β1-Adrenergic Receptor Association with PSD-95

Abstract

The β₁-adrenergic receptor (β₁AR) is the most abundant subtype of β-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal β₁AR-interacting proteins, we screened a rat brain cDNA library using the β₁AR carboxyl terminus (β₁AR-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 β₁AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the β₁AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length β₁AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between β₁AR and PSD-95 is mediated by the last few amino acids of the β₁AR, and mutation of the β₁AR carboxyl terminus eliminated the binding and disrupted the co-localization of the β₁AR and PSD-95 in cells. Agonist-induced internalization of the β₁AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the β₁AR or β₁AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the β₁AR andN-methyl-d-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific β₁AR binding partner that modulates β₁AR function and facilitates physical association of the β₁AR with synaptic proteins, such as theN-methyl-d-aspartate receptors, which are known to be regulated by β₁AR stimulation.