Abstract
CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.
Highlights
Anti-Ras protein but has discrete functions, notably in integrinmediated cell adhesion and spreading, formation of cell/cell contacts [6], superoxide formation, and cAMP-induced neurite outgrowth [1, 7]
CAPRI Harbors an Evolutionarily Conserved Helix Motif in Its C-terminal Tail—We aimed to investigate the mechanism of CAPRI switching between its RasGAP and Rap1GAP activities, which are catalyzed through the same GTPase-activating proteins (GAPs)-related domain
To search for potential functional motifs that may play a role in the switch between the dual GAP functions, we examined the amino acids (AA) sequences of CAPRI proteins from different vertebrate species, ranging from fish to humans
Summary
Anti-Ras protein but has discrete functions, notably in integrinmediated cell adhesion and spreading, formation of cell/cell contacts [6], superoxide formation, and cAMP-induced neurite outgrowth [1, 7]. A number of guanine nucleotide exchange factors and GAPs for Ras and Rap have been identified These are multidomain proteins regulated by receptor-mediated extracellular signals, either through second messengers or localized cues such as protein or lipid binding partners that serve as localization signals or scaffolds, controlling their activities in time and space [7, 9, 10]. The C2 and pleckstrin homology/Bruton tyrosine kinase domains that flank the Ras-GRD are required for Rap1GAP activity but not RasGAP activity, both Ras and Rap bind to the RasGRD of GAP1IP4BP (20 –22). Agonist-evoked intracellular Ca2ϩ mobilization leads to a rapid C2 domain-dependent translocation of CAPRI and Rasal to the plasma membrane With this plasma membrane translocation, both the RasGAP and Rap1GAP activities of CAPRI increase substantially [20, 25]. Membrane association is required for both the RasGAP and RapGAP activities of CAPRI, the RapGAP activity of Rasal is independent of membrane binding [20, 22]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
