-terminal domain, insertions that extended this domain, and a point mutation, F59A, that disrupted the interaction of this domain with the beta subunit were all affected in their ability to activate PLCbeta to varying degrees. All mutants, however, interacted equally effectively with the G(o)alpha subunit. The results indicate that the G protein gamma subunit plays a direct role in the modulation of effector function by the betagamma complex.