Dual Regulation of Phospholipase C-beta by G betagamma

Agonist-bound G protein coupled receptors (GPCRs) activate G protein heterotrimers by catalyzing release of GDP and binding of GTP to the G alpha subunit (Ga), releasing active Ga and G betagamma (Gbg) subunits. Activated alpha subunits of the Gq family and betagamma subunits of the Gi family stimulate phospholipase C-beta (PLC-b) isoforms to catalyze hydrolysis of phosphatidylinositol-1,2-bisphosphate (PIP2) generating the second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG). PLC-b isoforms are also GTPase activating proteins (GAPs) for Gaq, and Gbg subunits inhibit the GAP activity of PLC-b. Coordinated regulation of these activities is essential for sustained signaling at steady state. Regulation of PLC-b by Gaq and Gbg is well studied but details of the mechanism are still lacking. Activation of PLC-b simultaneously by G protein pathways has been suggested based on observations in cells, but it is not known if scaffolding proteins or other factors are necessary for simultaneous stimulation of PLC-b by G protein subunits. The binding interface between Gbg and PLC-b is unclear, and so is the mechanism of PLC-b GAP inhibition by Gbg. To enable the study of these mechanisms in vitro, I developed a new method to purify Gaq subunits based on observations from the Tall group. This method combined Ric8A-mediated enhancement of Gaq expression and the traditional method of using detergents to isolate functional Ga from membrane bound G protein heterotrimers, resulting in 3- to 4-fold increase in yields of Gaq. Using purified proteins and working with other members of the lab, I showed that the PLC-b3 isoform is synergistically activated by Gaq and Gbg subunits. The observed synergism is up to 10-fold, quantitatively consistent with cellular observations, thus establishing that no additional proteins or pathways are required. Next, I developed a FRET-based binding assay between Gbg and PLC-b and identified the pleckstrin homology (PH) domain in PLC-b as the Gbg binding site. Using structural and biochemical analyses, I showed that Gbg-PLC-b requires intrinsic motion of the PH domain. This led to the proposal for a new conformation of PLC-b not observed in crystal structures and a new model for Gbg-PLC-b binding. Subsequent studies suggested that Gbg inhibits PLC-b GAP activity by a mechanism that does not require Gbg-PLC-b binding.

Author dc:creator

• Kadamur Bhavani, Ganesh

Contributors dc:contributor

• Rosen, Michael K.
• Ross, Elliott M.
• Albanesi, Joseph P.
• Zhang, Xuewu

• GTP-Binding Protein beta Subunits
• GTP-Binding Protein gamma Subunits
• Phospholipase C beta

Language dc:language

en

Identifier

1038532922

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/5297