Leah S Bernstein

Emory University, Atlanta, Georgia, United States

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Publications (8)33.57 Total impact

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    Katherine H. Pedone · Leah S. Bernstein · Maurine E. Linder · John R. Hepler ·
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    ABSTRACT: Many eukaryotic signaling proteins are modified by the covalent attachment of long-chain lipids. These highly hydrophobic molecules bind target proteins and facilitate interaction with cellular membranes, lipid molecules and other proteins. Generally, there are three classes of lipids that modify target proteins: myristate, isoprenoids and palmitate (for reviews, see refs. 1, 2). Myristate, a 14-carbon saturated fatty acid, binds proteins at N-terminal glycine residues via an amide linkage. This usually occurs cotranslationally after removal of the initiating methionine, although it also can occur after proteolytic cleavage and exposure of an internal glycine. Long-chain isoprenoid lipids, including far-nesyl and geranylgeranyl groups, modify proteins posttranslationally and attach via a thioether linkage to C-terminal cysteine residues. Palmitate is a 16-carbon saturated fatty acid that modifies proteins posttranslationally through thioester incorporation at cysteines. Palmitate also can modify proteins at additional sites and by alternative mechanisms, as in proteins palmitoylated at serine and threo-nine residues via oxyester linkages and by amide-linked N-terminal cysteines and glycines. In addition, other fatty acid species form thioester linkages with proteins. For these reasons, the general term for protein lipid modification by thioester attachment is thioacylation, or S-acylation, and the term palmitoylation refers specifically to protein modification by palmitate. Proteins that undergo palmitoylation by thioester attachment at cysteine residues, or S-palmitoylation, represent the majority of protein targets of palmitoylation. This chapter will focus on methods for identifying protein substrates for thioester incorporation of palmitate.
    12/2008: pages 1623-1636;
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    ABSTRACT: Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsalpha, or the Gs-coupled beta2-adrenergic receptor (beta2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsalpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the beta2AR. Purified RGS2 selectively bound to the third intracellular loop of the beta2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.
    Cellular Signalling 04/2006; 18(3):336-48. DOI:10.1016/j.cellsig.2005.05.004 · 4.32 Impact Factor
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    ABSTRACT: Regulators of G-protein signaling (RGS) proteins act directly on Galpha subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.
    Journal of Biological Chemistry 08/2005; 280(29):27289-95. DOI:10.1074/jbc.M502365200 · 4.57 Impact Factor
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    ABSTRACT: RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.
    Journal of Biological Chemistry 06/2004; 279(20):21248-56. DOI:10.1074/jbc.M312407200 · 4.57 Impact Factor
  • Leah S Bernstein · Maurine E Linder · John R Hepler ·
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    ABSTRACT: Palmitoylation refers to the covalent attachment of a 16-carbon fatty acid to cysteine residues of proteins. This modification occurs on many intracellular signaling proteins including regulators of G protein signaling proteins (RGS). Palmitoylation mediates the interaction of proteins with membranes and other proteins and can control the biological activity of a protein. Palmitate attachment occurs through a labile thioester bond and is readily reversible in cells, thus providing a particularly important means for protein regulation. This chapter presents protocols for investigating RGS protein palmitoylation in mammalian cells. The RGS protein of interest is heterologously expressed in HEK293 cells, and cells are metabolically labeled with [3H]palmitate. The RGS protein is isolated from fractionated cells by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography to determine if [3H] has been incorporated. To confirm that the radiolabeled fatty acid is linked to the protein through a thioester bond, labeled proteins are treated with neutral hydroxylamine. Oxyester-linked palmitate, which is occasionally found on serine and threonine residues, is insensitive to this treatment, whereas thioesters are sensitive. To verify that incorporated radiolabel is palmitate, the protein is treated with base, which also cleaves thioester bonds. The resulting lipids are extracted from the sample, then analyzed by chromatography.
    Methods in Molecular Biology 02/2004; 237:195-204. · 1.29 Impact Factor
  • L S Bernstein · AA Grillo · S S Loranger · M E Linder ·
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    ABSTRACT: RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.
    Journal of Biological Chemistry 07/2000; 275(24):18520-6. DOI:10.1074/jbc.M000618200 · 4.57 Impact Factor
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    ABSTRACT: RGS (regulator of Gprotein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Gα subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward Gq versus Gi family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of Gq-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of Gi-mediated signaling. RGS2 mutants were identified that display increased potency toward Gi family members without affecting potency toward Gq. These mutations and the structure of RGS4-Giα1 complexes suggest that RGS2-Giα interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the α8-α9 loop of RGS2 and αA of Gi class α subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Gα subunits.
    Journal of Biological Chemistry 12/1999; 274(48):34253-9. DOI:10.1074/jbc.274.48.34253 · 4.57 Impact Factor
  • Source
    S P Srinivasa · L S Bernstein · K J Blumer · M E Linder ·
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    ABSTRACT: RGS4, a mammalian GTPase activating protein for G protein alpha subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (Delta1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (Delta1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green fluorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.
    Proceedings of the National Academy of Sciences 06/1998; 95(10):5584-9. DOI:10.1073/pnas.95.10.5584 · 9.67 Impact Factor

Publication Stats

718 Citations
33.57 Total Impact Points


  • 2004-2006
    • Emory University
      • Department of Pharmacology
      Atlanta, Georgia, United States
  • 1998-1999
    • Washington University in St. Louis
      • Department of Cell Biology and Physiology
      San Luis, Missouri, United States