G protein-coupled, 7-TM receptors mediate response to diverse ligands, including neurotransmitters, chemokines, and morphogens. Thus, understanding 7-TM receptor function has impact on a wide range of physiological processes. Dictyostelium has been an excellent and relevant system for such analysis. Our studies of the CARs were the first to link 7-TM receptors with chemotaxis and morphogenesis. As chemokine receptors, they have been a paradigm for understanding neutrophil chemotaxis; analyzing their role in morphogen signaling is equally instructive. In the metazoa, the Wnt ligand and Frizzled (Fz) 7-TM receptors regulate GSK3 signaling and mutation of many genes in this pathway results in embryonic lethality and tumorigenesis in mice and humans. The ability to analyze related pathway components in Dictyostelium may contribute significant understanding to these events. We have isolated the novel tyrosine kinase ZAK1, and shown that it functions in concert with CAR3 and GSK3 to direct posterior cell differentiation and inhibit anterior differentiation. ZAK1 tyrosine kinase activity is stimulated by cAMP/CAR3 and, in turn, ZAK1 is required to phosphorylate and activate GSK3. We have mapped the ZAK1 phosphorylation sites to the activation loop of Dictyostelium GSK3 and mammalian GSK3b and shown that these tyrosine phosphorylations are required for GSK3 activation in vivo and in vitro. Recent solution of the mammalian GSK3b crystal structure provides a view into potential molecular mechanisms for GSK3 activation by tyrosine phosphorylation and suggests a novel mode for GSK3b regulation in mammalian and other systems. CAR4 is required for anterior cell gene expression but is inhibitory to posterior fates. Developmental defects of car4-null cells can be partially rescued by inhibition of GSK3 activity in vivo, indicating that GSK3 is a downstream negative target of CAR4. We have now shown that car4-nulls have persistent tyrosine phosphorylation and hyper-activation of GSK3. CAR4 does not inhibit cAMP/CAR3 regulated activation of ZAK1. ZAK1 is regulated identically in wild-type and car4-null cells. Rather, using a phosphoGSK3 substrate, we have shown that CAR4 activates a protein tyrosine phosphatase (PTPase). These data are consistent with phosphorylation/de-phosphorylation as a mechanism for activation/de-activation of GSK3. We propose that the different levels of tyrosine phosphorylation of GSK3 that result from the antagonistic actions of the activating tyrosine kinase ZAK1 or the inhibitory CAR4-PTPase constitute the core of the regulatory machinery on GSK3 activity in the context of cell fate decision by the 7-TM CARs. zak1-null cells retain a low-level of phosphorylation and activation of GSK3. We have now identified a second tyrosine kinase, ZAK2, that also regulates GSK3 during development. ZAK2 and ZAK1 are 92% identical in their PTK domains but only 60% identical through the remainder of the proteins. zak2-nulls have significantly diminished activation of GSK3 and are severely compromised in posterior differentiation. Both ZAK1 and ZAK2 negatively regulate anterior fates, but they appear to have selective preference for two different subpopulations. In addition to establishing cell fate patterns, both Fz and CAR signaling regulate cell polarity organization. Wwe now show that ZAK1 and GSK3, but not ZAK2, are also required to establish cell polarity and chemotaxis in Dictyostelium . These data indicate further functional variance between ZAK1 and ZAK2. Although CARs and Fz receptors have different ligands, and Fz had been considered a strictly metazoan receptor family, the resemblance of their signaling pathways through GSK3 invited a more rigorous phylogenetic analysis. Fz family members, including Smoothened, are defined by the Fz structural domain within their transmembrane domains. Analyses of CAR3 and CAR4 sequences revealed a Fz domain through transmembrane domains 2-6 and alignment of the CARs and consensus Fz sequences confirm topological, spatial, and sequence preservation (E=9e-04) throughout the domains. The phylogenetic kinship among the Fz receptors and CARs, in concert with their common functional linkage to GSK3, suggest an ancient origin for this essential pathway for regulating cell fate decisions. Dictyostelium initiate a pulsatile release of cAMP during early development that directs chemotaxis via the CARs. Adenylyl cyclase (AC) is transiently activated by the cAMP signal and is then rapidly adapted (de-activated). While Gbg and the cytosolic factor CRAC are implicated in AC activation, adaptation mechanisms for AC remain unknown. We have now uncovered a novel inhibitory pathway regulated by the G protein subunit Ga9 which we suggest is a component of the adaptation pathway downstream from chemattractant signaling. We have defined this pathway genetically using """"""""loss-of-function"""""""" and """"""""gain-of-function"""""""" mutations. ga9-null cells develop faster, form more cAMP signaling centers, and show resistance to factors that inhibit cAMP signaling, consistent with the loss of an inhibitory protein in the cascade. Additionally, ga9-null cells initiate cAMP pulses more frequently and are more rapidly resensitized (de-adapted) to the cAMP signal. In contrast, cells expressing constitutively activated Ga9 form significantly fewer cAMP signaling centers, and are restricted in their ability to activate AC. Ga9 does not appear to inhibit AC directly, but rather seems to function in an upstream signaling event. Data now suggest that Ga9 regulates receptor adaptation/de-adaptation for chemotactic response. In mammalian cells, phosphorylation of 7-TM receptors is linked to adaptation, but while CAR phosphorylation is contemporaneous with AC adaptation in Dictyostelium, experiments with mutant CARs suggest that their phosphorylation is not required for adaptation. We provide strong evidence to support a functional synergism between Ga9 and receptor phosphorylation to adapt AC. Dictyostelium that lack Ga9 and that also only express phosphorylation-defective CARs, activate AC normally in response to cAMP stimulation, but do not adapt to the signal. Biochemical data indicate that the Ga9 may function partially to regulate the sub-cellular distribution of the AC activator CRAC and may be AC-specific; other CAR-dependent, CRAC-independent pathways, such as guanylyl cyclase activation and actin assembly, adapt normally in these Ga9/CAR mutant strains. Our data raise the possibility that Ga proteins in other systems may also mediate receptor adaptation. Animal cells contain lipid storage droplets (LSDs) for energy metabolism, steroid hormone synthesis, membrane biosynthesis and cell signaling. LSDs in most cells are coated with ADRP, but in adipocytes the droplets are coated with a structurally related protein, Perilipin (Peri). Stimulated adipocytes show PKA-dependent phosphorylation of Peri and the coincident translocation of Hormone Sensitive Lipase (HSL) from the cytosol to the droplet surface and activation of triacylglycerol hydrolysis. We have generated peri-null mice and shown that there adipose mass is <30% of WT, resulting from significantly elevated basal lipolytic rates and the consequent reduction of lipid stores. peri-null adipocytes also fail to translocate HSL to the droplet surface or significantly activate lipolysis. Thus, Peri functions both to inhibit lipolysis in quiescent cells and to permit maximally activated, HSL-dependent lipolysis during hormonal stimulation. We have identified additional proteins related to Peri and ADRP in mouse, Drosophila, and Dictyostelium and shown that these proteins also target exclusively to lipid droplets when expressed in mammalian cells These LSD proteins may be universal regulators for lipogenesis, lipolysis, or packaging and trafficking of neutral lipid storage droplets.
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