{"id":{"repo_id":"utswmed","oai_identifier":"oai:utswmed-ir.tdl.org:2152.5/5297"},"canonical_url":"https://search.dev.ndltd.org/etd/utswmed/oai:utswmed-ir.tdl.org:2152.5/5297","repository":{"repo_id":"utswmed","name":"University of Texas Southwestern Medical Center","base_url":"https://utswmed-ir.tdl.org/server/oai/request"},"display":{"title":"Dual Regulation of Phospholipase C-beta by G betagamma","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Kadamur Bhavani, Ganesh"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Rosen, Michael K.","Ross, Elliott M.","Albanesi, Joseph P.","Zhang, Xuewu"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-06-04T18:50:39Z","date_published":"2018-06-04T18:50:39Z","updated_at":"2026-07-24T05:52:26Z","subjects":["GTP-Binding Protein beta Subunits","GTP-Binding Protein gamma Subunits","Phospholipase C beta"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["1038532922"],"render_values":[{"text":"1038532922","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152.5/5297","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Rosen, Michael K.","Ross, Elliott M.","Albanesi, Joseph P.","Zhang, Xuewu"]},{"key":"dc:creator","label":"Author","values":["Kadamur Bhavani, Ganesh"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-06-04T18:50:39Z","2016-05","2016-03-29","May 2016","2018-06-04T18:42:18Z"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","text"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["GTP-Binding Protein beta Subunits","GTP-Binding Protein gamma Subunits","Phospholipase C beta"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2152.5/5297","1038532922"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Dual Regulation of Phospholipase C-beta by G betagamma"]}]}],"canonical_facts":{"dc:contributor":["Rosen, Michael K.","Ross, Elliott M.","Albanesi, Joseph P.","Zhang, Xuewu"],"dc:creator":["Kadamur Bhavani, Ganesh"],"dc:date":["2018-06-04T18:50:39Z","2016-05","2016-03-29","May 2016","2018-06-04T18:42:18Z"],"dc:description":["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."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2152.5/5297","1038532922"],"dc:language":["en"],"dc:subject":["GTP-Binding Protein beta Subunits","GTP-Binding Protein gamma Subunits","Phospholipase C beta"],"dc:title":["Dual Regulation of Phospholipase C-beta by G betagamma"],"dc:type":["Thesis","text"]},"updated_at":"2026-07-24T05:52:26Z"}