Abstract
Platelets are recruited to sites of vascular injury, where they are activated and aggregate to form a hemostatic plug. This process requires the activation of the small GTPase Rap1B by its cognate guanine nucleotide exchange factor CalDAG-GEFI. Studies on platelet function suggest that CalDAG-GEFI activity is regulated by changes in cytosolic calcium, but the exact molecular mechanism is poorly understood. Here we show that purified CalDAG-GEFI is autoinhibited and directly regulated by calcium. Substitutions of putative calcium-binding residues within the canonical EF hands of CalDAG-GEFI diminish its capacity to activate Rap1B. Structural differences between active (WT) and inactive (EF hand variant) CalDAG-GEFI protein were determined by hydrogen-deuterium exchange MS. The highest differential rates of deuterium uptake in WT over EF hand variant CalDAG-GEFI were observed in regions within the catalytic Cdc25 domain and a putative autoinhibitory linker connecting the Cdc25 and EF hand domains. Exchange activity in the EF hand variant was fully restored by an additional substitution, valine 406 to glutamate, which is thought to disrupt the interface between the autoinhibitory linker and the Cdc25 domain. Overall, our results suggest a model for how CalDAG-GEFI remains in an autoinhibited state when levels of cytosolic calcium in resting platelets are low. In response to cellular stimulation, calcium mobilization and binding to the EF hands causes conformational rearrangements within CalDAG-GEFI, including the autoinhibitory linker that frees the catalytic surface of CalDAG-GEFI to engage and activate Rap1B. The data from this study are the first evidence linking CalDAG-GEFI activity directly to calcium.
Highlights
Platelets are recruited to sites of vascular injury, where they are activated and aggregate to form a hemostatic plug
Previous studies using isothermal titration calorimetry determined that the isolated EF hands of CalDAG-GEFI bind calcium with very high affinity (Kd ϳ80 nM) [13]
Inset, stained gel of purified WTϩV406E after SDS-PAGE. They all possess a characteristic domain structure with an N-terminal Ras exchange motif (REM)/Cdc25 catalytic domain and a C-terminal regulatory domain consisting of a pair of EF hands and a C1 domain
Summary
Of ␣IIb3 integrins on the cell surface, a process that depends on the inside-out signaling to these receptors [1]. The most abundant Rap-GEF and Rap-GAP in platelets are calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) and Ras p21 protein activator 3 (RASA3, GAP1IP4bp), respectively [4, 5]. CalDAG-GEFI contains a Ras exchange motif (REM) domain with no known function, a catalytic Cdc domain, a pair of calcium-binding EF hands, and an atypical C1 domain with no known function. CalDAG-GEFI is a multidomain protein consisting of a REM domain (salmon), a catalytic Cdc domain (green), a putative autoinhibitory linker (red), two calcium-binding EF hands (blue), and an atypical C1 domain (yellow). Hydrogen– deuterium exchange MS studies further suggest that calcium binding induces global conformational changes within CalDAG-GEFI, most prominently in the EF hands and a putative autoinhibitory linker sequence connecting the EF hands and Cdc domain. Our work provides the first evidence that CalDAG-GEFI activity is directly regulated by calcium and that release of autoinhibition is the molecular mechanism underlying CalDAGGEFI activation
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