INTERACTION BETWEEN THE ORAI1-BASED CA2+ ENTRY MECHANISM AND THE RAS GTPASE SYSTEM AS SEEN BY FRET BETWEEN ORAI1 AND H-RAS AND MODULATION OF STORE-OPERATED CA2+ ENTRY (SOCE)

SEBASTIÁN SUSPERREGUY; KARINA FORMOSO; MARIA DE LA PAZ SARASOLA; LUTZ BIRNBAUMER

Congreso; LXIII Reunion Anual de la Sociedad Argentina de Investigación Clínica; 2018

Sociedad Argentina de Investigación Clínica

In test experiments, performed as part of training course in analysis of fluorescence resonance energy transfer (FRET), we co expressed Orai1-YFP and human CFP-HRas) in HEK293 cells hoping to get no FRET signal between them. Unexpectedly we discovered direct interaction between these two proteins at the level of the cellular plasma membrane.  This finding suggested that the Ras signaling cascade(s) and SOCE interacted, as Orai1 I the canonical SOCE channel. We decide to test for this interaction at the functional level and we quantified Ca2+ entry using the Fura2 method, which reports changes in cytosolic Ca2+ in response to release of Ca2+ from the endoplasmic reticulum (ER), activating STIM1 at RE and finally the assembly of the Orai1-based Ca entry channel (CRAC) allowing Ca to enter from the extracellular space.  We performed two types of experiments: In one we tested the effect of HRas expression on thapsigargin (Tg)-evoked store depletion and Ca2+ entry and on EGF-EGFR evoked SOCE and in the other we suppressed Tg-evoked SOCE by overexpression of Orai1 and asked whether expression of HRas would relieve this inhibition). HRas expression tested positive in both types of test: It inhibited Tg- and EGF-EGFR-evoked SOCE, and it suppressed inhibition of Tg-evoked SOCE by Orai1, the degree of suppression being dependent on relative expression levels of the transfected proteins, never reaching more than 60-70%.  We conclude that the Ras signaling and the Orai1-mediated Ca2+ entry mechanisms interact functionally, and propose that one or more steps of Ras activated signaling cascades are modulated by cytosolic Ca2+ changes.  We are currently testing for possible differences among H-Ras, K-Ras and N-Ras, and also for the impact that the activation state of Ras using dominant positive (G12V, Q61L) and dominant negative (S17N) mutants) may have in the above described assays.