Soluble Adenylyl Cyclase Mediates Nerve Growth Factor-induced Activation of Rap1

Abstract

Nerve growth factor (NGF) and the ubiquitous second messenger cyclic AMP (cAMP) are both implicated in neuronal differentiation. Multiple studies indicate that NGF signals to at least a subset of its targets via cAMP, but the link between NGF and cAMP has remained elusive. Here, we have described the use of small molecule inhibitors to differentiate between the two known sources of cAMP in mammalian cells, bicarbonate-and calcium-responsive soluble adenylyl cyclase (sAC) and G protein-regulated transmembrane adenylyl cyclases. These inhibitors, along with sAC-specific small interfering RNA, reveal that sAC is uniquely responsible for the NGF-elicited rise in cAMP and is essential for the NGF-induced activation of the small G protein Rap1 in PC12 cells. In contrast and as expected, transmembrane adenylyl cyclase-generated cAMP is responsible for Rap1 activation by the G protein-coupled receptor ligand PACAP (pituitary adenylyl cyclase-activating peptide). These results identify sAC as a mediator of NGF signaling and reveal the existence of distinct pathways leading to cAMP-dependent signal transduction.

“Soluble” adenylyl cyclase‐generated cyclic adenosine monophosphate promotes fast migration in PC12 cells

“…Interestingly, it has been suggested that Trk and p75 NTR may act either as subunits of a single receptor complex or as independent transducers of distinct neurotrophin functions (Chao, 1994). Although it is generally thought that the NGF-induced activation of Rap1, which we have shown to be mediated via sAC (Stessin et al, 2006), involves TrkA, rather than p75 NTR , we have also found sAC to mediate the activation of Rap1 by TNF in neutrophils (Han et al, 2005). Thus, the roles of TrkA and p75 NTR in the activation of sAC by NGF are a subject for future studies.…”

“…Application of a reagent such as the tmAC-specific activator forskolin yields very large changes in whole-cell cAMP, which may have little physiological relevance to a particular biological process. In contrast, the microdomain model of cAMP action dictates that subtle changes in whole-cell cAMP, as can be observed when using bicarbonate to stimulate endogenous sAC activity in an in vitro assay or during NGF treatment of PC12 cells (Stessin et al, 2006), can have profound physiological consequences. In the future, great care should be exercised when using pharmacological agents that alter cAMP.…”

“…Specifically, it is known that PMA exerts its effects via the activation of protein kinase C (PKC), which was independently shown to be essential for cell motility in primary (Choe et al, 2003). NGF has been shown to promote the survival and differentiation of multiple neuronal populations within the central nervous system (LeviMontalcini and Angeletti, 1968), but the signaling mechanisms involved in NGF-induced neuritogenesis remain controversial (Vaudry et al, 2002).We recently identified "soluble" adenylyl cyclase (sAC) as the source of cAMP downstream of NGF and a mediator of NGF-induced activation of the monomeric GTPase Rap1 in PC12 cells (Stessin et al, 2006). sAC is a ubiquitously expressed cAMP source in mammalian cells, distinct from the classically described trans-membrane adenylyl cyclases (tmACs) in its subcellular localization and biochemical profile.…”

“…We now show that cAMP pools, generated by different sources, do not possess the same function. Both PMA and NGF elicit cAMP production; NGF activates sAC (Stessin et al, 2006), whereas PMA stimulation of PKC activates tmACs. However, the two pools of cAMP have very different consequences.…”

“…It will be interesting to utilize modern biophysical methods for localizing cAMP generation in vivo to determine the subcellular distribution of these distinct pools. For example, it seems likely the sAC-generated pool of cAMP will be situated in a position to modulate relevant NGF targets, such as Rap1 (Stessin et al, 2006).As revealed in Figure 2, whole cell-based cAMP measurements can be misleading with regard to cellular processes controlled by cAMP. Application of a reagent such as the tmAC-specific activator forskolin yields very large changes in whole-cell cAMP, which may have little physiological relevance to a particular biological process.…”