Subcellular localization of Rap1 GTPase activator CalDAG‐GEFI is orchestrated by interaction of its atypical C1 domain with membrane phosphoinositides

Abstract

Background The small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG‐GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. Objective Here, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. Methods CDGI interaction with phosphoinositides was studied by lipid co‐sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. Results Lipid co‐sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)‐biphosphate (PIP2) and phosphatidylinositol (3,4,5)‐triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co‐sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. Conclusion Our studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)‐biphosphate and phosphatidylinositol (3,4,5)‐triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.

Differential Lipid Binding Specificities of RAP1A and RAP1B are Encoded by the Amino Acid Sequence of the Membrane Anchors

“…Taken together, these results strongly suggest that interactions with PS and PIP 3 are essential for RAP1A.GTP PM interactions. In this context, it is interesting to note that CalDAG-GEFI, a GEF for RAP1, directly associates with PIP 2 and PIP 3 through its C1 domain, 32 and lipid binding domains for these same lipids are found in the RAP1 effector RIAM (RAP1-GTP interacting adaptor molecule). 33,34 A similar mode of action also operates for another RAP1 effector, talin, during integrin activation.…”

The phosphoinositide code is read by a plethora of protein domains

“…These ~50 residue modules are composed of two β-sheets and an α-helix that form a fold stabilized by a pair of zinc coordination sites. Although there are no ligandbound structures yet available, the atypical C1 domain of RAS guanyl-releasing protein 2 utilizes a set of basic residues to specifically recognize PI(4,5P) 2 and PI(3,4,5)P 3 , thus directing the protein to the plasma membrane to activate downstream signaling [82]. These modules can bind diacylglycerol, PI lipids, and proteins, providing opportunities for coincidence detection.…”

RasGRP2 Structure, Function and Genetic Variants in Platelet Pathophysiology

“…The physiological importance of the C1 domain of RasGRP2 in platelets is highlighted by the dramatic effect of its loss on platelet function in vitro and in vivo in both mice and humans [41,84]. Lipid co-sedimentation assays and molecular dynamics simulations with cellular localization experiments demonstrate that the atypical C1 regulatory domain of RasGRP2 controls subcellular localization by interacting with the membrane phosphoinositides, phosphatidylinositol (4,5)-biphosphate (PIP2) and phosphatidylinositol (3,4,5)-triphosphate (PIP3) [85]. Specific C1 residues Arg508, Arg513, and Arg530 contribute to PIP2/3 specific binding, facilitating the recruitment of the membrane-associated Rap1 and allowing downstream αIIbβ3 integrin activation.…”