Functional characterization of the guanine nucleotide exchange factor (GEF) motif of GIV protein reveals a threshold effect in signaling.
Heterotrimeric G proteins are critical signal-transducing molecules controlled by a complex network of regulators. GIV (a.k.a. Girdin) is a unique component of this network and a nonreceptor guanine nucleotide exchange factor (GEF) that functions via a signature motif. GIV's GEF motif is involved in the regulation of critical biological processes such as phosphoinositide 3 kinase (PI3K)-Akt signaling, actin cytoskeleton remodeling, cell migration, and cancer metastasis. Here we investigated how the GEF function of GIV affects the wiring of its signaling pathway to shape different biological responses. Using a structure-guided approach, we designed a battery of GIV mutants with different Gαi-binding and -activating properties and used it to dissect the specific impact of changes in GIV's GEF activity on several cellular responses. In vivo signaling assays revealed a threshold effect of GEF activity for the activation of Akt by GIV in different cell lines and by different stimuli. Akt signaling is minimal at low GEF activity and is sharply increased to reach a maximum above a threshold of GEF activity, suggesting that GIV is a critical signal amplifier and that activation of Akt is ultrasensitive to changes in GIV's GEF activity. A similar threshold dependence was observed for other biological functions promoted by GIV such as remodeling of the actin cytoskeleton and cell migration. This functional characterization of GIV's GEF motif provides insights into the molecular interactions between nonreceptor GEFs and G proteins and the mechanisms that govern this signal transduction pathway.
Department of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093, USA.
Mikel
M
Garcia-Marcos
Patrick S
PS
Kietrsunthorn
Yelena
Y
Pavlova
Michelle A
MA
Adia
Pradipta
P
Ghosh
Marilyn G
MG
Farquhar
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002465:Cell Movement / Q000187:drug effects; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism*
C502451:CCDC88A protein, human; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D008246:Lysophospholipids; D008840:Microfilament Proteins; D050505:Mutant Proteins; D033921:Vesicular Transport Proteins; D051057:Proto-Oncogene Proteins c-akt; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C032881:lysophosphatidic acid