1: cAR1-mediated spatiotemporal dynamics of Ras signaling. GPCR-mediated Ras activation is a key signaling step for eukaryotic cells to generate temporal adaptation or to cause spatial amplification in response to spatially uniform stimuli or a chemoattractant gradient. It is not clear how a GPCR/G-protein machinery regulates spatiotemporal dynamics of Ras activation to achieve these cellular responses. Here, using quantitative live cell imaging methods, we measured spatiotemporal dynamics of RAs activation in Dictyostelium discoideum cells in response to various cAMP stimuli. Activation of cAR1-GPCR triggers a transient Ras activation, followed by an imperfect adaptation of Ras signaling. In response to a uniformly applied two-step cAMP stimulation, while G-protein activation displays a step-like increase, Ras activation shows two transient responses with imperfect adaptions, and PIP3 production shows two transient responses followed by perfect adaptions. A cAMP gradient induces a transient Ras activation that quickly adapts to lower levels around cell membrane. There is no clear spatial amplification at the step of Ras activation between the front and back of the cell, which is in clear contrast to the spatially amplified PIP3 response in the front. While regulators of Ras and other components have not been identified, our quantitative measurements demonstrate that different signaling events downstream of GPCR have distinct kinetic patterns, and provide a foundation for understanding how these events are linked to each other to produce chemotactic responses. (Xu et al., to be submitted). 2: We have discovered that arrestins are key components of the signaling circuit involved in the oscillatory cell-cell signaling in eukaryotes. We have found that cAR1 GPCR-mediated arrestin function regulates the period of transient ERK2 activation that controls the frequency of cAMP oscillations in D. discoideum. Oscillation of chemical signals is a common biological phenomenon but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6 min intervals, directing cell migration. Although the G protein-coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here, we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB-C-) display cAMP oscillations during the aggregation stage that are twice as frequent as wild type cells. The adcB-C- cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that AdcC associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB-C- cells, suggesting that arrestins are required to achieve the switch from high-affinity to low-affinity cAMP receptors that occurs after the aggregation stage of multicellular development (Cao et al., MBoC 2014 in press).