title: Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets.
abstract: Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a Gγ recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein γ subunit (Gγcyto) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the Gγ recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan.
authors.forename: Misato
authors.initials: M
authors.lastname: Kaishima
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Fukuda
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D003600:Cytosol / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D051380:GRB2 Adaptor Protein; D044388:GTP-Binding Protein gamma Subunits; D008565:Membrane Proteins; D011961:Receptors, Fc; D066246:ErbB Receptors