The highly conserved and ubiquitously expressed 14‐3‐3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14‐3‐3, we identified a novel transcriptional co‐activator, TAZ (transcriptional co‐activator with PDZ‐binding motif) as a 14‐3‐3‐binding molecule. TAZ shares homology with Yes‐associated protein (YAP), contains a WW domain and functions as a transcriptional co‐activator by binding to the PPXY motif present on transcription factors. 14‐3‐3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co‐activation through 14‐3‐3‐mediated nuclear export. The C‐terminus of TAZ contains a highly conserved PDZ‐binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ‐stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain‐containing protein NHERF‐2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14‐3‐3.

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Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

G protein-coupled receptors (GPCRs) mediate physiological responses to various ligands, such as hormones, neurotransmitters and sensory stimuli. The signalling and trafficking properties of GPCRs are often highly malleable depending on the cellular context. Such fine-tuning of GPCR function can be attributed in many cases to receptor-interacting proteins that are differentially expressed in distinct cell types. In some cases these GPCR-interacting partners directly mediate receptor signalling, whereas in other cases they act mainly as scaffolds to modulate G protein-mediated signalling. Furthermore, GPCR-interacting proteins can have a big impact on the regulation of GPCR trafficking, localization and/or pharmacological properties.

The Na ؉ ͞H ؉ exchanger regulatory factor (NHERF) binds to the tail of the ␤ 2 -adrenergic receptor and plays a role in adrenergic regulation of Na ؉ ͞H ؉ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the ␤ 2 receptor. Mutagenesis studies of the ␤ 2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S͞T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the ␤ 2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.PDZ domains are conserved protein modules that mediate protein-protein interactions (1-3). The term ''PDZ'' is derived from the first letters in the names of the three proteins in which these modules were originally characterized: PSD-95, Dlg, and ZO-1. PDZ domains bind to the C-terminal tails of target proteins, and the binding preferences of a number of PDZ domains have been characterized (4-7). Such characterization is useful because it allows for predictions regarding the set of proteins to which a given PDZ domain may potentially bind.A PDZ domain-containing protein, the Na ϩ ͞H ϩ exchanger regulatory factor (NHERF), has been recently characterized (8) and found to regulate the Na ϩ ͞H ϩ exchanger type 3 (NHE3) (8-9) via an interaction which does not involve binding of the NHE3 C terminus to the NHERF PDZ domains (9). The function and preferences of the two NHERF PDZ domains were unknown until NHERF was identified as a binding partner of the ␤ 2 -adrenergic receptor (10). The interaction of NHERF with the ␤ 2 receptor is mediated via binding of the first PDZ domain of NHERF to the last few amino acids of the ␤ 2 receptor tail (10). In the present study, we characterize this interaction via mutagenesis of the ␤ 2 receptor tail and saturation NHERF-binding studies. We furthermore demonstrate that NHERF-2, a close relative of NHERF, specifically binds to the ␤ 2 receptor tail and exhibits binding specificity similar to NHERF. Moreover, we demonstrate how the binding preferences of the first PDZ domain of the NHERF family of protein...

No abstract

GPR37 (also known as Pael-R) and GPR37L1 are orphan G protein-coupled receptors that are almost exclusively expressed in the nervous system. We screened these receptors for potential activation by various orphan neuropeptides, and these screens yielded a single positive hit: prosaptide, which promoted the endocytosis of GPR37 and GPR37L1, bound to both receptors and activated signaling in a GPR37- and GPR37L1-dependent manner. Prosaptide stimulation of cells transfected with GPR37 or GPR37L1 induced the phosphorylation of ERK in a pertussis toxin-sensitive manner, stimulated 35 S-GTPγS binding, and promoted the inhibition of forskolin-stimulated cAMP production. Because prosaptide is the active fragment of the secreted neuroprotective and glioprotective factor prosaposin (also known as sulfated glycoprotein-1), we purified full-length prosaposin and found that it also stimulated GPR37 and GPR37L1 signaling. Moreover, both prosaptide and prosaposin were found to protect primary astrocytes against oxidative stress, with these protective effects being attenuated by siRNA-mediated knockdown of endogenous astrocytic GPR37 or GPR37L1. These data reveal that GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ␤-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ␤-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.During the development of the cerebral cortex, neuronal precursors proliferate in the ventricular and subventricular zones that line the cerebral cavity and then migrate outward to make connections with other neurons. Given the billions of cells involved and the requirements for temporal and spatial precision, it is perhaps not surprising that many different types of problems can arise during this process. Abnormalities in cortical development can lead to a range of distinct neurodevelopmental disorders, some of which are caused by mutations to a single gene. For example, bilateral frontoparietal polymicrogyria is a condition in which patients exhibit profound cognitive abnormalities and seizures because of disordered cortical connectivity in the frontoparietal area. Bilateral frontoparietal polymicrogyria is an autosomal recessive syndrome that results from mutations in the orphan receptor GPR56 (1). Thus, insights into the natural function of GPR56 might shed light on the specific pathology underlying bilateral frontoparietal polymicrogyria and also lead to new insights about the fundamental mechanisms controlling cortical development.GPR56 is a member of the adhesion family of G proteincoupled receptors (GPCRs) 2 , which are characterized by extremely large extracellular N termini (NT) exhibiting homology to adhesion proteins (2). There are approximately 30 adhesion GPCRs, all of which are still considered to be orphan receptors. Almost all members of the adhesion GPCR family possess an N-terminal region known as a "GPCR proteolytic site" or GPS dom...