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Insights into G Protein βγ Regulation of Phospholipase Cε
Başlık:
Insights into G Protein βγ Regulation of Phospholipase Cε
Yazar:
Madukwe, Jerry Chuwuemeka, author.
ISBN:
9780355975017
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (109 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Alan Smrcka Committee members: Mark Dumont; Paul Kammermeier; Joshua Munger.
Özet:
PLCepsilon is involved in the regulation of multiple cellular signaling processes and it has been implicated in the development of heart failure. Due to its central importance, an intimate understanding of its regulation is critical for developing potential pharmacological therapies. To identify domains of Phospholipase Cepsilon (PLCepsilon) that interact with G protein betagamma (Gbetagamma), we designed various point mutations and truncations of wild-type PLCepsilon and tested them for Gbetagamma activation in transfected COS-7 cells. In contrast to activation by Ras, deletion of an individual domain in PLCepsilon is not sufficient to completely block Gbetagamma mediated activation. Simultaneous deletion of the carboxy-terminal RA2 domain and the amino-terminal (CDC25 and cysteine-rich domains) results in complete abrogation of PLCepsilon activation by Gbetagamma, while activation by Rho is retained. In vitro reconstitution experiments revealed that purified Gbetagamma directly interacts with a purified fragment of PLCepsilon (PLCepsilon-PH-RA2) to increase membrane phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis. Deletion of the RA2 domain decreased Gbetagamma binding and eliminated Gbetagamma-stimulation of PIP2 hydrolysis. Our data show that two independent domains of PLCepsilon are involved in its regulation by Gbetagamma and demonstrate direct interactions between Gbetagamma and PLCepsilon.
Furthermore, to clearly understand the mechanism of Gbetagamma regulation of Golgi compartmentalized PLCepsilon (Golgi-PLCepsilon) by plasma membrane (PM) endothelin receptor (ET-1AR) activation, which is required for cardiomyocyte hypertrophy in a neonatal rat ventricular myocyte (NRVM) model of cardiac disease, we overexpressed differentially translocating Ggamma in NRVMs using adenovirus. We tested if G protein gamma (Ggamma)-mediated differential translocation of Gbetagamma differentially modulates Gbetagamma dependent Golgi-PLCepsilon-mediated phosphatidylinositol 4-phosphate (PI4P) hydrolysis upon stimulation of the ET-1AR. Our data show that Ggamma differences in translocation characteristics do not confer any differential effect in Golgi-PLCepsilon-mediated PI4P hydrolysis upon ET-1AR activation.
Finally, we explored differential modulation by Ggamma of cardiac cell hypertrophy and protein kinase D (PKD); a downstream effector of Gbetagamma dependent Golgi-PLCepsilon signaling that regulates cardiac hypertrophy. Our data show that Ggamma-mediated differential translocation of Gbetagamma confers no differential effect on PKD activation and cellular hypertrophy.
Taken together, results of our studies provide the first evidence for the direct regulation of PLCepsilon by Gbetagamma and provide insights into the mechanism by which Gbetagamma subunits activate PLCepsilon downstream of ET-1AR in cardiac hypertrophy.
Notlar:
School code: 0188
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(680167.1) | 680167-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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