Reply to Tall et al.: Dictyostelium Ric8 does not have a chaperoning function during development and chemotaxis

In our recent paper, we conclude that Dictyostelium Ric8 is as a nonreceptor guanine nucleotide exchange factor (GEF) for the Gα protein important for amplification of G protein–coupled receptor (GPCR) signaling (1). In their letter, Tall et al. dispute this conclusion and suggest that a function of Dictyostelium Ric8 in folding of the Gα protein might better explain the phenotype than its function as GEF (2). However, we show that WT and ric8-null cells have similar steady-state expression levels of Gα. To test the functionality of the Gα2 protein, we analyzed the interaction between Gα2 and the cAR1 receptor. In vitro, this was measured as the inhibition of cAMP binding induced by full activation of Gα2 with nonhydrolysable GTPγS. The reduction in cAMP binding in ric8 null is similar to that in WT, indicating that a similar amount of Gα is functionally coupled to the receptor. Second, dissociation of Gα2βγ was monitored in vivo by measuring the cAMP-induced FRET change between the FRET pair Gα2-Cerulean and Gβ-Venus. The FRET signal before stimulation is similar in ric8-null cells compared with WT cells, indicating the presence of similar amounts of Gα2βγ complex. Furthermore, we show that Ric8 is not essential for the initial dissociation of heterotrimeric G proteins and subsequent activation of Ras by uniform chemoattractant. Consistent with its function as GEF, recombinant Ric8 stimulates nucleotide exchange of Gα2 in vitro, and the in vivo FRET assays showed that prolonged dissociation of Gα2βγ on uniform cAMP stimulation requires Ric8. Together, we conclude in the paper that these results provide no evidence that Ric8 could play a role as chaperone for Gα2 folding but clearly show that Ric8 functions as a GEF and amplifier of Gα2 signaling.

Recent studies have shown that mammalian Ric8-A may have different effects on individual Gα subunits; it acts as a a GEF for Gαq, Gαi1, and Gαo, whereas in the case of Gαi1, a chaperoning function has been proposed (3, 4). In our research, we mainly focused on the role of Ric8 in regulating chemotaxis and development. Of the 12 identified Gα subunits in Dictyostelium, Gα2 is coupled to the cAR1 receptor and is essential for cAMP-mediated development and chemotaxis, whereas Gα4 is essential for mediating chemotaxis toward folate. In addition to Gα2 and Gα4, Dictyostelium Ric8 also binds to Gα1, Gα7, Gα9, and Gα12. Further research is necessary to determine the function of Ric8 for these Gα subunits: GEFs and signal amplifiers as for Gα2 or a chaperoning function to assist folding of these Gα subunits.