Sustained GRK2-dependent CREB activation is essential for α2-adrenergic receptor-induced PC12 neuronal differentiation

Many aspects of neuronal development, such as neuronal survival and differentiation, are regulated by the transcription factor CAMP-response element-binding protein (CREB). We have previously reported that alpha(2)-adrenergic receptors (ARs), members of the G protein-coupled receptor (GPCR) superfamily, induce neuronal differentiation of rat pheochromocytoma (PC)-12 cells in a subtype-specific manner, i.e. alpha(2A) < alpha(2B) < alpha(2C). Herein, we sought to investigate CREB's involvement in this alpha(2)AR-dependent neurogenic process. We used a combination of gene reporter assays and immunoblotting analysis, coupled with co-immunoprecipitation and morphological analysis, in transfected PC12 cell lines. Chronic alpha(2B)- or alpha(2C)-AR activation results in sustained CREB activation, which is both necessary and sufficient for neuronal differentiation of PC12 cells expressing these two alpha(2)ARs. In contrast, chronic alpha(2A) activation only leads to transient CREB activation, insufficient for PC12 neuronal differentiation. However, upon CREB overexpression, alpha(2A)-AR triggers neuronal differentiation similarly to alpha(2B)- or alpha(2C)-ARs. Importantly, NGF (Nerve Growth Factor)'s TrkA receptor transactivation is essential for the sustained activation of CREB by all three alpha(2) subtypes in PC12 cells, whereas protein kinase A (PKA), the prototypic kinase that phosphorylates CREB, is not. Instead, TrkA-activated GPCR-kinase (GRK)-2 mediates the sustained CREB activation during alpha(2)AR-induced neuronal differentiation of PC12 cells. In conclusion, catecholaminergic-induced neuronal differentiation of PC12 cells through alpha(2)ARs uses a non-canonical pathway involving TrkA transactivation and subsequent GRK2-dependent, sustained phosphorylation/activation of CREB. These findings provide novel insights into catecholaminergic neurogenesis and suggest that alpha(2)AR agonists, combined with NGF analogs or GRK2 stimulators, may exert neurogenic/neuroprotective effects.