Proteomic analysis of β1-adrenergic receptor interactions with PDZ scaffold proteins

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta(1)-adrenergic receptor (beta(1)AR) interactions with PDZ scaffolds, the beta 1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta(1)AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta(1)AR- interacting proteins, SAP97 and MAGI-3, were also identified. The beta(1)AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta(1)AR being the key determinants of the interaction. Full-length beta(1)AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta(1)AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta(1)AR profoundly impaired beta(1)AR-mediated ERK1/2 activation but had no apparent effect on beta(1)AR-mediated cyclic AMP generation or agonist-promoted beta(1)AR internalization. These findings revealed that the interaction of MAGI-3 with beta(1)AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta(1)AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta(1)AR signaling and trafficking can be regulated in a cell-specific manner.