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

摘要

This thesis examines the construction and characterization of mutants of G protein a 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 a subunit, Go a X (Go a D273N/Q205L). Go a X switched nucleotide binding specificity; it bound xanthine nucleotides instead of guanine nucleotides. Go a X formed an heterotrimer with bg subunits when in the XDP form. Binding of XTP induced a conformational change in Go a X similar to that of the activated wild-type Go a and promoted its dissociation from the bg complex.; In chapter 3, we characterized the receptor interaction of Go a X. It was able to interact with G protein-coupled receptors effectively; the stimulated m2 muscarinic acetylcholne receptor catalyzed the XTP g S binding of Go a X, and the Go a X bg complex induced the high affinity ligand-binding state in the N-formyl peptide receptor. Interestingly, we found that the empty Go a 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 a and G16 a and found that G11 a X (G11 a X(G11 a D277N/Q209L) and G16 a X (G16 a D28ON/Q213L) bound XTP g S and not GTP g S when expressed in COS-7 cells. Empty G11 a and G16 a mutants also interacted with their cognate receptors and blocked their activity. Similar to Go a X, both G11 a X and G16 a 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 a X, and obtained NIH3T3 cell lines stably expressing the empty G16 a mutants by viral infection. We found that G16 a 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.