Chimeric G alpha i2/G alpha 13 proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha 13.
The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G alpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between G alpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G alpha(i) proteins. Using G alpha(i2), which does not bind RGL domains, as the backbone in which G alpha(13) sequences were swapped or mutated, we observed that the switch region of G alpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of G alpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of G alpha(13) and chimeric G alpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which G alpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.
Oral & Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
José
J
Vázquez-Prado
Hiroshi
H
Miyazaki
Maria Domenica
MD
Castellone
Hidemi
H
Teramoto
J Silvio
JS
Gutkind
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian; D051960:GTP-Binding Protein alpha Subunit, Gi2; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism; D017470:Receptors, Glutamate / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D013329:Structure-Activity Relationship; D014162:Transfection
C577234:ARHGEF11 protein, human; C577222:ARHGEF12 protein, human; C577223:Arhgef12 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D020710:RGS Proteins; D017470:Receptors, Glutamate; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498016:Gnai2 protein, mouse