A macromolecular complex of β ₂ adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA

Abstract

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and ␤2 adrenergic receptor (␤2AR) can bind ezrin͞radixin͞moesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that ␤2 is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. ␤2AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the ␤2AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-␤ 2AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

Mechanistic Insight into Control of CFTR by AMPK

“…39 and 40). In this model, regulation of CFTR is predicted to occur without any detectable changes in cytosolic second messenger concentration, because CFTR is intimately connected to signaling elicited by stimulation of A2B adenosine and ␤ 2 -adrenergic receptors (41)(42)(43). Moreover, type 2 lysophosphatidic acid receptors also form macromolecular complexes with CFTR that are mediated through a PDZ scaffolding protein (NHERF2).…”

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…Since defective recycling of the Gprotein-coupled receptor in the absence of NHERF binding is invariably associated with the accelerated degradation of the receptors (24,26,50), our observations, collectively, argue against the role of NHERF in CFTR recycling. Although the functional studies imply that the endogenous level of NHERF is insufficient to influence CFTR activity in BHK and PANC-1 cells, variations in the concentration and/or the compartmentalization of NHERF may ensure a regulatory role for NHERF in CFTR activation in other cells (64), including the sweat duct and Calu-3 epithelia (29,65).…”

A Chemokine Receptor CXCR2 Macromolecular Complex Regulates Neutrophil Functions in Inflammatory Diseases

“…4A (upper panel). Using this in vitro macromolecular complex assembly assay (29,30,36,42), we detected a complex consisting of CXCR2, PDZ scaffold proteins (NHERF1 or NHERF2), and PLC-␤2 C-tail (Fig. 4A).…”

“…1 and 2) does not provide information whether the interactions between CXCR2 or PLC-␤2 and NHERF1 are direct, as cell lysates contain large numbers of other proteins as well. To test if CXCR2 or PLC-␤2 binds NHERF1 directly or by other intermediary proteins, and to test the PDZ motif dependence, we performed a pairwise binding assay that detects a direct interaction between purified proteins in vitro (29,30,36,42). Purified His-S fusion proteins containing the C-tail fragments (last 45 amino acids) of CXCR2 WT, C-tail ⌬TTL (with PDZ motif TTL deleted), or C-tail AAA or ATA (with PDZ motif TTL mutated to AAA or ATA) were mixed with GST-NHERF1, and the mixtures were pulled down by S-protein-agarose (the S-protein can specifically bind to the S-tag within the fusion proteins).…”