Dictyostelium grows as single-cell vegetative amoebae. However, upon starvation, cells initiate a multicellular developmental program that is mediated through signal transduction pathways, many of which are controlled through serpentine G protein-coupled receptors. During aggregation, which leads to the formation of the multicellular organism, oscillatory pulses of extracellular cAMP bind cell-surface, serpentine receptors (cARs) that couple to the heterotrimeric G protein containing Galpha2. This stimulates signaling pathways to activate adenylyl cyclase, guanylyl cyclase, and gene expression. cAMP stimulation also leads to transient protein tyrosine phosphorylation of a number of proteins; analysis of phenotypes of mutants for protein tyrosine phosphatases (PTPs) indicates that reversible tyrosine phosphorylation also plays an important role in these processes. Multiple cycles of activation followed by a requisite period of adaptation are necessary to mediate the biological responses leading to the formation of the multicellular organism. After the formation of the mound, other signaling pathways direct the cell-type differentiation and morphogenesis that leads to the formation of a mature fruiting body. Recent results indicate that two MAP kinases (ERK1 and ERK2), PTPs, and Ras, as well as heterotrimeric G proteins, play integral roles in controlling these pathways. The activation and subsequent adaptation of adenylyl cyclase, which is required for relaying the chemotactic signal, is complex and requires both the Gbetagamma and ERK2. Molecular genetic analysis indicates that both aspects require the cAMP receptor, however, the activation of ERK2 is independent of known heterotrimeric G protein subunits. ERK1 and ERK2 are also required for responses during later development. This proposal is directed at using both biochemical and molecular genetic approaches to dissect and understand the mechanisms by which activation and adaption control some of the pathways during aggregation and in the later multicellular stages. We will use available mutants in signaling components to further dissect these pathways and determine how they are regulated. We will use REMI insertional mutagenesis to identify and then clone genes that are specifically involved in mediating receptor activation/adaptation of the MAP kinase cascades and cAMP pulse-induced gene expression. We will employ the yeast two-hybrid system to identify signaling components that lie downstream from the Galpha subunit Galpha2 and components that represent potential upstream activators and downstream substrates of ERK1 and ERK2. Analysis of the function of three PTPs will be directed at understanding their role during aggregation and mound formation and their function in regulating growth stimulation, a process that is similar to serum stimulation of quiescent mammalian cells. Combined genetic analysis and biochemical characterization of the signal transduction pathways should help us understand how they function to control aggregation and cell-type differentiation.
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