Membrane Targeting of C2GAP1 Enables Dictyostelium discoideum to Sense Chemoattractant Gradient at a Higher Concentration Range

Chemotaxis, which is G protein-coupled receptor (GPCR)-mediated directional cell migration, plays pivotal roles in diverse human diseases, including recruitment of leukocytes to inflammation sites and metastasis of cancer. It is still not fully understood how eukaryotes sense and chemotax in response to chemoattractants with an enormous concentration range. A genetically traceable model organism, Dictyostelium discoideum, is the best-studied organism for GPCR-mediated chemotaxis. Recently, we have shown that C2GAP1 controls G protein coupled receptor-mediated Ras adaptation and chemotaxis. Here, we investigated the molecular mechanism and the biological function of C2GAP1 membrane targeting for chemotaxis. We show that calcium and phospholipids on the plasma membrane play critical roles in membrane targeting of C2GAP1. Cells lacking C2GAP1 (c2gapA–) displayed an improved chemotaxis in response to chemoattractant gradients at subsensitive or low concentrations (<100 nM), while exhibiting impaired chemotaxis in response to gradients at high concentrations (>1 μM). Taken together, our results demonstrate that the membrane targeting of C2GAP1 enables Dictyostelium to sense chemoattractant gradients at a higher concentration range. This mechanism is likely an evolutionarily conserved molecular mechanism of Ras regulation in the adaptation and chemotaxis of eukaryotes

Direct Interaction between TalinB and Rap1 is necessary for adhesion of Dictyostelium cells

BACKGROUND: The small G-protein Rap1 is an important regulator of cellular adhesion in Dictyostelium, however so far the downstream signalling pathways for cell adhesion are not completely characterized. In mammalian cells talin is crucial for adhesion and Rap1 was shown to be a key regulator of talin signalling. RESULTS: In a proteomic screen we identified TalinB as a potential Rap1 effector in Dictyostelium. In subsequent pull-down experiments we demonstrate that the Ras association (RA) domain of TalinB interacts specifically with active Rap1. Studies with a mutated RA domain revealed that the RA domain is essential for TalinB-Rap1 interaction, and that this interaction contributes to cell-substrate adhesion during single-celled growth and is crucial for cell-cell adhesion during multicellular development. CONCLUSIONS: Dictyostelium Rap1 directly binds to TalinB via the conserved RA domain. This interaction is critical for adhesion, which becomes essential for high adhesive force demanding processes, like morphogenesis during multicellular development of Dictyostelium. In mammalian cells the established Rap1-talin interaction is indirect and acts through the scaffold protein - RIAM. Interestingly, direct binding of mouse Rap1 to the RA domain of Talin1 has recently been demonstrated

C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB− cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB(-) cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis

Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations

Ras of complex proteins (Roc) is a Ras-like GTP binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain, and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labeling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains, that in contrast are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-Kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis, and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum RocCOR interface significantly influence the structure and nucleotide-induced conformational changes of the Roc domains

GPCR-controlled membrane recruitment of negative regulator C2GAP1 locally inhibits Ras signaling for adaptation and long-range chemotaxis

Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficient c2gapA(-) cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects of c2gapA(-) cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis

Additional file 2: Figure S1. of Direct Interaction between TalinB and Rap1 is necessary for adhesion of Dictyostelium cells

Dissociation of mGppNHp from RasG in the absence and presence of 100 ΟM purified RA-TalB. (TIF 430 kb

Additional file 1: Table S1. of Direct Interaction between TalinB and Rap1 is necessary for adhesion of Dictyostelium cells

A subset of proteins identified in Mass Spectrometry analysis of Rap1 pull down sample. Proteins have been identified as present in the sample if a minimum of 2 unique peptides were identified in Rap1 pull down sample that were not present in the GST control sample. (DOCX 22 kb

Forty-five years of cGMP research in Dictyostelium: Understanding the regulation and function of the cGMP pathway for cell movement and chemotaxis

In Dictyostelium chemoattractants induce a fast cGMP response that mediates myosin filament formation in the rear of the cell. The major cGMP signaling pathway consists of a soluble guanylyl cyclase sGC, a cGMP-stimulated cGMP-specific phosphodiesterase and the cGMP-target protein GbpC. Here we combine published experiments with many unpublished experiments performed in the past 45 years on the regulation and function of the cGMP signaling pathway. The chemoattractants stimulate heterotrimeric Gαβγ and monomeric Ras proteins. A fraction of the soluble guanylyl cyclase sGC binds with high affinity to a limited number of membrane binding site, which is essential for sGC to become activated by Ras and Gα proteins. sGC can also bind to F-actin; binding to branched F-actin in pseudopods enhances basal sGC activity, whereas binding to parallel F-actin in the cortex reduces sGC activity. The cGMP pathway mediates cell polarity by inhibiting the rear: in unstimulated cells by sGC activity in the branched F-actin of pseudopods, in a shallow gradient by stimulated cGMP formation in pseudopods at the leading edge, and during cAMP oscillation to erase the previous polarity and establish a new polarity axis that aligns with the direction of the passing cAMP wave

C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB- cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB - cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis