The Stargazin-Related Protein γ7 Interacts with the mRNA-Binding Protein Heterogeneous Nuclear Ribonucleoprotein A2 and Regulates the Stability of Specific mRNAs, Including Ca_V2.2

Abstract

The role(s) of the novel stargazin-like ␥-subunit proteins remain controversial. We have shown previously that the neuron-specific ␥7 suppresses the expression of certain calcium channels, particularly Ca V 2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of ␥7 on Ca V 2.2 expression is via an increase in the degradation rate of Ca V 2.2 mRNA and hence a reduction of Ca V 2.2 protein level. Furthermore, exogenous expression of ␥7 in PC12 cells also decreased the endogenous Ca V 2.2 mRNA level. Conversely, knockdown of endogenous ␥7 with short-hairpin RNAs produced a reciprocal enhancement of Ca V 2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed ␥7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of ␥7 is essential for all its effects, and we show that ␥7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in Ca V 2.2 mRNA, and is associated with native Ca V 2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances Ca V 2.2 I Ba , and this enhancement is prevented by a concentration of ␥7 that alone has no effect on I Ba . The effect of ␥7 is selective for certain mRNAs because it had no effect on ␣2␦-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in Ca V 2.2 mRNA. Our results indicate that ␥7 plays a role in stabilizing Ca V 2.2 mRNA.

Ribonucleoprotein HNRNPA2B1 Interacts With and Regulates Oncogenic KRAS in Pancreatic Ductal Adenocarcinoma Cells

“…44,45 Although a membrane-bound fraction has also been previously identified 44,46 the interaction with RAS proteins has not been previously described. Using cell fractionation and Western blotting, we found a substantial amount of HNRNPA2B1 protein in the membrane fraction (P100) (Fig.…”

Stargazin-related protein γ7 is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth

“…As before, we used a mixture of three shRNAs specifically complementary to mRNA encoding rat  7 , and used Drosophila gnu shRNA as a control (Ferron et al, 2008). We found that transfection of rat  7 shRNAs markedly decreased the length of PC12 neurites, compared with the control shRNA, when measured 9 days after transfection and 3 days after the start of differentiation with NGF (Fig.…”

“…Our conclusion was that  7 was not a subunit of these Ca 2+ channels. In a subsequent study, we showed that  7 interacts with the RNA binding protein hnRNP A2 and increases the rate of degradation of certain mRNAs, including that of Ca V 2.2 (Ferron et al, 2008). We concluded that by sequestering hnRNP A2,  7 reduces the binding of this RNA binding protein to specific mRNAs, therefore reducing their stability.…”

“…Furthermore, in our previous study we showed that it was possible to knock down endogenous  7 in cultured neurons with shRNA (Ferron et al, 2008). Therefore, to examine whether endogenous  7 has a role in neurite outgrowth and morphology in sympathetic neurons, we transfected SCGs with a mixture of three  7 shRNAs (Ferron et al, 2008), and found that this resulted in a reduction in levels of endogenous  7 (Fig.…”

“…To determine whether endogenous  7 , which we had previously shown to have a punctuate distribution (Ferron et al, 2008), was also colocalised with endosomal markers, we examined its colocalisation with EEA1 in SCG neurons (Fig. 7).…”