Phenotypic and Biochemical Characterization of Cells Expressing a Gas/Gai Chimeric Protein: a Thesis

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Doctoral Dissertation


Graduate School of Biomedical Sciences, Department of Biochemistry


Adenylate Cyclase; Biochemistry; Academic Dissertations


G-proteins are heterotrimeric complexes composed of α, β, and τ subunits and are involved in coupling receptor and effector functions during signal transduction across plasma membranes. G-proteins Gs and Gi are stimulatory and inhibitory to the catalytic subunit of adenylyl cyclase, respectively. A chimeric G-protein α subunit cDNA was constructed from the complete 5' untranslated region of Gαs52 (the 52 kD α subunit of Gs), the first 356 codons of the rat Gαs52, and the last 36 codons and 428 bp of the 3' untranslated region of the rat Gai2 (the α subunit of Gi2) cDNA. Expression of the chimeric G-protein alpha subunit (Gαs/i(38)) causes a constitutive increase in adenylyl cyclase activity in three different fibroblast cell lines. In turn, the elevated cyclase activity leads to higher levels of basal cyclic AMP and protein kinase A activity. The effect of Gαs/i(38) on cyclase does not seem to be through an inhibiton of Gαi function, but instead appears to be the consequence of direct action on the catalytic subunit, resulting in both a decreased time required for maximal cyclase activation and a greater maximal activation as well. Such alterations are not noted in cells expressing exogenous, wild-type Gαs. This data is based primarily on reconstitution assays using cholate extracts from fibroblast Gαs/i(38) clones and membranes derived from the S49 murine T-cell lymphoma (cyc- variant). Endogenous G-protein steady-state changes were detected by immunoblot analysis, but do not appear to account for the observed phenotypic alterations in Gαs/i(38) expressing clones. Furthermore, the validity of the above findings is unequivocally demonstrated through the use of amethopterin-mediated amplification of the chimeric Gαs/i(38) gene transcript and the consequent activation of adenylyl cyclase.


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