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PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

The α2C-adrenergic receptor (α2CAR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of α2CAR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of α2CAR with more than 25 related GPCRs revealed that only coexpression with the β2-adrenergic receptor (β2AR) increased the surface localization of α2CAR in human embryonic kidney-293 cells. Coimmunoprecipitation of α2CAR with β2AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that α2CAR expressed alone was mainly intracellular, whereas α2CAR coexpressed with β2AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in α2CAR binding sites upon coexpression with β2AR, with no apparent change in affinity for α2AR ligands. Functional assays with the α2AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of β2AR with α2CAR enhanced α2CAR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced α2CAR internalization in response to α2AR agonists when α2CAR and β2AR were coexpressed. In addition, substantial cointernalization of α2CAR in response to βAR agonists was observed when α2CAR was coexpressed with β2AR. These data reveal that α2CAR can interact with β2AR in cells in a manner that regulates α2CAR surface expression, internalization, and functionality.

The alpha(2C)-adrenergic receptor (alpha(2C)AR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of alpha(2C)AR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of alpha(2C)AR with more than 25 related GPCRs revealed that only coexpression with the beta(2)-adrenergic receptor (beta(2)AR) increased the surface localization of alpha(2C)AR in human embryonic kidney-293 cells. Coimmunoprecipitation of alpha(2C)AR with beta(2)AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that alpha(2C)AR expressed alone was mainly intracellular, whereas alpha(2C)AR coexpressed with beta(2)AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in alpha(2C)AR binding sites upon coexpression with beta(2)AR, with no apparent change in affinity for alpha(2)AR ligands. Functional assays with the alpha(2)AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of beta(2)AR with alpha(2C)AR enhanced alpha(2C)AR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced alpha(2C)AR internalization in response to alpha(2)AR agonists when alpha(2C)AR and beta(2)AR were coexpressed. In addition, substantial cointernalization of alpha(2C)AR in response to betaAR agonists was observed when alpha(2C)AR was coexpressed with beta(2)AR. These data reveal that alpha(2C)AR can interact with beta(2)AR in cells in a manner that regulates alpha(2C)AR surface expression, internalization, and functionality.

Heterologous expression of α1D-adrenergic receptors (α1D-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with α1B-ARs promotes surface localization of α1D-ARs. Here, we report that the α1B-/α1D-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of α1B-ARs and epitope-tagged α1D-ARs in both human embryonic kidney 293 and DDT1MF-2 cells, α1D-AR binding sites were not detectable with the α1D-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect α1D-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the α1B-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in α1B/α1D-AR-expressing cells, revealing that α1D-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in α1B/α1D-AR-coexpressing cells. Stable coexpression of an uncoupled mutant α1B-AR (Δ12) with α1D-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that α1B/α1D-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why α1D-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with α1B-ARs.

The α1D-adrenergic receptor (α1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote α1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of α1D-AR at the cell surface: α1B-AR and β2-AR. Confocal imaging confirmed that coexpression with β2-AR resulted in translocation of α1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that α1D-AR and β2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total α1D-AR binding sites upon coexpression with β2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with β2-AR significantly enhanced the coupling of α1D-AR to norepinephrine-stimulated Ca2+mobilization. Heterodimerization of β2-AR with α1D-AR also conferred the ability of α1D-AR to cointernalize upon β2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other’s activity. These findings demonstrate that the selective association of α1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between α1- and β2-ARs that is mediated through heterodimerization and cross-internalization.

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

The potential role of dimerization in controlling the expression and pharmacological properties of alpha1-adrenoceptor subtypes was examined using coimmunoprecipitation of epitope-tagged receptors. Human alpha1-adrenoceptor subtypes (alpha1A, alpha1B, alpha1D) were tagged at their amino-termini with Flag or hemagglutinin epitopes and transfected into human embryonic kidney 293 cells. Homodimerization of all three subtypes was observed by coimmunoprecipitation of receptors with different tags and was not altered by norepinephrine treatment. Heterodimer formation between hemagglutinin-tagged alpha1B-adrenoceptors and Flag-tagged alpha1A- or alpha1D-adrenoceptors was also observed. However, no alpha1A/alpha1D-adrenoceptor heterodimers were observed, suggesting that dimerization is subtype-specific. The extent of heterodimerization was also unaltered by norepinephrine treatment. alpha1-Adrenoceptor truncation mutants lacking carboxyl or amino-terminal sequences formed homo- and heterodimers similarly to full-length receptors, suggesting that these domains play little or no role in dimerization. Biotinylation with a membrane-impermeable agent showed that monomers and homo- and hetero-oligomers of all three subtypes are expressed on the cell surface. Radioligand binding studies showed that heterodimerization did not alter the affinity of alpha1-adrenoceptors for norepinephrine, prazosin, or subtype-selective antagonists, suggesting that dimerization does not result in pharmacologically distinct subtypes. However, coexpression of alpha1B-adrenoceptors significantly increased both binding site density and protein expression of alpha1A- and alpha1D-adrenoceptors, and increased cell surface expression of alpha1D-adrenoceptors, suggesting a functional role for heterodimerization. Conversely, coexpression of alpha1A-with alpha1D-adrenoceptors, which did not heterodimerize, had no effect on receptor density or protein. These studies demonstrate subtype-selective heterodimerization of alpha1-adrenoceptors, which does not change their pharmacological properties but seems to have functional consequences in regulating receptor expression and trafficking.

