A structural determinant that renders G alpha(i) sensitive to activation by GIV/girdin is required to promote cell migration.
Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G alpha(i3) necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G alpha(i) subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G alpha(i3) is a better substrate for GIV than the highly homologous G alpha(o). We identified Trp-258 in the G alpha(i) subunit as a novel structural determinant for GIV binding by comparing GIV binding to G alpha(i3)/G alpha(o) chimeras. Mutation of Trp-258 to the corresponding Phe in G alpha(o) decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G alpha-binding partners, i.e. G betagamma, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of G alpha(i3) by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G alpha(i3) W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G alpha(i3) by GIV is essential for biological functions associated with G alpha(i3) activation. In conclusion, we have discovered a novel structural determinant on G alpha(i) that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G alpha(i) and that represents an attractive target site for designing small molecules to disrupt the G alpha(i)-GIV interface for therapeutic purposes.
Garcia-Marcos
Mikel
M
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA.
Ghosh
Pradipta
P
Ear
Jason
J
Farquhar
Marilyn G
MG
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000502:physiology; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding; D020710:RGS Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020730:Guanine Nucleotide Dissociation Inhibitors; D008840:Microfilament Proteins; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C502451:CCDC88A protein, human; C525615:GPSM1 protein, human; C498915:Gpsm1 protein, mouse; C498913:Gpsm1 protein, rat; C527550:Ccdc88a protein, mouse; C097187:regulator of G-protein signalling 19