Different assay technologies are available that allow ligand occupancy of G protein coupled receptors to be converted into robust functional assay signals. Of particular interest are universal screening systems such that activation of any GPCR can be detected with a common assay end point. The promiscuous G protein Galpha16 and chimeric G proteins are broadly used tools for setting up almost universal assay systems. Many efforts focused on making G proteins more promiscuous, however no attempts have been made to make promiscuos G proteins more sensitive by interfering with their cellular protein distribution. As a model system, we used a promiscuous G protein alphaq subunit, that lacks the highly conserved six amino acid N-terminal extension and bears four residues of alphai sequence at its C-terminus replacing the corresponding alphaq sequence (referred to as delta6qi4). When expressed in COS7 cells, delta6qi4 undergoes palmitoylation at its N-terminus. Cell fractionation and immunoblotting analysis indicated localization in the particulate and cytosolic fraction. Interestingly, introduction of a consensus site for N-terminal myristoylation (the resulting mutant referred to as delta6qi4myr) created a protein that was dually acylated and exclusively located in the particulate fraction. As a measure of G protein activation delta6qi4 and delta6qi4myr were coexpressed (in CHO cells) with a series of different Gi/o coupled receptors and ligand induced increases in intracellular Ca2+ release were determined with the FLIPR technology (Fluorescence plate imaging reader from Molecular Devices Corp.). All of the receptors interacted more efficiently with delta6qi4myr as compared with delta6qi4. It could be shown that increased functional responses of agonist activated GPCRs are due to the higher content of delta6qi4myr in the plasma membrane. Our results indicate that manipulation of subcellular localization of G protein alpha subunits-moving them from the cytosol to the plasma membrane-potentiates signaling of agonist activated GPCRs. It is concluded that addition of myristoylation sites into otherwise exclusively palmitoylated G proteins is a new and sensitive approach and may be applicable when functional assays are expected to yield weak signals as is the case when screening extracts of tissues for biologically active GPCR ligands.