The activation of the muscarinic acetylcholine receptor (mAChR) family, consisting of five subtypes (M1-M5), produces a variety of physiological effects throughout the central nervous system. However, the role of each individual subtype remains poorly understood. To further elucidate signal transduction pathways for specific subtypes, we used the most divergent portion of the subtypes, the intracellular third (i3) loop, as bait to identify interacting proteins. Using a brain pull-down assay, we identify elongation factor 1A2 (eEF1A2) as a specific binding partner to the i3 loop of M4, and not to M1 or M2. In addition, we demonstrate a direct interaction between these proteins. In the rat striatum, the M4 mAChR colocalizes with eEF1A2 in the soma and neuropil. In PC12 cells, endogenous eEF1A2 co-immunoprecipitates with the endogenous M4 mAChR, but not with the endogenous M1 mAChR. In our in vitro model, M4 dramatically accelerates nucleotide exchange of eEF1A2, a GTP-binding protein. This indicates the M4 mAChR is a guanine exchange factor for eEF1A2. eEF1A2 is an essential GTP-binding protein for protein synthesis. Thus, our data suggest a novel role for M4 in the regulation of protein synthesis through its interaction with eEF1A2.

Na+/H+exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing adaptor protein known to bind to various receptors, channels, cytoskeletal elements, and cytoplasmic signaling proteins. We report here that the phosphorylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosphorylated at other points of the cell cycle. This mitosis phase-dependent phosphorylation of NHERF-1 could be blocked by roscovitine, consistent with phosphorylation by cyclin-dependent kinases. In vitrostudies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robustly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the NHERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 possesses two serines (Ser279and Ser301) that conform to the SP X(K/R) motif preferred for phosphorylation by Cdc2. Mutation of either of these serines reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation of both residues together completely abolished Cdc2-mediated phosphorylation. When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser279and Ser301to aspartate, to mimic Cdc2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 from lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase HeLa cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associate with Pin1, a WW domain-containing peptidylprolyl isomerase that does not detectably bind to NHERF-1 in interphase lysates. The association of NHERF-1 with Pin1 facilitated dephosphorylation of NHERF-1, as shown in experiments in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser279and Ser301and that this phosphorylation regulates NHERF-1 oligomerization and association with Pin1.

The β1-adrenergic receptor (β1AR) is known to be localized to synapses and to modulate synaptic plasticity in many brain regions, but the molecular mechanisms determining β1AR subcellular localization are not fully understood. Using overlay and pull-down techniques, we found that the β1AR carboxyl terminus associates with MAGI-2 (membrane-associated guanylate kinase inverted-2), a protein also known as S-SCAM (synaptic scaffolding molecule). MAGI-2 is a multidomain scaffolding protein that contains nine potential protein-protein interaction modules, including 6 PDZ domains, 2 WW domains, and a guanylate kinase-like domain. The β1AR carboxyl terminus binds with high affinity to the first PDZ domain of MAGI-2, with the last few amino acids of the β1AR carboxyl terminus being the key determinants of the interaction. In cells, the association of full-length β1AR with MAGI-2 occurs constitutively and is enhanced by agonist stimulation of the receptor, as assessed by both co-immunoprecipitation experiments and immunofluorescence co-localization studies. Agonist-induced internalization of the β1AR is markedly increased by co-expression with MAGI-2. Strikingly, this result is the opposite of the effect of co-expression with PSD-95, a previously reported binding partner of the β1AR. Further cellular experiments revealed that MAGI-2 has no effect on β1AR oligomerization but does promote association of β1AR with the cytoplasmic signaling protein β-catenin, a known MAGI-2 binding partner. These data reveal that MAGI-2 is a specific β1AR binding partner that modulates β1AR function and facilitates the physical association of the β1AR with intracellular proteins involved in signal transduction and synaptic regulation.

ABSTRACTPlatelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na+/H+exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the ß2-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.

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 Ser289in 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.

In this article, the author discusses his tenure as president in the North Dakota Music Educators Association (NDMEA) and mentions acknowledgments for the board of directors for their support, appointment of new presidents Darcy Brandenburg and Brian Saylor and his role as advocate for music.

The author discusses music profession and its association with emotions and feelings including secure a mentor through the licensing board Education Standards and Practices Board (ESPB), a conference of North Dakota Music Educators Association, and selection and arrangement of music.

The author discusses activities at the North Dakota Music Educators Association (NDMEA) including its new leaderships, mentoring availabilities, and the NDMEA/All-State Conference.