It is becoming increasingly clear that signaling via G proteincoupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that 2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1͞2 thus behaving as dual efficacy ligands. ERK1͞2 activation by dual efficacy ligands was not affected by ADP-ribosylation of G␣i and could be observed in S49-cyc ؊ cells lacking G␣s indicating that, unlike the conventional agonist isoproterenol, these drugs induce ERK1͞2 activation in a Gs͞i-independent manner. In contrast, this activation was inhibited by a dominant negative mutant of -arrestin and was abolished in mouse embryonic fibroblasts lacking -arrestin 1 and 2. The role of -arrestin was further confirmed by showing that transfection of -arrestin 2 in these knockout cells restored ICI118551 promoted ERK1͞2 activation. ICI118551 and propranolol also promoted -arrestin recruitment to the receptor. Taken together, these observations suggest that -arrestin recruitment is not an exclusive property of agonists, and that ligands classically classified as inverse agonists rely exclusively on -arrestin for their positive signaling activity. This phenomenon is not unique to 2-adrenergic ligands because SR121463B, an inverse agonist on the V2 vasopressin receptor-stimulated adenylyl cyclase, recruited -arrestin and stimulated ERK1͞2. These results point to a multistate model of receptor activation in which ligand-specific conformations are capable of differentially activating distinct signaling partners.
Phosphoinositide 3-kinase (PI3K), PTEN and localized phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ] play key roles in chemotaxis, regulating cell motility by controlling the actin cytoskeleton in Dictyostelium and mammalian cells. PtdIns(3,4,5)P 3 , produced by PI3K, acts via diverse downstream signaling components, including the GTPase Rac, Arf-GTPases and the kinase Akt (PKB). It has become increasingly apparent, however, that chemotaxis results from an interplay between the PI3K-PTEN pathway and other parallel pathways in Dictyostelium and mammalian cells. In Dictyostelium, the phospholipase PLA2 acts in concert with PI3K to regulate chemotaxis, whereas phospholipase C (PLC) plays a supporting role in modulating PI3K activity. In adenocarcinoma cells, PLC and the actin regulator cofilin seem to provide the direction-sensing machinery, whereas PI3K might regulate motility.
Phosphoinositide 3-kinase (PI3K)γ and Dictyostelium PI3K are activated via G protein–coupled receptors through binding to the Gβγ subunit and Ras. However, the mechanistic role(s) of Gβγ and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gβ null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P3. Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras–guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.
Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein–coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase–activating protein acting as a global inhibitor.
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