GSBS Dissertations and Theses

Title

Mutations of the Alpha-Subunit of G-Proteins: A Thesis

Approval Date

September 1988

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Biochemistry

Subjects

GTP-Binding Protein alpha Subunits; Signal Transduction; Academic Dissertations; Dissertations, UMMS

Abstract

Signal transduction by G-proteins (a heterotrimer membrane protein composed of an α, β, and γ subunit) requires that the α-subunit undergoes a transition from a GDP-bound inactive state to an activated GTP-bound state. The exchange of GDP for GTP leads to a conformational change in the α-subunit that results in the loss of affinity for the βγ subunits. We predicted that appropriate genetic manipulation of key regions of the α-subunit could result in the induction of the active conformation that would mimic at least in part the activated GTP-bound state. We have demonstrated that the substitution of the 38 amino acid residue carboxyl termimus of Gαs with the last 36 amino acid residues of Gαi2 resulted in a chimeric Gα-subunit (C4) that exhibits a constitutively active Gαs-like activity. Similarly, the substitution of the amino terminal 61 amino acid residues of Gαs with the first 54 residues of Gαi2 also resulted in a chimeric Gα-subunit that is persistently active (Gs like). We have also generated point mutations in the Gαs subunit that are comparable to the activating mutations in the ras protein. Our results suggest that point mutations in the signature sequence of the A (Val 49) and C (Thr 225) homologous regions that are implicated in regulating the GTPase activity of the molecule also resulted in the activation of the subunit. The present study has identified four key regions of the α-subunit that are critical for the activity and regulation of the Gs protein.

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