GRK Regulation of Beta2 Adrenergic Receptor Mediated Gene Transcription: Impacts on Learning and Memory

Abstract

BackgroundAdrenergic signaling plays critical roles in health and disease both peripherally and centrally. In the central nervous system, beta adrenergic receptors are broadly expressed and respond to norepinephrine (NE) signaling from neurons projected from the Locus Coeruleous (LC). NE is known to regulate attention, arousal, and acuity as well as fear learning in health and is implicated in neurodegenerative disease and post‐traumatic stress disorder (PTSD). Beta2 adrenergic receptors (B2ARs) are known to be involved in long term potentiation through Gs mediated activation of protein kinase A (PKA) which regulates calcium channels or through forming complexes with AMPA receptors. Recently published work from our lab has shown that functionally distinct and selectively phosphorylated subpopulations of B2ARs exist in a single neuron. These subpopulations of B2ARs are exclusively phosphorylated at either a PKA (S261/262) or g‐protein receptor kinase (GRK) (S355/356) site on the c‐terminus of the receptor and display distinct agonist‐induced receptor intracellular trafficking. Here we investigate the distinct signals associated with the selective phosphorylation of B2ARs at the GRK and PKA site and seek to elucidate the functional implications of the signals in vitro and in vivo.HypothesisWe hypothesize that stimulation of B2AR causes phosphorylation of the receptor at either PKA or GRK phosphorylation sites. Phosphorylation of the GRK site leads to receptor internalization and delivers signaling to the nucleus, which can regulate gene transcription while phosphorylation of the PKA site does not.MethodsOverexpression of wild type B2AR or receptors with serine to alanine mutations of the GRK (S355/356A) or PKA (S261/262A) phosphorylation sites in neurons as well as pharmacologic inhibitors of PKA or GRK were used to study receptor trafficking and signaling. Förster resonance energy transfer (FRET) biosensors ICUE3 expressed at the plasma membrane or the nucleus were used to detect cAMP signaling. Knockin mice expressing mutant receptor were used to examine learning and memory in the Morris water maze paradigm.ResultsStimulation of B2AR leads to distinct membrane trafficking based on phosphorylation status of the receptor. Phosphorylation of the GRK but not PKA site on B2AR is necessary for internalization of the receptor and this internalized receptor is in an active conformation. GRK phosphorylation of B2AR is also necessary for increased cAMP levels in the nucleus and transcription of cFOS, FOSB, RHOB, and OLIG2, genes associated with learning and memory. Knockin mice expressing B2AR with a S355/356A mutation showed decreased spatial learning and memory.Conclusion and SignificanceThese data demonstrate that GRK‐dependent B2AR signaling can regulate gene transcription and that loss of this internal signaling affects learning and memory in mice. These data offer new mechanistic insight into B2AR signaling in the brain that could have implications for both our understanding of learning and memory and for neurological diseases and PTSD.Support or Funding InformationThis work was funded by NIH T32GM099608 and GM GM129376