RasGRP in membranes of activated T cells.

(A) In a time-course experiment, Jurkat T cells were stimulated with OKT3 for various times and aliquots were assayed for Ras-GTP and total Ras using immunoblotting methods. (B) Homogenates of Jurkat T cells from the various time points were separated into particulate (P) and soluble (S) fractions and RasGRP was immunoblotted with the H176 anti-RasGRP peptide antibody. (C) Membranes from untreated cells, or from cells treated for 10 minutes with OKT3, were assayed for their ability to promote transfer of exogenous guanyl nucleotide to membrane-bound Ras and the sensitivity of this reaction to antibodies raised against the catalytic domain of RasGRP was determined. Membranes were preincubated with IgG prepared from immune (I) or preimmune (pre-I) serum for 12 minutes at 30°C, then [α³²P]GTP was added for 1 minute. Ras was then extracted with detergent and precipitated with an anti-Ras monoclonal antibody. Coprecipitated, Ras-associated guanyl nucleotides were resolved by chromatography and quantified. Representative chromatographic data are shown. The graphed values are the average of triplicate assays. Values were normalized to the value obtained when full association was achieved by incubation with EDTA, followed by addition of excess MgCl₂ to stabilize the Ras-guanyl nucleotide complex before immune-precipitation. (D) In vitro Ras guanyl nucleotide exchange reactions were performed with recombinant Sos and RasGRP in the presence of IgG to demonstrate the specificity of the J32 antibodies.