A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1
- University of California, San Francisco, United States
- UMC Utrecht, Utrecht University, Netherlands
- University of California, United States
- Lawrence Berkeley National Laboratory, United States
Figures
Figure 1
Altered basal RasGRP1 signaling by a human His 212 Tyr SNV.
(A) The REM, Cdc25, EF hands, C1 and coiled-coil are known protein domains of RasGRP1. Indicated are residues in different domains that have been shown to impact RasGRP1 activity (Thr 184, Arg 271, …
Figure 2 with 1 supplement
A neutral charge on Histidine 212 in RasGRP1 enhanced basal activity.
(A) Alignment of RasGRP isoforms and RasGRP1 sequences in different species to determine conserved residues. His 212 conservation is indicated by grey color. (B–G) Low RasGRP1-expressing Jurkat …
Figure 2—figure supplement 1
Analyses of RasGRP1 level-dependent effects on ERK- and S6-kinase pathway activation.
(A–F) His 212 was mutated into neutrally charged residues (His 212 Ala, His 212 Tyr), or into positively charged residues (His 212 Arg, His 212 Lys). RasGRP1 signaling was determined by levels of …
Figure 3 with 1 supplement
A constitutive positive charge on His 212 in RasGRP1 reduces kinase signaling and prevents RasGRP1 plasma membrane recruitment.
(A) Assay to determine RasGRP1 activity in receptor stimulated cells using RasGRP1-/-RasGRP3-/- DT40 cells, BCR stimulation, and gating on low levels of transfected RasGRP1-EGFP. (B–E) Effects of …
Figure 3—figure supplement 1
Analyses of cellular RasGRP1 localization as a function of His 212 variation.
(A) Shown are representative examples of microscopy analyses of RasGRP1-EGFP low expressing DT40 RasGRP1−/− RasGRP3−/− cells without and with B cell receptor (BCR) stimulation. The top row shows the …
Figure 4
His 212 in RasGRP1 is a pH sensor.
(A) Intracellular pH (pHi) influences the charge of histidines (His). Low pHi shifts equilibrium towards protonated histidine (His+), while in high pHi conditions equilibrium shifts towards …
Figure 5 with 1 supplement
RasGRP substrate specificity and active GEF structures.
(A) Cartoon showing the four RasGRP isoforms, with key conserved regulatory residues indicated. (B) RasGRP isoform specificity for GTPases HRas, Rap1B, and Rap2A was tested by in vitro nucleotide …
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Figure 5—source data 1
Crystallographic table (RasGRP).
- https://doi.org/10.7554/eLife.29002.011
Figure 5—figure supplement 1
Sequence comparison of RasGRP homologs.
(A) The phylogenetic tree of RasGRP homologs was generated by MUSCLE (Please cite Li et al. 2015, Nucleic Acids Research, 43, W580-4.). Each gene is annotated by the organism common name followed by …
Figure 6
A conserved, stabilizing salt-bridge maintains RasGRP1 activity.
(A) The RasGRP2:Rap1B complex structure is shown with the same orientation as Figure 5D. Important structural elements surrounding His 161 are highlighted (REM-cdc25 linker; red, helix J and K; …
Figure 7 with 1 supplement
His 212 – Glu 404 pair and transition from inactive to active RasGRP1.
(A, B) Schematic diagram of RasGRP1 conformations. Color scheme is the same as Figure 6A,C and E. In the autoinhibited form (A), the cdc25-EF linker sits in the Ras binding site and the EF domain …
Figure 7—figure supplement 1
Basal signaling activity of RasGRP1 WT and Glu 404 (A,B), or Glu208 (C,D) variants in RasGRP1−/−RasGRP3−/− DT40 cells.
Shown are average median levels of P-ERK (A, C) and P-S6 (B, D), corrected for untransfected cells for each experiment. Friedman test was used, with post-test Dunn's multiple comparisons, comparing …
Figure 8
Model of RasGRP1 regulation.
(Left) In resting cells, RasGRP1's basal activity is autoinhibited through a dimer; the Cdc25-EF linker (red) blocks the Ras binding site, and the EF hands (purple) prevent C1 domains (cyan) from …
Tables
Table 1
Estimated pKa values.
https://doi.org/10.7554/eLife.29002.015| Estimated pKa values | ||||||
|---|---|---|---|---|---|---|
| Residue no. | wild-type | E208A | E211A | E404A | E205A, E207A, E208A, E211A | E205A, E207A, E208A, E211A, E404A |
| His 212 | 6.90 | 6.81 | 6.71 | 6.71 | 6.44 | 6.12 |
| His 286 | 6.56 | 6.55 | 6.55 | 6.55 | 6.53 | 6.52 |
| His 303 | 6.83 | 6.83 | 6.83 | 6.83 | 6.82 | 6.82 |
| His 315 | 6.17 | 6.16 | 6.16 | 6.17 | 6.15 | 6.15 |
| His 318 | 6.26 | 6.26 | 6.26 | 6.26 | 6.26 | 6.26 |
| His 358 | 6.57 | 6.57 | 6.56 | 6.57 | 6.56 | 6.56 |
| His 381 | 6.27 | 6.27 | 6.27 | 6.27 | 6.27 | 6.27 |
| His 389 | 6.58 | 6.58 | 6.58 | 6.58 | 6.58 | 6.58 |
| His 411 | 6.23 | 6.22 | 6.22 | 6.22 | 6.21 | 6.20 |
