Dual Regulation of Phospholipase C-beta by G betagamma
| dc.contributor.advisor | Rosen, Michael K. | en |
| dc.contributor.committeeMember | Ross, Elliott M. | en |
| dc.contributor.committeeMember | Albanesi, Joseph P. | en |
| dc.contributor.committeeMember | Zhang, Xuewu | en |
| dc.creator | Kadamur Bhavani, Ganesh | en |
| dc.creator.orcid | 0000-0002-7019-1485 | |
| dc.date.accessioned | 2018-06-04T18:50:39Z | |
| dc.date.available | 2018-06-04T18:50:39Z | |
| dc.date.created | 2016-05 | |
| dc.date.issued | 2016-03-29 | |
| dc.date.submitted | May 2016 | |
| dc.date.updated | 2018-06-04T18:42:18Z | |
| dc.description.abstract | 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. | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 1038532922 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/5297 | |
| dc.language.iso | en | en |
| dc.subject | GTP-Binding Protein beta Subunits | en |
| dc.subject | GTP-Binding Protein gamma Subunits | en |
| dc.subject | Phospholipase C beta | en |
| dc.title | Dual Regulation of Phospholipase C-beta by G betagamma | en |
| dc.type | Thesis | en |
| dc.type.material | text | en |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Molecular Biophysics | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |