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Abstract
RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.
Highlights
The Ras family of small G-proteins, including Ras and Rap, are molecular switches that transmit signals when bound to GTP (Rojas et al, 2012)
We identified all single nucleotide variants (SNVs) reported for RasGRP1 in publicly available databases, and took a shotgun approach to test a panel of these SNVs for their ability to alter RasGRP1 regulation (Figure 1C)
To assess the potential signaling impact of these SNVs, we used RasGRP1À/ÀRasGRP3À/À DT40 cells genetically deleted for RasGRP1 and RasGRP3 and reconstituted these cells via transfection with wildtype EGFP-RasGRP1 (WT) or a catalytically inactive RasGRP1 (Arg271Glu) as before (Iwig et al, 2013), or with a panel of RasGRP1 SNVs. This assay allows for activity assessment of RasGRP1-ERK signaling (Iwig et al, 2013), and of RasGRP1-mTORC1-p70S6 kinase signaling resulting in phosphorylation of ribosomal protein S6 (P-S6)
Summary
The Ras family of small G-proteins, including Ras and Rap, are molecular switches that transmit signals when bound to GTP (Rojas et al, 2012). GEFs activate Ras-family members by triggering the release of GDP and its replacement by GTP (Wittinghofer and Vetter, 2011) These GEFs, of which there are several distinct families in humans, activate Ras-family members in response to a variety of signals, and thereby initiate downstream kinase signaling cascades (Bos et al, 2007; Cherfils and Zeghouf, 2013). One family of these GEFs is comprised of the Ras guanine-nucleotide releasing proteins (RasGRPs) that can activate Ras or Rap, of which there are four in humans.
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