Regulation of the G protein βγ subunits through the covalent modification of Gβ

Abstract

The βγ‐subunits of G protein (Gβγ) dissociate from the a‐subunit (Ga) upon activation of G protein‐coupled receptors. Once free, Gβγ is able to regulate many target proteins, including phospholipase Cβ2 (PLCβ2), phosphoinositide 3‐kinase γ (PI3Kγ) and G protein‐coupled receptor kinase 2 (GRK2). Because overactivation of Gβγ signaling has been implicated in the pathophysiology of several diseases, inhibitors of Gβγ might be very valuable from a pharmacological point of view. Our group has used a combination of virtual and manual screening to find small molecules that bind to a protein‐protein interaction "hot spot" on the β subunit. These compounds inhibit the binding of a phage displaying the SIGKAFKILGYPDYD peptide (SIGK) to Gβγ in an enzyme‐linked immunosorbent assay (ELISA) at concentrations in the nM to µM range. Recently, we discovered that some of the small molecules, like cystamine and selenocystamine, disrupt Gβγ function by a redox mechanism. Their effect can be reversed by the addition of dithiothreitol (DTT). Mass spectrometry indicates that selenocystamine forms a selenylsulfide bridge at preferentially one position, and mutagenesis studies suggest that both cystamine and selenocystamine act on cysteine 204 of the βsubunit. This is the first evidence that protein‐protein interactions with Gβγ can be regulated in a reversible redox manner.This work was supported by NIH grant GM60286‐08.