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First published online 3 May 2006
doi: 10.1242/dev.02399

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GABA induces terminal differentiation of Dictyostelium through a GABA_B receptor

Center for Molecular Genetics, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0368, USA.

View larger version (12K): [in a new window]   Fig. 1. GABA induction of spore formation and production of SDF-2. (A) KP cells developing as monolayers were treated with various concentrations of GABA and the proportion of spores determined after 1 hour (circles). They were also incubated with antibodies to AcbA for 30 seconds before adding various concentrations of GABA (squares). Each experiment was repeated three to five times; the error bars represent one s.d. (B) 10 nM GABA was added to KP cells (squares) and dhkA–/K cells (circles) developing as monolayers. Aliquots of the surrounding buffer were collected at the times indicated and stored frozen. SDF-2 activity was determined in serial dilutions of the samples using the KP cell bio-assay as previously described (Anjard et al., 1998). The amount of SDF-2 is given as units per 103 cells. The lower limit in this assay is five units per 103 cells.

View larger version (28K): [in a new window]   Fig. 2. Spore formation in wild type and grlE– strains. Early culminants of the wild-type strain AX4 (white columns) and the grlE– null strain (hatched columns) were disaggregated, washed and deposited at 105 cells per well in 2 ml buffer. GABA (100 nM); 10 pM SDF2; 100 nM GABA and 10 µM glutamate; or 10 pM SDF-2 and 10 µM glutamate were then added to the wells as indicated. The proportion of spores was determined 1 hour later and compared with cells that received no additions. Each experiment was repeated three to five times; the error bars represent 1 s.d.

View larger version (24K): [in a new window]   Fig. 3. Inhibition of spore induction. (A) KP cells developing as monolayers were treated with various concentrations of GABA and glutamate, and the proportion of spores determined after 1 hour: 1 nM GABA (black circles), 10 nM GABA (triangles), 100 nM GABA (squares), 1 µM GABA (white circles). Glutamate was added simultaneously with GABA at the concentrations indicated. (B) SDF-2 was added to the cells at 0.1 pM (circles); 10 pM SDF-2 (triangles); 10 nM (squares). Glutamate was added simultaneously at the concentrations indicated. (C) GABA and CGP 55845 were added at various concentrations and the proportion of spores determined after 1 hour; 1 nM GABA (black circles), 10 nM GABA (triangles), 100 nM GABA (squares), 1 µM GABA (white circles). CGP 55845 was added simultaneously at the concentrations indicated. (D) SDF-2 was added to the cells at 0.1 pM (circles); 10 pM SDF-2 (triangles); 10 nM (squares). CGP 55845 was added simultaneously at the concentrations indicated.

View larger version (32K): [in a new window]   Fig. 4. Time course of GrlE expression. (A) Total RNA was extracted from wild-type AX4 cells harvested at the indicated time of development and electrophoretically separated on a 1.2% agarose gel before being transferred to a nylon membrane. The membrane was hybridized with a probe for grlE (see Materials and methods). (B) Samples were collected from wild-type and grlE– null cells that had developed for 0, 4 and 16 hours, and probed for grlE mRNA. Consistent loading in each lane was confirmed by staining for rRNA.

View larger version (13K): [in a new window]   Fig. 5. Model of signal transduction pathways during terminal differentiation. When GABA is bound to GrlE on the surface of prespore cells, the signal is transduced via PI3 kinase and PkbR1 to result in release of AcbA, the precursor of SDF-2. The same pathway in prestalk cells results in the exposure of the protease domain of TagC, such that it can process AcbA into SDF-2. When glutamate is bound to GrlE, a distinct signal transduction pathway results in the inhibition of release of AcbA in response to SDF-2. When SDF-2 binds its receptor DhkA, phospho-relay to RdeA is inhibited and may be reversed, resulting in a decrease in the activity of the internal cAMP phosphodiesterase RegA. The subsequent increase in cAMP generated by the adenylyl cyclase ACR activates PKA which triggers release of AcbA, as well as rapid encapsulation of prespore cells. Inhibition of phosphorelay to RegA in prestalk cells results in exposure of the protease domain of TagC.