POTENTIATION OF GPCR-SIGNALING VIA MEMBRANE TARGETING OF G PROTEIN α SUBUNITS

ABSTRACT

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 Gα₁₆ 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 α_q subunit, that lacks the highly conserved six amino acid N-terminal extension and bears four residues of α_i sequence at its C-terminus replacing the corresponding α_q sequence (referred to as Δ6qi4). When expressed in COS7 cells, Δ6qi4 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 Δ6qi4myr) created a protein that was dually acylated and exclusively located in the particulate fraction. As a measure of G protein activation Δ6qi4 and Δ6qi4myr 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 Δ6qi4myr as compared with Δ6qi4. It could be shown that increased functional responses of agonist activated GPCRs are due to the higher content of Δ6qi4myr in the plasma membrane. Our results indicate that manipulation of subcellular localization of G protein α 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.

Key Words: :

• G protein coupled receptor
• G protein
• Promiscuous
• Gα
• ₁₆
• Myristoylation
• Signal transduction