Selection of gbeta subunits with point mutations that fail to activate specific signaling pathways in vivo: dissecting cellular responses mediated by a heterotrimeric G protein in Dictyostelium discoideum.
In Dictyostelium discoideum, a unique Gbeta subunit is required for a G protein-coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Galpha2, and Gbeta genes completely impair all these responses. To dissect specificity in Gbetagamma signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gbeta genes and isolated Gbeta alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gbeta subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gbetagamma by making point mutations on Gbeta. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gbetas and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtbetagamma interacts with its regulators, Galpha and phosducin.
Jin
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T
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Amzel
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M
Devreotes
P N
PN
Wu
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L
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002633:Chemotaxis; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation
D046911:Macromolecular Substances; D015800:Protozoan Proteins; D011953:Receptors, Cyclic AMP; D011994:Recombinant Proteins; C068125:cyclic AMP receptor cAR1; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases