RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

Abstract

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4−CD8− ‘double negative’ (DN) thymocytes, pass through a checkpoint termed “β-selection” before maturing into CD4+CD8+ ‘double positive’ (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

“…For example, examination of the EF domain sequence of RasGRP4 suggests that this protein does not bind calcium, or does so in a manner distinct from classical EF hands. It is perhaps due to these distinctions that loss of RasGRP1 is so detrimental to thymocyte development (Dower et al, 2000) and only minimally compensated for by RasGRP3 or 4 (Zhu et al, 2012; Golec et al, 2013). RasGRP1 also selectively appears as a common integration site in murine leukemia virus screens (Mikkers et al, 2002; Suzuki et al, 2002; Akagi et al, 2004), resulting in increased expression of RasGRP1, which is not observed for other Ras-specific exchange factors (Hartzell et al, 2013).…”

Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

“…Non-antigen receptor triggered pathways that are typically not associated with DAG production have been implicated in RasGRP1 membrane localization. Specifically, RasGRP1 but not RasGRP3 signals downstream of the CXCR4 chemokine receptor in thymocytes (123) and a heterodimer of TCR/CXCR4 has been described to recruit the PLC enzymes essential in this pathway (124). How different receptor systems couple to DAG and RasGRP and may be able to synergistically trigger this pathway is an interesting concept for future research.…”

“…Perhaps surprisingly, other RasGEFs, be it of the RasGRP-, Rasgrf-, or SOS-type, do not effectively compensate for the loss of RasGRP1 in thymocytes. The fact that there is only minimal compensation for loss of RasGRP1 coming from RasGRP3 or RasGRP4 (123, 127) makes one wonder about the underlying mechanism. Is it purely the relative abundance of RasGRP1 that bestows its unique function in thymocytes and would expression of RasGRP3 from the RasGRP 1 promoter be able to compensate for the loss of RasGRP1?…”

RasGRP1 and RasGRP3 Are Required for Efficient Generation of Early Thymic Progenitors

“…Consistent with roles for RasGRP1 and RasGRP3 downstream of CCR9, thymi from RasGRP1/3-deficient mice showed similar reductions in ETP numbers as those observed in CCR9-deficient animals. Because CCR9 is a G protein-coupled receptor and RasGRP family members have been implicated in signaling events downstream of several such receptors of this family (28,42,43), it is reasonable to think that RasGRP1 and RasGRP3 may operate downstream of CCR9. Also, given that RasGRP1 and RasGRP3 regulate Ras activation, it is tempting to speculate that RasGRP1 and RasGRP3 regulate the Ras-ERK signaling cascade after CCR9 stimulation.…”

“…The RasGRP family consists of four RasGEFs, RasGRP1-4. Work from our laboratory and others has shown that RasGRP1-deficient mice exhibit impaired transition through the b-selection checkpoint (28,29). Furthermore, RasGRP1-deficient mice show a dramatic defect in thymocyte-positive selection and invariant NK T cell development (30)(31)(32).…”