- Identification of Gαi2-interacting proteins via affinity pull-down and quantitative mass-spectrometry analysis.

ABSTRACT: In studying patients with activating GNAI2 mutations, we observed T cell hyperresponsiveness that was independent of cAMP suppression. Therefore, we hypothesized that Gαi2 utilizes alternative signal transduction pathways which might be delineated by identifying its interacting proteins in T cells. To accomplish this, we performed affinity pulldown followed by quantitative mass-spectrometry analysis. Using this approach, we identified both well-known and previously reported interactors of Gαi2 as well as 128 previously unreported interactors including RASA2. We validated RASA2, a GTPase-activating protein for RAS, and PP2A-A/C as Gαi2 effector targets by several methods: overexpression-coimmunoprecipitations using 293T cells, endogenous coimmunoprecipitations from patient T cells, and by mixing-pulldown experiments using purified recombinant proteins. Additional imaging experiments confirmed Gαi2-RASA2 interactions within living cells and delineated that activating mutations of Gαi2 sequester RASA2 toward the plasma membrane to promote RAS activation for T cell growth and proliferation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): T Cell, Blood

SUBMITTER: David Anderson  

LAB HEAD: Helen C. Su

PROVIDER: PXD048980 | Pride | 2024-09-20

REPOSITORIES: Pride