Proteomic Analysis of β1-Adrenergic Receptor Interactions with PDZ Scaffold Proteins

Abstract

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...

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

“…GST and GST-CRFR1 fusion proteins bound to the matrix were washed extensively in PBS-containing 0.3% Triton X-100. 100 nM GST and GST-CRFR1 in blot buffer (2% nonfat dry milk, 0.1% Tween 20, 50 mM NaCl, 10 mM HEPES, pH 7.4) were incubated with gridded nylon membranes that were spotted with His/S-tagged PDZ domain fusion proteins (1 g/bin) for 1 h at room temperature (23,24). The arrays were then washed three times with blot buffer and incubated with a horseradish peroxidase-conjugated anti-GST antibody (1:3000).…”

“…We have investigated the capacity of the CRFR1 carboxylterminal tail to bind to a proteomic array of 96 class I PDZ domains (23,24). We found that the CRFR1 carboxyl-terminal tail binds to a selective subset of class I PDZ domains on the array.…”

SAP97 promotes the stability of Nax channels at the plasma membrane

“…The PDZ-array overlay assay was performed as described [10,12]. Briefly, a nylon membrane spotted with a series of His/Stagged PDZ domain proteins was pre-treated with a blocking buffer containing 2% non-fat dry milk and 0.1% Tween-20 in 150 mM NaCl and 10 mM phosphate buffer, pH 7.3 (PBS) for 30 min, then overlaid with the GST-fused proteins (15 nM) in the blocking buffer.…”

“…We screened for potential interactions between Na x and PDZ proteins using the PDZ-array overlay assay [10,12] with the GSTfused protein with the C-terminal region of Na x (GST-Na x -C-term) (Fig. 1).…”

“…Because the C-terminal sequence of Na x (-Q-T-Q-I for rat and mouse, and -Q-S-Q-I for human) fits this 'non-canonical' PDZ-binding motif, we hypothesized that the Na x channel may be regulated by PDZ-scaffold proteins. In the present study, we took advantage of a proteomic PDZ-domain array [10], containing 96 distinct PDZ domains, to screen for PDZ proteins that might interact with Na x . Among the PDZ proteins thus identified, we found that SAP97 (also known as DLG1) is coexpressed with Na x in the SFO, and contributes to the stability of Na x channels in the plasma membrane.…”