title: Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.
abstract: GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gαi3on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function.
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Kshitij
authors.initials: K
authors.lastname: Parag-Sharma
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Anthony
authors.initials: A
authors.lastname: Leyme
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Vincent
authors.initials: V
authors.lastname: DiGiacomo
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Arthur
authors.initials: A
authors.lastname: Marivin
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Stefan
authors.initials: S
authors.lastname: Broselid
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 mgm1@bu.edu.
authors.forename: Mikel
authors.initials: M
authors.lastname: Garcia-Marcos
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D006367:HeLa Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C502451:CCDC88A protein, human; D008840:Microfilament Proteins; D033921:Vesicular Transport Proteins; D020962:Heterotrimeric GTP-Binding Proteins
keywords: G protein-coupled receptor (GPCR); bioluminescence resonance energy transfer (BRET); guanine nucleotide exchange factor (GEF); heterotrimeric G protein; receptor tyrosine kinase