Rapid neuronal signaling cascades initiated by corticosterone

Besides inducing transcription by activating nuclear receptors,
corticosterone (CORT) acts rapidly to alter cellular activity
through multiple signaling cascades. Pharmacology-based experiments
presented here show that nanomolar doses of CORT activate several
pathways in primary hippocampal cultures within 20 minutes, a
time-frame that excludes genomic mechanisms mediated by classical
glucocorticoid (GR) and mineralocorticoid (MR) nuclear receptors.
Moreover, none of these effects were subject to inhibition by
either a series of structurally-unrelated antagonists of the
classical GR and MR or by inhibitors of translation. Accordingly, a
major aim of this work was to identify mechanisms proximal to the
cell membrane that could potentially mediate these rapid actions of
CORT. Initial time-course studies showed that 10 nM CORT rapidly
triggers protein tyrosine kinase (PTK) activation that can be
blocked by the small molecule inhibitor, PP2. It was also found
that Src kinase is rapidly activated by CORT through tyrosine
phosphorylation at Y416 and dephosphorylation at Y527. These events
were subject to regulation by another key PTK member, Pyk2
(proline-rich tyrosine kinase 2). A series of experiments designed
to study the potential mechanisms that regulate Pyk2, revealed that
CORT increases the phosphorylation status of at least three
tyrosine sites within Pyk2, namely, Y402, Y579/580 and Y881.
Further, pharmacological probing uncovered requisite roles for
other signaling pathways, such as the classical PLC-PKC pathway and
the “novel” PKA and PKB pathways. Interestingly, further
investigations revealed that the rapid CORT-induced activation of
Pyk2/Src occurs in a G-protein dependent manner. This, together
with the observation that membrane-impermeable BSA-conjugated CORT
(CORT-BSA) produces similar responses profile to that obtained with
CORT, led to pilot experiments to probe whether GPR30, a
recently-described G protein-coupled receptor that appears to
transduce signals from other steroid ligands, might be the putative
receptor for CORT. Support for this view was provided by the
finding that the rapid actions of CORT and CORT-BSA on Pyk2/Src
activation could be blocked by a novel inhibitor of GPR30.
Nevertheless, further studies will be needed to establish the role
of GPR30 more firmly. Other studies sought to identify downstream
targets of Pyk2/Src. Results show that Pyk2/Src regulates
activation of c-Abl (another PTK) and RhoA, both of which are
regulators of a number of cellular processes, including actin
cytoskeleton remodeling. Furthermore, it was found that CORT
triggers phosphorylation of the NR2B subunit of NMDAR, increases
surface expression of NMDAR and activates downstream MAP kinases;
all of these events depend on Src, one of whose direct substrates
is the NR2B subunit. In a summary, the evidence presented in this
dissertation suggests that, by acting via a G protein-coupled
receptor, rather than through classical nuclear receptor
mechanisms, CORT rapidly activates a series of intracellular
signaling cascades that lie proximal to the neuronal plasma
membrane; Pyk2/Src are early kinases involved and beyond these
divergent pathways come into play, ultimately influencing functions
ranging from rearrangements of the actin cytoskeleton, spine
structure, scaffold protein clustering and function, to synaptic
plasticity and transcriptional regulation.