Dataset Information

Ggamma in dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients.

ABSTRACT: G-protein-mediated signal transduction pathways play an essential role in the developmental program of the simple eukaryotic organism Dictyostelium discoideum. Database searches have yielded 11 Galpha-subunits, a single Gbeta-subunit, but no Ggamma-subunits. We report here the purification, cDNA isolation, and functional analysis of a Ggamma-subunit. Like Gbeta, the Ggamma appears to be unique and hybridization studies show that Ggamma and Gbeta are expressed in parallel during development. Species-wide sequence comparisons of Ggamma-subunits and gamma-like domains of RGS proteins reveal short stretches of highly conserved residues as well as the common CXXL motif at the COOH-terminal of Ggammas that target Gbetagammas to plasma membrane. Overexpression of a CSVL-deleted Ggamma (GgammaDelta) in wild-type cells shifts Gbetagamma to the cytosol and selectively impairs certain G-protein-mediated signal transduction pathways. These cells are able to respond to increments in the stimulus, but are unable to sense chemoattractant gradients. They neither move directionally nor recruit PH-domains to their leading edge. Thus, a full complement of membrane-tethered Gbetagamma is required for sensing shallow gradients, but is not essential for responses to increments in extracellular stimuli.

SUBMITTER: Zhang N

PROVIDER: S-EPMC60167 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Ggamma in dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients.

Zhang N N Long Y Y Devreotes P N PN

Molecular biology of the cell 20011001 10

G-protein-mediated signal transduction pathways play an essential role in the developmental program of the simple eukaryotic organism Dictyostelium discoideum. Database searches have yielded 11 Galpha-subunits, a single Gbeta-subunit, but no Ggamma-subunits. We report here the purification, cDNA isolation, and functional analysis of a Ggamma-subunit. Like Gbeta, the Ggamma appears to be unique and hybridization studies show that Ggamma and Gbeta are expressed in parallel during development. Spec ...[more]

PMID: 11598203

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Project description:BackgroundRas proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.ResultsRasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein.ConclusionThe data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.

Dataset Information

Ggamma in dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients.

Publications

Ggamma in dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients.

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