Differentiation‐induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line

Abstract

Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D 2L dopamine receptor markedly potentiated both forskolin-and A 2 -adenosine receptor-stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca 2+ to inhibit drug-stimulated cAMP accumulation was enhanced following differentiation, as was D 2L dopamine receptor-mediated inhibition of Ga s -stimulated cAMP accumulation. Differentiation altered basal and drug-stimulated phosphorylation of the cAMP-response element-binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.

Protein Kinase C and Epidermal Growth Factor Stimulation of Raf1 Potentiates Adenylyl Cyclase Type 6 Activation in Intact Cells

“…In their undifferentiated form, CAD cells express robust levels of both AC6 and AC9, and PMA does not potentiate significantly forskolin or G␣ s -stimulated cyclic AMP accumulation in these cells (Johnston et al, 2004; functional data not shown). However, upon removal of serum from the growth medium, the CAD cells differentiate, develop neuronal-like processes, lose AC9 expression almost completely, and retain robust AC6 expression (Johnston et al, 2004). In these differentiated CAD cells, PMA potentiated both forskolin and adenosine 2A receptorstimulated cyclic AMP accumulation (Fig.…”

“…The ability of PMA to modulate drug-stimulated cyclic AMP of endogenous AC6 was initially explored in a novel neuronal cell model, Cath.a differentiated (CAD) cells (Johnston et al, 2002(Johnston et al, , 2004. In their undifferentiated form, CAD cells express robust levels of both AC6 and AC9, and PMA does not potentiate significantly forskolin or G␣ s -stimulated cyclic AMP accumulation in these cells (Johnston et al, 2004; functional data not shown).…”

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…First and foremost, there is a dearth of well-characterized isoform-specific antibodies (Iwamoto et al, 2003;Antoni et al, 2006;Sadana and Dessauer, 2009;Göttle et al, 2009). In some cases, antibodies have been used along with other molecular (mRNA measures) or biochemical (functional assays) approaches revealing partially consistent results Johnston et al, 2004;Liu et al, 2004;Göttle et al, 2009). A second factor concerning quantitative expression studies is the generally low levels of AC protein expression (Alousi et al, 1991;Seifert et al, 1998).…”

“…The majority of studies employed a RT-PCR approach to assess mRNA levels (Varga et al, 1998;Johnston et al, 2004;Liu et al, 2004), but microarray approaches have been used as well (Atwood et al, 2011;Zhu and Oxford, 2011). HEK293 cells are often used for examining both endogenous and exogenous AC signaling.…”

Fluorescent protein complementation assays: new tools to study G protein-coupled receptor oligomerization and GPCR-mediated signaling

“…In a recent study using BRET, it was observed that AC2 and AC5 can heteromerize, and that heteromerization confers unique features to the AC2-AC5 complex that differ from the corresponding homomers (Baragli et al 2008). Further, in a study of both endogenously expressed and overexpressed ACs in a neuronal cell model, it was observed that increased expression of AC9 correlates with the inhibition of AC6 activity, suggesting that AC9 may be a negative regulator of AC6, possibly by heteromer formation (Johnston et al 2004). The finding that AC9 may function as a negative regulator is in agreement with an earlier report (Hacker et al 1998).…”