Anne Theibert

Chapter 17 Transmembrane Signaling in Dictyostelium

Publisher Summary Dictyostelium provides a biochemically and genetically accessible system for studies of transmembrane signaling. Current techniques to control cellular sensitivity and monitor chemotaxis, cyclic adenosine monophosphate (cAMP) signaling, and adenylate cyclase activation are outlined. Receptor-binding assays are reviewed, and a protocol for receptor purification is offered. The assays are brought together in a flow sheet for characterization of transmembrane signaling mutants. Transmembrane signaling mutants can be simply isolated by screening for morphological aberrations during development. The differentiated phenotype is reversible so that, once isolated, the mutants can be grown and propagated as the wild type. A cell surface cAMP receptor is identified and purified, and a specific antiserum is raised. Evidence suggests that this receptor may regulate chemotaxis, activation of adenylate cyclase, and gene expression. An extensive, cAMP-mediated phosphorylation of the receptor may cause adaptation of the physiological responses. The recent demonstration of in vitro guanylnucleotide regulation of the adenylate cyclase suggests that control mechanisms, similar to those found in vertebrates, may operate in this primitive eukaryotic organism.

Demonstration of inositol 1,3,4,5-tetrakisphosphate receptor binding

Inositol 1,3,4,5-tetrakisphosphate (InsP4) is produced rapidly upon stimulation of the phosphoinositide system and may serve as a second messenger in hormone and neurotransmitter action. In this report we demonstrate specific binding sites for [3H]InsP4 in rat tissue membranes. In cerebellar membranes, [3H]InsP4 binding sites are displaced both by InsP4 and inositol 1,4,5-trisphosphate (InsP3) with similar potency (IC50 approximately equal to 300 nM) whereas several other inositol phosphates are much weaker. We have distinguished the InsP4 binding site from the InsP3 receptor binding site by differences in brain regional and tissue distribution, affinity for InsP4 and InsP3, and sensitivity to calcium.

The neuronal Arf GAP centaurin α1 modulates dendritic differentiation

Centaurin α1 is an Arf GTPase-activating protein (GAP) that is highly expressed in the nervous system. In the current study, we show that endogenous centaurin α1 protein is localized in the synaptosome fraction, with peak expression in early postnatal development. In cultured dissociated hippocampal neurons, centaurin α1 localizes to dendrites, dendritic spines and the postsynaptic region. siRNA-mediated knockdown of centaurin α1 levels or overexpression of a GAP-inactive mutant of centaurin α1 leads to inhibition of dendritic branching, dendritic filopodia and spine-like protrusions in dissociated hippocampal neurons. Overexpression of wild-type centaurin α1 in cultured hippocampal neurons in early development enhances dendritic branching, and increases dendritic filopodia and lamellipodia. Both filopodia and lamellipodia have been implicated in dendritic branching and spine formation. Following synaptogenesis in cultured neurons, wild-type centaurin α1 expression increases dendritic filopodia and spine-like protrusions. Expression of a GAP-inactive mutant diminishes spine density in CA1 pyramidal neurons within cultured organotypic hippocampal slice cultures. These data support the conclusion that centaurin α1 functions through GAP-dependent Arf regulation of dendritic branching and spines that underlie normal dendritic differentiation and development.

[25] Identification and ligand-induced modification of the cAMP receptor in Dictyostelium

Abstract Photoaffinity labeling with 8-N3[32P]cAMP and in vivo phosphorylation with 32PO4 provide sensitive methods for the identification of the surface cAMP receptor. Photoaffinity labeling achieves high specificity while in practice, the in vivo32P labeling affords considerably higher sensitivity. Both methods allow fairly direct assays of cellular responses to an extracellular cAMP stimulus at the level of the receptor—a shift in electrophoretic mobility and a rise in phosphorylation. Quantification of the shift in mobility, assayed by photolabeling, has shown a strong correlation between modification of the receptor and adaptation. These labelling techniques will also help to elucidate the role of the concomidant changes in receptor phosphorylation, which may cause the change in electrophoretic mobility. Furthermore, photolabeling and 32P labeling will allow study of the possible multiple affinity classes of the cAMP receptor and of the possible expression of cAMP receptors in later stages of development.

Surface Receptor Mediated Activation and Adaptation of Adenylate Cyclase in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum offers an attractive model system for studying a variety of physiological processes such as Chemotaxis, inter- and intra-cellular communication, gene expression, pattern formation and morphogenesis. Early in the developmental phase of the organisms’ life cycle up to 106 identical unicellular amoebae are induced to migrate chemotactically towards a central region. This aggregation process occurs in response to cyclic AMP secreted by one or more centrally situated cells. Cyclic AMP acts not only as a chemotactic signal but also induces the synthesis and secretion of cAMP from the responding cells. The net result is a concomitant inward migration of cells and outward propagation of cAMP waves throughout the population. The rate of cAMP-induced cAMP synthesis is not constant but oscillates with a frequency of 5–10 minutes. This occurs because cAMP-stimulated adenylate cyclase activity adapts or becomes desensitized in the presence of cAMP.

SECOND MESSENGER RECEPTORS IN THE PHOSPHOINOSITIDE CYCLE

Abundant evidence suggests that the phosphoinositide (PI) system is a prominent and prevalent second messenger system. In addition to mediating short term changes in humoral transmission, the PI cycle may regulate long term alterations evoked by growth factors, hormones and in learning and memory, as is exemplified by long term potentiation. The PI cycle influences cellular function through the generation of diacylglycerol (DAG) and inositol trisphosphate (IP3) as well as other inositol phosphates. DAG stimulates protein kinase C (PKC), whereas the primary function of IP3 is to release calcium from intracellular stores. It is easy to conceptualize how IP3-induced release of calcium would activate short term alterations such as neuronal firing or muscle contraction. One of the challenges of PI research is to determine mechanisms whereby growth, differentiation and synaptic plasticity might be modulated by this Second messenger system. Another challenge is to explain how discrete processing of information can be handled by a single second messenger system which is responsive to so many different primary messengers. Neurotransmitters achieve their selectivity, in part, by acting at subtypes of receptors. Accordingly, one might expect heterogeneity among the various enzymes and binding proteins involved in the PI cycle (for a comprehensive review of the PI system, see Berridge 1987).