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    EPR Center Presentation II - Membrane Proteins

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    taherhusenihakim
    Membrane proteins

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    EPR Center Presentation II - Membrane Proteins

    Uploaded by

    taherhusenihakim
    0 views31 pages
    Membrane proteins

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    © © All Rights Reserved

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    Membrane proteins

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    0 views31 pages

    EPR Center Presentation II - Membrane Proteins

    Uploaded by

    taherhusenihakim
    Membrane proteins

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 31

    Receptors

    • Activation Æ conformational change that transfers


    information across the cell membrane
    • Allow the cell to sense and respond to environmental
    conditions and stimuli
    • Three classes:
    – Ligand-gated ion channels (nicotinic acetylcholine receptor)
    – Enzyme-linked receptors (epidermal growth factor receptor)
    – G-protein coupled receptors (many hormones, peptides, biogenic
    amines)
    Ligand-gated ion channels
    (Ionotropic receptors)

    • Neuronal receptors for acetylcholine, glycine,


    serotonin, GABA
    • Very large (~ 300 kDa) glycoproteins
    • Heteropentamers (α γ α β δ)
    • Ligand binding Æ opens channel allowing rapid
    flow of ions down concentration gradient
    • Either cation or anion selective
    Nicotinic acetylcholine receptor
    (Torpedo marmorata)

    4.0 Å resolution reconstructed from cryoelectron microscopy

    N. Unwin, J. Mol. Biol. 346, 967 (2005)


    Subunits symmetrically arranged around central pore
    Two Ach binding sites at the α-δ and α-γ subunit interfaces
    Each monomeric subunit

    N-terminal extracellular domain:


    two β-sheets joined through
    a disulfide bond

    TM domain: four α-helices per


    subunit, arranged symmetrically
    with the five M2 helices lining
    the central pore

    Cytoplasmic domain: each subunit


    contributes one α-helix to a
    “vestibule”

    Torpedo AchR α-subunit


    Electrostatic potential of the
    outer and inner vestibules
    - highly negative
    - many Glu and Asp residues

    (AchR is cation-selective, allowing


    passage of both K+ and Na+ ions)

    Unwin, J. Mol. Biol. 346, 967 (2005)


    TM helices M1, M3, M4 shield pore
    helices from the lipid bilayer
    Hyrophobic constriction keeps pore closed
    Rotation of α-subunit M2 helices proposed
    as channel-opening mechanism
    G-protein coupled receptors (GPCRs)
    • Largest group of cell surface receptors found in
    nature (several hundred identified, account for ~ 3%
    of the human genome)
    • Present in all eukaryotes (including plants, yeast, on
    up to mammals)
    • Found in essentially all tissues and cell types
    • Are targets of ~ 40% of all drugs currently in clinical
    use
    GPCRs signal by activating heterotimeric guanine
    nucleotide-binding proteins (G-proteins) that consist
    of α, β, and γ subunits

    N-termini of
    Gα and Gγ are
    lipid-modified

    Transducin

    Binding to GPCR Æ a conformational change in Gα


    and dissociation of Gα from Gβγ
    Overview of the G protein cycle. Gα, blue; Gβ, brown; Gγ, gray; GDP, yellow, with smaller
    circles representing the two phosphates; GTP, green, with smaller circles representing
    three phosphates; RGS (regulator of G protein signaling), magenta.
    Major subclasses of Gα
    • Gαs activate adenylyl cyclase to increase cAMP
    (β-adrenergic receptors)

    • Gαq/11 activate phospholipase C: consequent


    hydrolysis of PI lipids produce IP3 and DAG
    leading to Ca2+ release and activation of PKC
    (Adrenergic-α2 receptors)

    • Gαi/o inhibit adenylyl cyclase to decrease cAMP,


    Gβγ subunit opens K+ channels
    (metabotropic epinephrine, serotonin, dopamine receptors,
    muscarinic acetylcholine receptor)

    • Gt cGMP phosphodiesterase, decreased cGMP


    (Rhodopsin)

    • Gα12/13 RAS, Src, PKC, phospholipase D


    (lysophosphatidic acid, thromboxine A2 receptors)
    Mammalian trimeric G proteins

    A given Gα subclass may associate with more than


    one receptor

    Only 1 Gαs identified so far, but many Gαq and Gαi


    proteins have been found

    Overall, ~ 20 Gα, 6 Gβ, and 11 Gγ proteins have been


    identified

    Signaling specificity lies in the recognition of a


    GPCR cytoplasmic surface by the Gα subunit
    GPCR structure

    Seven transmembrane α-helices


    (GPCRs are sometimes referred to as 7TM receptors)

    Cytoplasmic loop 3 (between TM helices 5 and 6) and the


    C-terminal tail involved in signal transduction
    Chimeric GPCRs expressed
    in Xenopus oocytes
    Æ Critical role of TMH5
    and CL3

    Kobika et al., Science 240, 1310 (1998)


