Ric-8: un nuevo GEF con estructura tipo "armadillo"

RIC-8 is a highly conserved cytosolic protein (63 KDa) initially identified in C. elegans as
an essential factor in neurotransmitter release and asymmetric cell division. Two different
isoforms have been described in mammals, RIC-8A and RIC-8B; each possess guanine
nucleotide exchange activity (GEF) on heterotrimeric G-proteins, but with different Gα subunits
specificities. To gain insight on the mechanisms involved in RIC-8 cellular functions it is
essential to obtain some information about its structure. Therefore, the aim of this thesis was to
study the relationship between structure and function on RIC-8, using as model RIC-8 from X.
laevis. Analysis in its primary structure did not give us information about the function of xRIC-8
and RIC-8 proteins were showed as a single family without similarity with others. For this
reason, to obtain a 3D model of xRIC-8, different bioinformatics approaches that include protein
folding and structure prediction were used. The RIC-8 structural model is composed of 10
armadillo folding motifs, organized in a right-twisted alpha-alpha super helix. In order to validate
the structural model, a His-tag fusion construct of RIC-8 was expressed in E. coli, purified by
affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD)
and thermostability studies. This model together with the comparison among RIC-8 proteins that
shows a high conservation in the carboxy region, deletion mutants that remove the last three
armadillo domain were created in order to search a loss of the GEF function. The mutants could
not be expressed in bacteria for in vitro assays, but their expression in HEK293T culture showed
that all of them preserved the GEF activity. Furthermore, confocal microscopy showed that all
the mutants could translocate to plasmatic membrane under stimulation with isoproterenol, and
have the capacity to interact with Gs. The results of this thesis has allowed present the first 3D
model for a RIC-8 protein, as well as classify RIC-8 as a new member of the protein family with armadillo repeats. Functionally, the carboxi terminal region, the most conserved among RIC-8,
did not show has the GEF activity, and did not alter its behavior in HEK293T cell cultures while
was stimulated with isoproterenol.