Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Gα_q family link activation of G protein-coupled receptors (GPCRs) to the stimulation of phospholipase Cβ (PLCβ) 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 Gα_q family members are independent of PLCβ activation. Novel binding partners of Gα_q subunits have also been reported. However, little is known about proteins that interact with other members of the Gα_q family, such as the hematopoietic specific Gα₁₆. The research aim of this study is to identify Gα₁₆-interacting proteins and the roles of protein complexes in Gα...[ Read more ]

Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Gα_q family link activation of G protein-coupled receptors (GPCRs) to the stimulation of phospholipase Cβ (PLCβ) 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 Gα_q family members are independent of PLCβ activation. Novel binding partners of Gα_q subunits have also been reported. However, little is known about proteins that interact with other members of the Gα_q family, such as the hematopoietic specific Gα₁₆. The research aim of this study is to identify Gα₁₆-interacting proteins and the roles of protein complexes in Gα₁₆-mediated signaling. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Gα_q/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 Gα₁₆ (Gα₁₆QL). Interestingly, overexpression of p63RhoGEF inhibited Gα₁₆QL-induced IP₃ production in a concentration-dependent manner. The binding of PLCβ₂ to Gα₁₆QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Gα₁₆QL, leading to a suppression of Gα₁₆QL-induced Ras activation. In the presence of p63RhoGEF, Gα₁₆QL-induced STAT3 phosphorylation was significantly reduced and Gα₁₆QL-mediated SRE transcriptional activation was attenuated. In addition, it was found that multiple regions of Gα₁₆ are responsible for binding with p63RhoGEF. Taken together, these results suggest that p63RhoGEF binds to activated Gα₁₆ and inhibits its signaling pathways. Apart from p63RhoGEF, our co-immunoprecipitation assay also suggested that Gα₁₆ could interact with class IA PI3Ks but not class IB PI3K. In contrast to the observation in Gα₁₆/p63RhoGEF complex; both Gα₁₆ and Gα₁₆QL can effectively form signaling complexes with class IA PI3Ks. Differential characteristics between Gα₁₆ and Gα_q were observed in terms of the selectivity of PI3K isoforms. Gα₁₆ bound to both p110α and p110β but not p110δ. Signaling complexes of Gα₁₆/p110α could be found in hematopoietic cells which endogenously express Gα₁₆. It was also found that overexpression of class IA PI3Ks did not affect Gα₁₆QL-mediated PLC activation. In addition, the immunoprecipitation assay showed that overexpression of p85/p110α or p85/p110β did not interfere with the binding with Gα₁₆ and PLCβ₂. Furthermore, Gα₁₆QL attenuated PI3K-induced Akt phosphorylation and PIP₃ level. Collectively, the present study provides evidence that Gα₁₆ interacts with signaling molecules other than PLCβ and modulates different signaling pathways.