Detection of phospholipase C-B2 activation by G-protein subunits

Suzanne Scarlata; Loren Runnels; Mario Rebecchi

doi:10.1117/12.307051

1 May 1998 Detection of phospholipase C-β ₂ activation by G-protein subunits

Suzanne Scarlata, Loren Runnels, Mario Rebecchi

Proceedings Volume 3256, Advances in Optical Biophysics; (1998) https://doi.org/10.1117/12.307051
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

Many neurotransmitters and hormones convey their signals into cells via transmembrane receptors that activate heterotrimeric G proteins which in turn active phospholipase C-(beta) (PLC- (beta) ). Activation of PLC-(beta) by the (alpha) _q and (beta) (gamma) subunits of G proteins results in activation of protein kinase C and release of Ca²⁺ from intracellular stores, which in turn results in a multitude cellular changes. We have recently found that activation of PLC-(beta) by G proteins occurs by lateral association on the membrane surface. Here, we have measured the affinity of the membrane- bound species by fluorescence energy transfer and have conducted time-resolved studies to assess the lifetime of the PLC-G protein complexes in order to understand PLC signaling inside the cell. To better interpret these results, we outline methods to convert the two-dimensional dissociation constant measured for the membrane-bound proteins to a three dimensional one. We also detail calculations to determine the concentrations at which non-specific protein-protein interactions occurs due to membrane crowding. To differentiate between the physical association of PLC-G complexes and activation, we have developed a real-time fluorescence-based PLC-(beta) activity to determine the length of time that PLC- (beta) remains active after G protein dissociation. A model for activation will be presented.

Citation Download Citation

Suzanne Scarlata, Loren Runnels, and Mario Rebecchi "Detection of phospholipase C-β ₂ activation by G-protein subunits", Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); https://doi.org/10.1117/12.307051

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