The Identification and Characterization of G-alpha16-Interacting Proteins

Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Galpha q family link activation of G protein-coupled receptors (GPCRs) to the stimulation of phospholipase Cbeta (PLCbeta) and calcium mobilization. However, they differ markedly in biochemical properties as well as tissue distribution. Recent findings also showed that some of the cellular activities of Galphaq family members are independent of PLCbeta activation. Novel binding partners of Galphaq subunits have also been reported. However, little is known about proteins that interact with other members of the Galphaq family, such as the hematopoietic specific Galpha 16. The research aim of this study is to identify Galpha16-interacting proteins and the roles of protein complexes in Galpha16-mediated signaling. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Galphaq/11 proteins and thus provides linkage to RhoA activation. In the present study, we employed co-immunoprecipitation studies in HEK293 cells to demonstrate that p63RhoGEF can form a stable complex with the constitutively active mutant of Galpha16 (Galpha 16QL). Interestingly, overexpression of p63RhoGEF inhibited Galpha 16QL-induced IP3 production in a concentration-dependent manner. The binding of PLCbeta2 to Galpha16QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Galpha16QL, leading to a suppression of Galpha 16QL-induced Ras activation. In the presence of p63RhoGEF, Galpha 16QL-induced STAT3 phosphorylation was significantly reduced and Galpha 16QL-mediated SRE transcriptional activation was attenuated. In addition, it was found that multiple regions of Galpha16 are responsible for binding with p63RhoGEF. Taken together, these results suggest that p63RhoGEF binds to activated Galpha16 and inhibits its signaling pathways. Apart from p63RhoGEF, our co-immunoprecipitation assay also suggested that Galpha16 could interact with class IA PI3Ks but not class IB PI3K. In contrast to the observation in Galpha16/p63RhoGEF complex; both Galpha16 and Galpha16QL can effectively form signaling complexes with class IA PI3Ks. Differential characteristics between Galpha16 and Galphaq were observed in terms of the selectivity of PI3K isoforms. Galpha16 bound to both p110alpha and p110beta but not p110delta. Signaling complexes of Galpha16 /p110alpha could be found in hematopoietic cells which endogenously express Galpha16. It was also found that overexpression of class IA PI3Ks did not affect Galpha16QL-mediated PLC activation. In addition, the immunoprecipitation assay showed that overexpression of p85/p110I+/- or p85/p110beta did not interfere with the binding with Galpha16 and PLCbeta2. Furthermore, Galpha16QL attenuated PI3K-induced Akt phosphorylation and PIP3 level. Collectively, the present study provides evidence that Galpha16 interacts with signaling molecules other than PLCbeta and modulates different signaling pathways.