Mesenchymal stem cells are able to trans-di¡erentiate into nonmesodermal lineage cells. Here, we identi¢ed downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels.

Although abundant Go has been found in nervous tissues and it has been implicated in neuronal differentiation, the mechanism of how Go modulates neuronal differentiation has not been defined. Here, we report that the ␣ subunit of Go (␣o) modulates neurite outgrowth by interfering with the signaling pathway initiated by cyclic AMP (cAMP). In F11 cells, cAMP induced neurite outgrowth and activated cAMP-responsive element binding protein (CREB). Specific inhibition of cAMPdependent protein kinase reduced both CREB activity and neurite outgrowth (NOG). Interestingly, cAMP reduced phosphorylation of extracellular signal-regulated kinase (Erk). Neither a dominant negative form nor an active form of Ras altered neurite outgrowth. Expression of ␣o (␣o wt ) decreased the average length of neurites but increased the number of neurites per cell. An active mutant, ␣o Q205L , which lost GTPase activity and thus could not bind to G␤␥, gave similar results, suggesting that the effect of ␣o is not mediated through G␤␥. Expression of ␣o wt or ␣o Q205L also prohibited CREB activation. Thus, activation of Erk may not be essential for neuronal differentiation in F11 cells and ␣o may cause changes in NOG by inhibiting CREB activation.

Go, a member of the Go/i family, is the most abundant heterotrimeric G protein in brain. Most functions of G o are mediated by the G␤␥ dimer; effector(s) for its ␣-subunit have not been clearly defined. Here we report that G o␣ interacts directly with cAMP-dependent protein kinase (PKA) through its GTPase domain. This interaction did not inhibit the kinase function of PKA but interfered with nuclear translocation of PKA while sparing its cytosolic function. This regulatory mechanism by which G o bifurcates PKA signaling may provide insights into how G o regulates complex processes such as neuritogenesis, synaptic plasticity, and cell transformation.o is the most abundant heterotrimeric G proteins expressed in the brain (1) and is classified as a member of the G i /G o family. G i and G o proteins are activated by a common set of receptors that include ␣2 adrenergic, D2 dopamine, opioid, 5HT1, somatostatin (SST), and the muscarinic M2 and M4 receptors (2). To date, unlike G i , which inhibits adenylyl cyclase, most functions of G o can be interpreted through the actions of a common pool of G ␤␥ dimers, and specific functions of G o␣ have yet to be defined. Several indirect lines of evidence suggest that G o␣ does function independent of G ␤␥ . The most compelling of these are that constitutively active G o␣ promotes oncogenic transformation of NIH 3T3 cells (3) and that overexpression of G o␣ is sufficient to promote neuritogenesis in neuroblastoma cell lines including PC12 (4), N1E-115 (4), Neuro2A (5), and, as we reported earlier, F11 cells (6). In this latter study we had found that both the wild-type G o␣ and the Q205L mutant, which cannot interact with G ␤␥ , promote an increase in the number of cAMP-induced neurites at the expense of neurite extension. We had also found that this effect of G o␣ is accompanied by a concomitant decrease in cAMP response element binding protein (CREB)-mediated gene expression, suggesting a cross-talk between G o and cAMP-dependent PKA.Functions of PKA isoforms are directly regulated by intracellular concentration of cAMP and expression of A kinase anchoring proteins (AKAPs). cAMP binds the regulatory (R) subunits and causes the release of catalytic (C) subunits (7). AKAPs interact with RII isoforms and direct the compartmentalization of PKA signaling (8). For example, PKAI isoforms with the RI regulatory subunits are soluble and widely expressed, whereas most PKAII isoforms with the RII subunits are associated with the particulate fractions of homogenates through interaction with various AKAPs (8). The predominant PKA isoform and principal mediator of cAMP action in the mammalian central nervous system is the RII␤-containing PKAII␤ (9).In the present study we report that G o␣ has a previously unappreciated scaffolding role in the cytosolic compartment that prevents translocation of PKA into the nuclear compartment. ResultsTo determine the interaction of G o and PKA we incubated rat brain microsomal proteins with GST-G o␣ fusion proteins and probed with antibodies against the p...

BETA2/NeuroD, a basic helix-loop-helix (bHLH) transcription factor, has been shown to play important roles in the development of the nervous system and the maintenance and formation of pancreatic and enteroendocrine cells. The gain of function of BETA2/NeuroD in neurogenesis has been shown in Xenopus embryos. In this study, we investigated the neurogenic potential of BETA2/NeuroD using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To induce or block the expression of BETA2/NeuroD, expression vectors for the full-length and a C-terminal deletion mutant of BETA2 were constructed and their transactivation potential was verified using reporter genes containing the insulin promoter sequences. Overexpression of BETA2 with full-length construct induced neurite outgrowth in F11 cells in the absence of cAMP. In contrast, the C-terminal deletion mutant, BETA2(1--233), which has dominant negative activity, inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that BETA2/NeuroD plays an important role in terminal differentiation of neuroblastoma cells. They also imply that BETA2/NeuroD or related bHLH factors plays an essential role for differentiation of F11 neuroblastoma cells.

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.