    Membrane topology of the human h2 adrenergic receptor

    Highly conserved residues found in each TM helix


    Two highly conserved disulfide bonds
    Palmitoylation site in C-terminal tail (Cys341)
    ~ 48,000 rhodopsin molecules per μm2
    (70% of total cell protein)
    Membrane topology of rhodopsin

    cytoplasm

    extracellular

    Helices 1,2,5,6,7 contains bends


    CL3 is α-helical based on EPR and more recent crystal data
    Altenbach et al., Biochemistry 40, 15493 (2001)
    Bovine rhodopsin @ 2.2 Å (1U19)

    Okada et al., J. Mol. Biol. 342, 571 (2004)


    Okada et al., J. Mol. Biol. 342, 571 (2004)
    Structural model of bovine rhodopsin

    Schematic representation of a GPCR in the cell membrane based on the


    packing arrangement of TMHs observed in the crystal structure of rhodopsin
    (Okada et al., J. Mol. Biol. 342, 571 (2004) pdb code 1U19).
    Proposed mechanism of rhodopsin activation

    Tilting and rotation of TMH6. A change in the relative orientations of TM


    helices 3 and 6 produces a conformational change at the cytoplasmic surface

    Farrens, Altenbach, Yang, Hubbell, and Khorana Science 274, 768 (1996)
    Interaction of switch helix II in the Gα subunit of transducin
    with the cytoplasmic face of activated rhodopsin
    Æ G-protein dissociation and activation

    Van Eps et al., PNAS 44, 16194 (2006)


    Interaction surfaces of rhodopsin and transducin

    Preininger and Hamm, Science STKE (2004)


    GPCR families based on sequence homology in TM domain

    Agonist/antagonist binding sites identified through mutagenesis, some cross-linking studies


    AFM of rhodopsin dimers in native disk membranes

    Organization and topography of the cytoplasmic surface of rhodopsin. a, Topograph obtained


    using atomic-force microscopy, showing the paracrystalline arrangement of rhodopsin dimers in
    the native disc membrane. b, Angularly averaged powder-diffraction pattern, showing peaks at
    (8.4 nm)11, (4.2 nm)11 and (3.8 nm)11. c, Magnification of a region of the topograph in a,
    showing rows of rhodopsin dimers, as well as individual dimers (inside dashed ellipse), and
    occasional rhodopsin monomers (arrowheads). Scale bars: a, 50 nm; inset, (5 nm)11; c, 15 nm.

    Fotiadis et al., Nature 421, 127 (2003)


    Evidence for GPCR dimers
    - co-IP
    - FRET and LRET
    - Co-expression of active
    and inactive isoforms
    - Binding cooperativity
    - Cross-linking

    Model of rhodopsin dimer


    complexed with arrestin

    Park et al., Biochemistry 43, 15643 (2004)


    Shutting down GPCR signaling

    • Deactivation of receptors occurs through


    phosphorylation and subsequent binding of β-arrestin
    (this leads clathrin-mediated endocytosis of the
    arrestin-receptor complex)
    • Deactivation of G-proteins occurs through intrinsic
    GTPase activity of the Gα subunit and subsequent
    reassembly of the inactive hetero-trimeric complex
    • Deactivation of second messengers occurs in part
    through arrestin-mediated recruitment of enzymes
    that degrade (or metabolize) the second messenger
    Residues in the human h2 adrenergic receptor that are important for receptor regulation. Possible sites for phosphorylation by
    protein kinases are indicated in gray circles. A C-terminal PDZ interaction motif is indicated that mediates interactions with
    NHERF1, NHERF2, and NSF scaffolding proteins. The highly conserved reference residue within each TMH is indicated with
    black circles and white text.
    Typically activation of a GPCR leads to (1) activation and inhibition of specific signaling pathways in the cell,
    (2) short-term desensitization mediated by phosphorylation of GPCRs by GRKs followed by β-arrestin
    binding to the GPCR, (3) endocytosis of the receptor, followed by postendocytic sorting of the receptor either
    (4) back to the plasma membrane or (5) to lysosomes for degradation.
    Arrestins also stimulate degradation of second-messenger molecules

    Following muscarinic receptor


    activation:
    - activation of the G-protein leads
    to production of DAG and IP3
    - arrestin was essential for
    conversion of DAG to PA
    - required recruitment of the
    arrestin-DAG kinase complex to the
    activated GPCR
    Nelson et al., Science 315 663 (2007)

    Previously shown that that arrestins


    recruit PDE4 to activated β-
    adrenergic receptors, stimulating
    degradation of cAMP
    Perry et al., Science 298 834 (2002)
    GPCRs
    • Gateway to a wide variety of G-protein
    coupled second messenger systems
    • Contain 7 TM helices
    • Extracellular N-terminal domain, cytoplasmic
    C-terminal tail
    • May function as dimers
    • Signaling mediated through rigid-body
    rearrangements of TM helices (esp. TMH6)

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