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 ␤ 1 -adrenergic receptor (␤ 1 AR) interactions with PDZ scaffolds, the ␤ 1 AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed ␤ 1 AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel ␤ 1 AR-interacting proteins, SAP97 and MAGI-3, were also identified. The ␤ 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 ␤ 1 AR being the key determinants of the interaction. Full-length ␤ 1 AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the ␤ 1 AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with ␤ 1 AR profoundly impaired ␤ 1 AR-mediated ERK1/2 activation but had no apparent effect on ␤ 1 AR-mediated cyclic AMP generation or agonist-promoted ␤ 1 AR internalization. These findings revealed that the interaction of MAGI-3 with ␤ 1 AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of ␤ 1 AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which ␤ 1 AR signaling and trafficking can be regulated in a cell-specific manner.Cellular responses to a given hormone or neurotransmitter can vary markedly between different types of cells. In many cases, such cellular differences are because of differential expression of receptor subtypes. However, even cells expressing exactly the same receptor subtypes can exhibit distinct responses to a given ligand, because receptors often behave quite differently in distinct cellular environments. The trafficking and signaling properties of G protein-coupled receptors (GPCRs), 3 which comprise the largest family of cell surface receptors, are known to be especially influenced by cellular context. The activity of a GPCR depends not only on the complement of downstream effectors expressed in a given cell but also on the set of expressed G proteins, kinases, and scaffold proteins that directly interact with the receptor to shape its signaling capability.PDZ scaffolds comprise a key class of GPCR-interacting proteins that can strongly influence receptor trafficking and signaling. The term PDZ is derived from the names of the first three proteins in which these domains were first identified: the post-synaptic density protein PSD-95, the Drosophila protein Discs-large, and the tight junction protein ZO-1. PDZ domains are ϳ90 amino acids in length and bind to specific carboxyl-terminal motifs on their target proteins. There are three general classes of PDZ domain...

G protein-coupled receptors such as the ␤1-adrenergic receptor (␤1AR) must be trafficked to the plasma membrane in order to bind with their extracellular ligands and regulate cellular physiology. By using glutathione S-transferase pull-down techniques, we found that the ␤1AR carboxyl terminus directly interacts with the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL; also known as PIST, GOPC, and FIG), a protein known to be primarily localized to the Golgi apparatus. CAL contains two predicted coiled-coil domains and one PSD-95/Discs-large/ZO-1 homology (PDZ) domain. The ␤1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the ␤1〈R-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the ␤1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the ␤1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length ␤1AR robustly associates with CAL, and this interaction is abolished by mutation of the terminal valine to alanine of the receptor (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. Consistent with observations that CAL is a Golgi-associated protein, overexpression of CAL reduces surface expression of ␤1AR. Interaction with CAL promotes retention of ␤1AR within the cell, whereas PSD-95, another ␤1AR-associated PDZ domaincontaining protein, competitively blocks ␤1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel ␤1AR-binding partner, modulates ␤1AR intracellular trafficking, thereby revealing a new mechanism of regulation for ␤1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane. ␤-Adrenergic receptors (␤ARs)1 are G protein-coupled receptors (GPCRs) that play critical roles in mediating physiological responses to the hormone epinephrine and the neurotransmitter norepinephrine. Noradrenergic stimulation of ␤1-adrenergic receptor (␤1AR) in the brain potently regulates memory formation and synaptic plasticity (1-6), and stimulation of ␤1AR in the heart profoundly regulates both the rate and force of cardiac contractions (7-9). Neither norepinephrine nor epinephrine can easily cross the plasma membrane; thus, ␤1AR must be localized at the cell surface in order to be activated by its ligands. The regulation of the trafficking and surface expression of ␤1AR and other GPCRs is therefore a topic of considerable physiological importance.Intracellular trafficking of GPCRs has been extensively studied, with most of this work focused on the events involved in receptor internalization and recycling. Within seconds or minutes of agonist stimulation, ␤ARs are phosphorylated by G protein-coupled receptor kinases and cAMP-dependent protein kinases (10, 11). Subsequently, in the cases of ␤1AR and ␤2AR, this phosphoryl...