An engineered mutant of G protein [alpha] subunit that binds xanthine nucleotide and not guanine nucleotide

Citation

Yu, Bo (1999) An engineered mutant of G protein [alpha] subunit that binds xanthine nucleotide and not guanine nucleotide. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/K78Q-9K42. https://resolver.caltech.edu/CaltechETD:etd-06172008-140047

Abstract

This thesis examines the construction and characterization of mutants of G protein [alpha] subunits that bind xanthine nucleotides, but not guanine nucleotides. G proteins play a critical role in transducing extracellular signals across the cell membrane. The mechanisms of G protein-mediated signal transduction are reviewed in chapter 1.

Chapter 2 describes the characterization of the first engineered xanthine nucleotide binding mutant of a G[alpha] subunit, Go[alpha]X (Go[alpha]D273N/Q205L). Go[alpha]X switched nucleotide binding specificity; it bound xanthine nucleotides instead of guanine nucleotides. Go[alpha]X formed an heterotrimer with [beta][gamma] subunits when in the XDP form. Binding of XTP induced a conformational change in Go[alpha]X similar to that of the activated wild-type Go[alpha] and promoted its dissociation from the [beta][gamma] complex.

In chapter 3, we characterized the receptor interaction of Go[alpha]X. It was able to interact with G protein-coupled receptors effectively; the stimulated m2 muscarinic acetylcholne receptor catalyzed the XTP[gamma]S binding of Go[alpha]X, and the Go[alpha]X[beta][gamma] complex induced the high affinity ligand-binding state in the N-formyl peptide receptor. Interestingly, we found that the empty Go[alpha]X, in the nucleotide-free state, formed a stable complex with receptor and inhibited the activity of Go-coupled receptors in COS-7 cells.

In chapter 4, we extended this study to two other G proteins. We constructed similar xanthine nucleotide binding mutant proteins in G11[alpha] and G16[alpha] and found that G11[alpha]X (G11[alpha]D277N/Q209L) and G16[alpha]X (G16[alpha]D280N/Q213L) bound XTP[gamma]S and not GTP[gamma]S when expressed in COS-7 cells. Empty G11[alpha] and G16[alpha] mutants also interacted with their cognate receptors and blocked their activity. Similar to Go[alpha]X, both G11[alpha]X and G16[alpha]X retained the receptor specificity of their wild-type proteins and can be used to inhibit subsets of G protein-coupled receptors.

In chapter 5, we constructed recombinant retroviruses encoding G16[alpha]X, and obtained NIH3T3 cell lines stably expressing the empty G16[alpha] mutants by viral infection. We found that G16[alpha]X blocked the activation of the endogenous thrombin and lysophophatidic acid (LPA) receptors in NIH3T3 cells. These experiments proved that retroviral gene expression can be an effective technique for delivering empty G protein mutants into cells.

Thesis Files