PATHWAYS OF RECEPTOR SIGNALING AND CHEMOTAXIS

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Chemotactic cells orient and move directionally in very shallow chemical gradients. As cells polarize, distinct structural and signaling components become spatially restricted to the leading edge or rear of cells. Feedback loops downstream of receptor signaling integrate both activating and inhibiting pathways to establish cell polarity within such gradients. While much effort has focused on defining activating pathways, inhibitory networks have been largely unexplored. Many receptor-mediated pathways in eukaryotes adapt/deactivate to persistent stimulation. While the MAP kinase ERK2 in Dictyostelium has been assumed to adapt to continuous engagement of cAMP with its 7-transmembrane, cell surface receptor CAR, we have shown, to the contrary, that ERK2 remains active under such conditions. The upstream phosphorylation pathway that is responsible for ERK2 activation transiently responds to CAR stimulation, whereas ERK2 de-phosphorylation (deactivation) is inhibited by continuous stimulation. We argue that the net result is persistently active ERK2 when the extracellular cAMP concentration is constant and that the oscillating production/destruction of secreted cAMP in chemotaxing cells accounts for the observed oscillatory activity of ERK2. We also showed that CAR-dependent pathways that control ERK2 activation/deactivation function independently of G proteins and of ligand-induced production of intracellular cAMP and the consequent activation of PKA. This regulation enables ERK2 to function both in an oscillatory manner, critical for chemotaxis, and in a persistent manner, necessary for gene expression, as secreted cAMP increases during later development. This work redefines mechanisms of ERK2 regulation by CAR signaling in Dictyostelium and establishes new implications for control of signal-relay during chemotaxis. 7-TMRs activate multiple downstream signaling cascades via heterotrimeric G protein-dependent and -independent pathways and control a wide range of biological processes. Upon ligand binding, 7-TMRs become phosphorylated at cytoplasmic serine and threonine residues by specific receptor kinases, functioning to uncouple heterotrimeric G protein pathways from receptor signaling and to activate G protein-independent pathways. In Dictyostelium, CAR signaling regulates chemotaxis and both G protein-dependent and -independent signaling cascades. Since the CARs become phosphorylated upon cAMP binding, we analyzed the ability of cells that only express non-phosphorylatable CAR variants to respond to cAMP. These cells were defective in adenylyl cyclase adaptation and in cell polarization and chemotaxis. While receptor phosphorylation is known to uncouple certain G protein-mediated signaling pathways from 7-TMRs in mammalian cells, our studies are the first to show that such mechanisms also function in pathways that regulate chemotaxis.