β1-Adrenergic Receptor Association with PSD-95

Abstract

The ␤ 1 -adrenergic receptor (␤ 1 AR) is the most abundant subtype of ␤-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal ␤ 1 AR-interacting proteins, we screened a rat brain cDNA library using the ␤ 1 AR carboxyl terminus (␤ 1 AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the ␤ 1 AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the ␤ 1 AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length ␤ 1 AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between ␤ 1 AR and PSD-95 is mediated by the last few amino acids of the ␤ 1 AR, and mutation of the ␤-Adrenergic receptors (␤ 1 AR, ␤ 2 AR, and ␤ 3 AR) 1 are heptahelical G-protein-coupled receptors that mediate physiological responses to the hormone epinephrine and the neurotransmitter norepinephrine. ␤ 1 AR and ␤ 3 AR exhibit high affinity for both epinephrine and norepinephrine, whereas ␤ 2 AR binds with high affinity only to epinephrine. The tissue distributions of the three receptors are distinct: ␤ 2 AR is highly expressed in many tissues, ␤ 3 AR is expressed at high levels only in adipose tissue, and ␤ 1 AR is expressed at high levels in the heart and brain and lower levels elsewhere (1). In the brain, ␤ 1 AR exhibits a predominantly neuronal expression pattern, whereas ␤ 2 AR is expressed mainly in glial cells (2-4). ␤ 1 AR is thus considered to be the "synaptic" ␤-adrenergic receptor, because electrophysiological experiments with specific antagonists demonstrate that ␤-adrenergic modulation of hippocampal neuronal activity exhibits a ␤ 1 AR-like pharmacological profile (5-7).Noradrenergic stimulation of synaptic ␤ 1 -adrenergic receptors is known to potently regulate memory formation and synaptic plasticity. Emotionally charged events often lead to the creation of vivid memories (8), and the formation of such emotional memories is due in large part to a surge in noradrenaline release and consequent stimulation of brain ␤-adrenergic receptors (8 -13). The powerful effects of ␤-adrenergic stimulation on memory formation correlate well with electrophysiological experiments demonstrating profound effects of ␤-adrenergic stimulation on the development of long term potentiation (LTP), an enhancement of synaptic responses that underlies some forms of memory (14). In both the hippocampus (15)(16)(17)(18)(19)(20)(21)(22) and the amygdala (23-25), two brain regions known to play key roles in the formation of emotional memories, LTP is markedly enhanced by ␤-adrenergic stimulation. This ␤-adrenergic modulation of LTP is mediated exclusively by ␤ 1 -adrenergic receptors, because hippocampal slices prepared from mice lacking ␤ 1 -adrenergic receptors do not exhibit ...

A Library of 7TM Receptor C-terminal Tails

“…3A). Nevertheless, if the binding to GST alone was used as the strict negative control, most of the tail proteins would in fact be considered to be positive hits because the weak bands, although Not known rmGlu 5a Not known rmGlu 8 Not known…”

“…58. were from Siv Hjorth (Novo Nordisk A/S). Rat metabotropic glutamate receptors mGlu 1a , mGlu 1b , mGlu 2 , mGlu 3 , mGlu 4 , mGlu 5a , mGlu 6 , mGlu 7 , and mGlu 8 were from Hans Brä uner-Osborne (Danish University of Pharmaceutical Sciences, Copenhagen, Denmark). Rat GABA, type B1A receptor was from Jean-Philippe Pin (Centre National de la Recherche Scientifique, Montpellier, France).…”

“…As shown in Table I, the four proteins, EBP50, NSF, SNX1, and GASP, which have been proposed to function as adaptor proteins involved in the post-endocytotic sorting of 7TM receptors, are structurally very different and have been implicated in various other cellular functions. Here, these proteins are probed for their ability to bind to the C-terminal tails of 59 different 7TM receptors as determined by GST pull-down assays, which routinely have been used to confirm protein interactions identified by co-immunoprecipitation and yeast two-hybrid screening (8,23,(35)(36)(37). In selected cases, interactions were further studied by surface plasmon resonance (SPR) technology or the interaction was characterized in more detail through gradual deletion mutagenesis of the tail protein.…”

G Protein-coupled Receptor Kinase 5 Regulates β1-Adrenergic Receptor Association with PSD-95

“…PSD-95 is thought to play critical roles in assembling these and other molecules into signaling complexes and regulating their signal transduction (20). Association with PSD-95 decreases agonist-induced ␤ 1 AR internalization, but has no effect on receptor desensitization or receptor-induced cAMP accumulation (15). This association provides a physical linkage between ␤ 1 ARs and other synaptic proteins, such as N-methyl-D-aspartate receptors, which are known to be regulated by ␤ 1 AR stimulation (21).…”

“…Using yeast two-hybrid screening, we recently identified PSD-95, a postsynaptic density-enriched protein, as a novel binding partner of ␤ 1 AR (15). PSD-95 belongs to the large family of membrane-associated guanylate kinase domain proteins that contain multiple PDZ domains.…”