Abstract
SummaryMigratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.
Highlights
The ability of eukaryotic cells to migrate directionally within extracellular chemoattractant gradients is essential for numerous biological processes
Receptor phosphorylation regulates chemotaxis To evaluate the role of CAR1 phosphorylation during chemotaxis, we compared the functions of wild-type CAR1 (WT-CAR1) and a non-phosphorylatable CAR1 mutant (CM1234), where all C-terminal serine residues were removed by either deletion or substitution with alanine or glycine (Hereld et al, 1994)
These CAR1 variants were constitutively expressed at comparable levels in a strain that lacks both CAR1 and CAR3 (Caterina et al, 1994), the cAMP receptors that mediate early chemotactic response during Dictyostelium development (Saxe et al, 1991; Insall et al, 1994b)
Summary
The ability of eukaryotic cells to migrate directionally within extracellular chemoattractant gradients is essential for numerous biological processes. Many processes that regulate receptor-mediated chemotactic movement are remarkably conserved through evolution. Such mechanistic universality and ease of experimental manipulation have made Dictyostelium a premier system for the discovery and analyses of regulatory signaling networks that are common to most migratory cells, including human neutrophils and macrophages (Jin et al, 2009). As a population of Dictyostelium depletes nutrients within its local environment, starved cells enter a cooperative developmental. Cells secrete cAMP, which acts as the extracellular chemoattractant to coordinate directed cell movement. Adapted cells remain transiently refractory to additional stimulation until they de-adapt (resensitize) for another round of cAMP signal relay and movement
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