Corticotropin releasing factor-induced CREB activation in striatal neurons occurs via a novel Gβγ signaling pathway

Abstract

The peptide corticotropin-releasing factor (CRF) was initially identified as a critical component of the stress response. CRF exerts its cellular effects by binding to one of two cognate G-protein coupled receptors (GPCRs), CRF receptor 1 (CRFR1) or 2 (CRFR2). While these GPCRs were originally characterized as being coupled to Gα(s), leading to downstream activation of adenylyl cyclase (AC) and subsequent increases in cAMP, it has since become clear that CRFRs couple to and activate numerous other downstream signaling cascades. In addition, CRF signaling influences the activity of many diverse brain regions, affecting a variety of behaviors. One of these regions is the striatum, including the nucleus accumbens (NAc). CRF exerts profound effects on striatal-dependent behaviors such as drug addiction, pair-bonding, and natural reward. Recent data indicate that at least some of these behaviors regulated by CRF are mediated through CRF activation of the transcription factor CREB. Thus, we aimed to elucidate the signaling pathway by which CRF activates CREB in striatal neurons. Here we describe a novel neuronal signaling pathway whereby CRF leads to a rapid Gβγ- and MEK-dependent increase in CREB phosphorylation. These data are the first descriptions of CRF leading to activation of a Gβγ-dependent signaling pathway in neurons, as well as the first description of Gβγ activation leading to downstream CREB phosphorylation in any cellular system. Additionally, these data provide additional insight into the mechanisms by which CRF can regulate neuronal function.

Figure 1. CRF rapidly stimulates CREB phosphorylation.

(A) Immunolabeled confocal images of cultured striatal neurons from 1- to 2-day old rat pups with MAP2 (red) and pCREB (green). Neurons stimulated with CRF (40 nM) for 15 min exhibited increased CREB phosphorylation (Scale Bar  =  20 µm). (B) Quantification of immunostaining, revealing CRF-mediated CREB phosphorylation (p<0.0001). (C) CRF induced a rightward shift in the plot of pCREB fluorescence intensity of approximately 80% of striatal neurons (D) CRF increased CREB phosphorylation in a concentration-dependent manner, with EC₅₀  =  0.3 nM. Concentrations ≥ 4 nM induced a signal that differed with statistical significance from vehicle-stimulated (NS) neurons. (E) Time course of 40 nM CRF-induced CREB phosphorylation, with τ  =  3.5 min. Statistically different groups are denoted by different alphabetical characters in corresponding bar graphs in this and subsequent figures. P-values <0.05 were considered a priori as significant.

Figure 2. CRFR1 mediates CRF-induced CREB phosphorylation.

(A) CRF-mediated CREB phosphorylation was blocked by the non-specific CRFR antagonist astressin (50 nM; F = 20.19), (B) and the CRFR1-specfic antagonist CP154526 (100 nM; F = 17.40). (C) CRF-induced CREB phosphorylation was mimicked by the CRFR1 agonist stressin-1 (STR; 70 nM). STR-induced CREB phosphorylation was also blocked by CP154526 (F = 15.99). (D) The CRFR2-specific antagonist antisauvagine-30 (100 nM) had no effect on CRF-induced CREB phosphorylation (F = 17.10).

Figure 3. CRF-induced CREB phosphorylation occurs independently of the AC/cAMP/PKA pathway.

(A) A PKA-specific concentration of the protein kinase inhibitor H89 (2 µM) failed to block CRF-induced CREB phosphorylation, but attenuated forskolin (FSK)-induced CREB phosphorylation (F = 35.78). (B) In the presence of the phosphodiesterase inhibitor IBMX (75 µM), CRF (40 nM) failed to increase cAMP during the time-course in which it increases pCREB. The β-adrenergic receptor agonist isoproterenol (ISO; 10 µM) was used as a positive control (F = 3226). (C) Several concentrations of CRF up to and including 400 nM failed to increase cAMP accumulation (in the presence of IBMX). (D) Inhibiting AC activity with SQ22536 (90 µM) completely blocked ISO-induced cAMP accumulation (F = 101.4). (E) Inhibition of AC with SQ22536 had no effect on CRF-induced CREB phosphorylation (F = 17.10).

Figure 4. CRF-induced CREB phosphorylation occurs via a MEK/MAPK-dependent mechanism.

(A) The CaMK inhibitor KN-93 (2 µM) did not affect CRF-induced CREB phosphorylation (F = 17.65). (B) CRF-induced CREB phosphorylation was blocked by the MEK antagonist U0126 (10 µM; F = 11.97). (C) The L-type Ca²⁺ channel blocker nifedipine (5 µM) had no effect on CRF-induced CREB phosphorylation (F = 18.50). (D) Chelating intracellular calcium with BAPTA-AM (30 min incubation with 5 µM) failed to block CRF-induced CREB phosphorylation (F = 12.72).

Figure 5. CRF-induced CREB phosphorylation occurs via a Gβγ-dependent mechanism.

(A) The Gβγ-specific blocker gallein (75 µM) eliminated CRF-induced CREB phosphorylation (F = 6.13). (B) In contrast, gallein had no effect on CREB phosphorylation induced by the β-adrenergic receptor agonist, isoproterenol (F = 16.83). (C) An additional Gβγ blocker (M119; 5 µM) also eliminated CRF-induced CREB phosphorylation (F = 27.96). (D) Neurons transfected mCherry and the Gβγ inhibitory peptide Grk2i failed to exhibit CRF-induced CREB phosphorylation (F = 11.26).

Figure 6. CRFR1 couples to Gα_s in striatal neurons.

(A) Pre-treatment with pertussis toxin (PTX; 500 ng/mL) did not affect CRF-induced pCREB (F = 11.10). (B) CTX blocked CRF-induced CREB phosphorylation, but had no effect on MAPK-dependent CREB phosphorylation induced by depolarization (60 K; F = 35.15). (C) CRF (4 µM) increased cAMP concentrations in the presence of the cholesterol chelator methyl-β-cyclodextrin (MβCD; 10 mM).

Figure 7. Proposed mechanism of CRF-induced CREB phosphorylation in striatal neurons.

CRF binds to Gα_s-coupled CRFR1, leading to a Gβγ-dependent activation of MEK and MAPK, resulting in downstream CREB phosphorylation.