Randy A. Hall

The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange.

Stimulation of β2-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 β2-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 β2-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 β2-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 ofthe final residue of the β2-adrenergic receptor from leucine toalanine abolishes the receptors interaction with NHERF andalso markedly alters β2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent β2-adrenergic receptor binding of NHERF plays a role in β2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins

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.

Heterodimerization of G Protein-Coupled Receptors: Specificity and Functional Significance

G protein-coupled receptors (GPCRs) are cell surface receptors that mediate physiological responses to a diverse array of stimuli. GPCRs have traditionally been thought to act as monomers, but recent evidence suggests that GPCRs may form dimers (or higher-order oligomers) as part of their normal trafficking and function. In fact, certain GPCRs seem to have a strict requirement for heterodimerization to attain proper surface expression and functional activity. Even those GPCRs that do not absolutely require heterodimerization may still specifically associate with other GPCR subtypes, sometimes resulting in dramatic effects on receptor pharmacology, signaling, and/or internalization. Understanding the specificity and functional significance of GPCR heterodimerization is of tremendous clinical importance since GPCRs are the molecular targets for numerous therapeutic drugs.

Fine-tuning of GPCR activity by receptor-interacting proteins

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.

Regulation of G protein-coupled receptor signaling by scaffold proteins.

The actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.

THE G PROTEIN-COUPLED RECEPTOR KINASE 2 IS A MICROTUBULE-ASSOCIATED PROTEIN KINASE THAT PHOSPHORYLATES TUBULIN

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulinin vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied β2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.

Differential Surface Expression and Phosphorylation of the N-Methyl-D-aspartate Receptor Subunits NR1 and NR2 in Cultured Hippocampal Neurons*

The trafficking and phosphorylation of the NR1 and NR2 subunits of the N-methyl-D-aspartate-type glutamate receptor complex were studied in cultured rat hippocampal neurons. Surface expression was examined by modifying surface receptors via treatment of intact neurons with either the protease chymotrypsin or the cross-linking reagent bis(sulfosuccinimidyl)suberate, followed by quantification of anti-NR1 and anti-NR2B Western blot immunostaining. These studies revealed that only 40-50% of total NR1 immunoreactivity is found at the cell surface, as compared to more than 90% of total NR2B immunoreactivity. Metabolic labeling of the cultures with 32P revealed that NR2 subunits are highly phosphorylated under basal conditions, whereas basal phosphorylation of NR1 subunits is barely detectable. Following stimulation of the cultures with glutamate/glycine or phorbol esters, NR1 phosphorylation was found to be enhanced by 3-5-fold, whereas phosphorylation of NR2 subunits was enhanced by less than 2-fold. To determine whether the difference in the basal NR1 versus NR2 phosphorylation could be due to tyrosine phosphorylation of NR2, phosphoamino acid analyses of NR2 were performed. These analyses revealed phosphorylation on serine but not on threonine or tyrosine; immunoprecipitation and deglycosylation experiments using anti-phosphotyrosine antibodies confirmed that NR2 subunits in the primary hippocampal cultures are not detectably phosphorylated on tyrosine residues. These results demonstrate that the NR1 and NR2 subunits, which assemble into heteromeric complexes to form functional N-methyl-D-aspartate receptors, are trafficked in neurons with differential efficiency to the plasma membrane and exhibit different levels of basal versus stimulated serine phosphorylation.

G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction.

The Na+/H+exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser289, and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser289 in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous “NHERF kinase” activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G Protein–Coupled Receptors

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein–coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

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 β1-adrenergic receptor (β AR) interactions with PDZ scaffolds, the β1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed β1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel β1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The β1AR 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 β1AR being the key determinants of the interaction. Full-length β1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the β1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with β1AR profoundly impaired β1AR-mediated ERK1/2 activation but had no apparent effect on β1AR-mediated cyclic AMP generation or agonist-promoted β1AR internalization. These findings revealed that the interaction of MAGI-3 with β1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of β1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which β1 AR signaling and trafficking can be regulated in a cell-specific manner.