The coiled-coil region of the G protein beta subunit. Mutational analysis of Ggamma and effector interactions.
The beta and gamma subunits of the heterotrimeric G proteins remain tightly associated throughout the signaling cycle as the betagamma dimer interacts with Galpha, receptors, and effectors. A coiled-coil structure involving alpha-helical segments at the N termini of the beta and gamma subunits contributes to the dimerization interface and has been implicated in effector signaling in yeast. Scanning mutagenesis of the coiled-coil region of the mammalian beta1 subunit was performed to examine the effect of point mutations on betagamma assembly and effector signaling in COS cell cotransfection assays. In addition to the E10K mutation described previously, mutations A11E, L14E, and I18E in beta1 were found to block betagamma association, as evidenced by the failure of the Gbeta mutants to undergo cytosolic translocation with cotransfected nonisoprenylated Ggamma. Although none of 14 beta1 point mutations prevented the betagamma-dependent activation of the c-Jun N-terminal kinase (JNK) effector pathway, the D20K point mutation enhanced JNK but not phospholipase C-beta2 activation. These findings implicate the coiled-coil region of Gbeta in JNK signaling, provide further evidence that the structural features of the betagamma complex mediating effector regulation may differ among effectors, and identify single codons in the mammalian beta subunit where mutation might yield a phenotype of defective signal transduction.
Pellegrino
S
S
Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zhang
S
S
Garritsen
A
A
Simonds
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D007700:Kinetics; D046911:Macromolecular Substances; D020928:Mitogen-Activated Protein Kinases; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054799:Phospholipase C beta; D010944:Plants; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D012568:Schizosaccharomyces; D016415:Sequence Alignment; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins