Small G-proteins are molecular switches that cycle between an inactive GDP- and active GTP-bound state. Activation is dependent on helper proteins that facilitate release and exchange of bound nucleotide: GEFs (Guanine nucleotide Exchange Factors), while its deactivation is regulated by GAPs (GTPase activating proteins) that stimulate the naturally low GTPase activity [1
]. Only in the active GTP bound form, the switch region of small G-proteins can interact with Ras-associating (RA) or Ras binding domains (RBD) of effector proteins [2
]. In this way Small G-proteins provide tight control over the cell response to extracellular stimuli by translating the signaling cascades inside the cell [4
In Dictyostelium discoideum
small G-proteins are essential for a wide variety of processes, including regulation of the cytoskeleton, chemotaxis, cell division and multicellular development [5
]. Due to its genetic tractability and high conservation of many important signaling pathways Dictyostelium
has proven to be an excellent model for studying small G-protein signaling. During the vegative state, Dictyostelium
are single-celled amoeba that feed on bacteria. Upon starvation, cells undergo a tightly regulated developmental process in which they secrete and chemotax toward cAMP, resulting in multicellular fruiting bodies [7
Rap proteins belong to the Ras superfamily of small G-proteins. In mammalian cells Rap is important for cellular adhesion, differentiation and cell proliferation [9
RapA is essential and is implicated in processes throughout the life cycle. In vegetative and chemotaxis competent cells RapA induces pseudopod formation by activating PI3K [11
], and it regulates substrate attachment and myosin II disassembly via the serine/threonine kinase Phg2 [12
]. Strains with a deletion of rapGAP2
showed defects in cell patterning and morphogenesis [14
], indicating that RapA is also important in late development. However so far nothing is known about downstream effectors of RapA that play a role in the progression of multicellular development.
Here we show that activation of RapA results in increased level of activated Rac proteins. To gain further insight into the link between these pathways, we have used a proteomic approach. Recombinant RapA was used as bait in pull-down screens and interacting proteins were identified by mass-spectroscopy. One of the binding partners, GxcC provides a potential link between Rap and Rac activation and deletion studies reveal that this pathway is involved in regulating development of Dictyostelium cells.