Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goa as a mediator of opioid receptor-induced extracellular signalregulated kinase activation in neural cells. The current study found that, in neuroblastoma · glioma NG108-15 hybrid cells, activation of extracellular signal-regulated kinase through delta opioid receptors was mediated by pertussis toxin-sensitive G protein and independent of Gbc subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goa 1 , but not Gia 2 , completely blocked delta opioid receptor-induced extracellular signal-regulated kinase activity. Decreasing Goa expression by RNA interference greatly reduced delta opioid receptor-induced extracellular signal-regulated kinase activity and extracellular signal-regulated kinase-dependent gene expression, while knocking down Gia 2 did not. By taking advantage of differences between human and mouse Goa gene sequences, we simultaneously knocked down endogenous Goa expression and expressed exogenous human Goa subunits. We found that both human Goa 1 and Goa 2 could mediate delta opioid receptor-induced extracellular signal-regulated kinase activation. This study suggests that one of the functions of Goa in the brain is to mediate extracellular signal-regulated kinase activation by G protein-coupled receptors.

Kif5b-driven anterograde transport and clathrin-mediated endocytosis (CME) are responsible for opposite intracellular trafficking, contributing to plasma membrane homeostasis. However, whether and how the two trafficking processes coordinate remain unclear. Here, we show that Kif5b directly interacts with clathrin heavy chain (CHC) at a region close to that for uncoating catalyst (Hsc70) and preferentially localizes on relatively large clathrin-coated vesicles (CCVs). Uncoating in vitro is decreased for CCVs from the cortex of kif5b conditional knockout (mutant) mouse and facilitated by adding Kif5b fragments containing CHC-binding site, while cell peripheral distribution of CHC or Hsc70 keeps unaffected by Kif5b depletion. Furthermore, cellular entry of vesicular stomatitis virus that internalizes into large CCV is inhibited by Kif5b depletion or introducing a dominant-negative Kif5b fragment. These findings showed a new role of Kif5b in regulating large CCV-mediated CME via affecting CCV uncoating, indicating Kif5b as a molecular knot connecting anterograde transport to CME.