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Characterizing the regulation of RasGRPI and CalDag1 : two C1 domain-containing ras guanine nucleotide exchange factors Anthony, Kira


RasGRP1 and CalDAG1 are two guanine nucleotide exchange factors (GEFs) for the Ras family of GTPases that share a high degree of sequence identity. However, RasGRP1 is a GEF for the classical Ras GTPases and CALDAG1 activates Rap1. These effector specificity differences suggest that there may be specific differences in the regulation of each of these GEFs. Activation of Ras occurs via recruitment of its GEFs to membranes, where the C1 domains of RasGRP1 and CalDAG1 are the most likely candidates to mediate this recruitment. The C1 domains of these GEFs are homologous to those of PKCs and may be classified into typical or atypical depending on whether or not they can bind diacylglycerol (DAG) and highly potent DAG analogues such as phorbol esters. Within both NIH 3T3 cells and T28 cells, it was determined that the C1 domain of RasGRP1 mediated its translocation to membranes in response to DAG and phorbol esters. Conversely, the isolated CalDAG1 C1 was not recruited to the plasma membrane in response to either phorbol ester stimulation in NIH 3T3 cells or T cell receptor stimulation in T28 cells. This indicates that the CalDAG1 C1 domain may not be a direct target of DAG in NIH 3T3 cells and that the RasGRPI and CalDAG1 C1 domains are biochemically distinct. Nonetheless, both RasGRP1 and CalDAG1 demonstrated an equivalent C1 domain-dependent plasma membrane localization pattern upon stimulation of the T cell receptor with the natural ligand anti- CD3[sub ε] in T28 cells. Unlike CalDAG1, RasGRP1 contains an alpha (a) helix motif that is predicted to dimerize to form a leucine zipper. In order to test whether or not RasGRP1 homodimerizes at the a helix both co-immunoprecipitation and a modified colocalization assay were employed. Taken together, the results from each of these two approaches indicate that RasGRP1 homodimerizes. Furthermore, although the results are compatible with the mediation of this protein-protein interaction by the RasGRP1 α helix, they do not unambiguously prove this hypothesis.

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