Go/i-coupled receptor effects through Galphao-GRIN interactions to regulate Sprouty2's inhibition of growth factor signaling

The heterotrimeric G protein Galphao/i communicates signals from many hormones and neurotransmitters. Galphao/i interacts with the protein GRIN (G protein-regulated inducer of neurite outgrowth). The GRIN protein family contains two forms: a large form (GRIN1) and a small form (GRIN2). The yeast two-hybrid system was used to further study the GRIN proteins and to determine their function in signaling pathways by identifying interacting partners. The N-terminal region of human GRIN2 (hGRIN2) was employed as the bait to screen a human brain cDNA library (the prey) to identify direct interacting proteins. Sprouty, a general inhibitor of the receptor tyrosine kinase pathway, was identified as an interactor. Co-immunoprecipitation and GST-pull-down experiments confirmed that hGRIN2 binds to Sprouty and Galpha o. A homologous mouse gene, mGRIN2, and the larger family member, mGRIN1, were cloned and shown to interact with Sprouty and Galphao as well. The consequences of the GRIN-Sprouty interactions in Galphao -signaling were characterized.; Mouse GRIN2 truncation co-immunoprecipitation experiments showed that the region where Sprouty and where Galphao bind to on mGRIN2 are one and the same. In addition, competitive co-immunoprecipitation experiments revealed that Galphao and Sprouty compete for GRIN2 binding. Imaging experiments demonstrated that a higher fraction of mouse GRIN2 proteins localized to the plasma membrane upon expression of GTP-Galphao but not with Sprouty. This suggests that GRIN proteins translocate to the plasma membrane upon active Galphao signaling. Meanwhile, Sprouty truncation experiments showed that Sprouty associated with GRIN through the conserved C-terminal cysteine-rich domain of Sprouty. I hypothesized that GRIN proteins and perhaps Galphao signaling can modulate Sprouty effects on MAP-Kinase signaling. Overexpression of mGRIN2 potentiated FGF activation of MAPK in Neuro2A cells and decreased tyrosine phosphorylation of Sprouty by FGF stimulation. Tyrosine phosphorylation of Sprouty has been shown to be important for its inhibitory activity and to regulate its degradation.; Using Neuro2A cells as an endogenous system to study the effects of the Galphao-GRIN-Sprouty relationship on MAPK activation, I demonstrated that pretreatment with Gi/o-coupled CB1 receptor agonists attenuated FGF activation of MAPK. Treatment with mGRIN1 siRNA both dampened FGF activation of MAPK in Neuro2A cells and blocked the effect of Gi/Go-coupled receptor attenuation of FGF activation of MAPK. A model is proposed to explain how Galphaobinding to GRIN releases Sprouty to attenuate growth factor stimulation of MAPK. This is a potential novel mechanism by which heterotrimeric G proteins affect receptor tyrosine kinase signaling.