Abstract
The Ras-guanyl nucleotide exchange factor RasGRP1 plays a critical role in T cell receptor-mediated Erk activation. Previous studies have emphasized the importance of RasGRP1 in the positive selection of thymocytes, activation of T cells, and control of autoimmunity. RasGRP1 consists of a number of well-characterized domains, which it shares with its other family members; however, RasGRP1 also contains an ∼200 residue-long tail domain, the function of which is unknown. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain. Further analysis of these knock-in mice showed that thymocytes lacking the tail domain of RasGRP1 underwent aberrant thymic selection and, following TCR stimulation, were unable to activate Erk. Furthermore, the deletion of the tail domain led to enhanced CD4+ T cell expansion in aged mice, as well as the production of autoantibodies. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, we have revealed the physiological relevance of the tail domain in RasGRP1 function and control of Erk signaling.
Highlights
The Ras family proteins are critical components of many signaling pathways and serve to regulate a variety of cell functions, such as proliferation and differentiation
Generation of RasGRP1d/d mice To assess the function of the RasGRP1 tail domain, we designed a targeting construct to generate knock-in mice with the tail domain deleted, which we designated as RasGRP1d/d (d = deleted) mice
Rasgrp1 consists of seventeen exons; we inserted a stop codon, TGA, into exon 15 to ensure retention of the C1 domain but deletion of the tail
Summary
The Ras family proteins are critical components of many signaling pathways and serve to regulate a variety of cell functions, such as proliferation and differentiation. The most well-characterized signaling pathway regulated by Ras is the Erk (extracellular signal-regulated kinases)-MAPK (mitogen activated protein kinase) pathway. In T cells, Ras is activated by two families of Ras GEFs (guanine exchange factors), RasGRP (Ras guanyl releasing protein) and Sos (son of sevenless). Studies analyzing mice deficient in either RasGRP1 or Sos indicate that both GEFs are critical in thymocyte development [2,3]. These proteins may have disparate roles in the different stages of thymocyte development as the ratio of Sos to RasGRP1 expression changes dramatically upon the DN3 to DP transition [3]
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