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  • PURIFICATION OF THE G PROTEIN-COUPLED RECEPTOR RHODOPSIN

    FOR STRUCTURAL STUDIES

    David Salom4, Ning Li4, Li Zhu1,3, Izabela Sokal1, and Krzysztof Palczewski1,2,3

    Departments of Ophthalmology1, Pharmacology2, and Chemistry3, University of

    Washington, Seattle, WA 98195, USA 4Novasite Pharmaceuticals Inc., San Diego, CA 92121, USA

    Key Words: G protein-coupled receptors, membrane proteins, photoreceptor cells,

    rhodopsin, purification of membrane proteins

    Correspondence to:

    Krzysztof Palczewski, Ph.D.

    University of Washington, Department of Ophthalmology

    Box 356485, Seattle, WA 98195-6485, USA

    Phone: +206-543-9074; Fax: +206-221-6784; E-mail: palczews@u.washington.edu

    and

    David Salom, Ph.D.

    Novasite Pharmaceuticals, Inc.

    11095 Flintkote Ave.

    San Diego, CA 92121, USA

    Phone: +858-638-8671; Fax: +858-597-4943; E-mail: dsalom@novasite.com

    Abbreviations used: BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; DM, n-dodecyl--

    D-maltoside; DTT, 1,4-dithio-DL-threitol; GPCR, G protein-coupled receptor, mAb, monoclonal

    antibody, NG, nonyl--D-glucoside; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate

    buffered saline; ROS, rod outer segment(s); SDS, sodium dodecyl sulphate; THM,

    transmembrane helix..

    1

    mailto:palczews@u.washington.edu

  • 1. INTRODUCTION

    2. RHODOPSIN AND OTHER GPCRs

    3. WORKING WITH RHODOPSIN

    4. ROD OUTER SEGMENT ISOLATION

    A. Materials

    B. Stock solutions

    C. Sucrose gradient procedure

    5. IMMUNOAFFINITY CHROMATOGRAPHY

    A. Coupling of antibody to solid support

    a. Purification of monoclonal antibodies

    b. Materials and solutions

    c. Coupling procedure

    B. Chromatographic purification from ROS extract

    a. Materials and solutions

    b. Chromatographic procedure

    C. Chromatographic purification from the whole retinal extract

    a. Buffers

    b. Purification

    6. NON-CHROMATOGRAPHIC PURIFICATION OF RHODOPSIN

    A. Stock solutions

    B. Precipitation of contaminant proteins

    C. DEAE-Sepharose chromatography

    7. CONCLUSIONS

    2

  • 1. INTRODUCTION

    Rhodopsin, the visual pigment expressed in rod photoreceptor cells of the vertebrate

    retina (Fig. 1A), consists of the apoprotein opsin and the chromophore 11-cis-retinal

    (Ebrey and Koutalos 2001; Hargrave 2001; Filipek et al. 2003b). The chromophore is

    bound to opsin via the protonated Schiff base linkage (Wang et al. 1980; Hubbard and

    Wald 1999). In vertebrates, rhodopsin is synthesized in the rod inner segment and

    vectorially transported to the rod outer segments (ROS) (Molday 1998; Papermaster

    2002). Perception of light starts with the photoisomerization of the chromophore 11-cis-

    retinal to all-trans-retinal and the formation of signaling metarhodopsin II (Meta II)

    (Okada et al. 2001). Catalytic activation of the G protein, transducin (Gt), by Meta II

    leads to a cascade of biochemical reactions termed phototransduction, resulting in a

    hyperpolarization of the plasma membrane of the rod cell (Polans et al. 1996; Fain et al.

    2001). Ultimately, these events within the rod cell lead to the reduced rate of the

    neurotransmitter release at the synaptic terminal, resulting in changes in the signaling

    state to the secondary retinal neurons.

    The cellular signaling upon binding of extracellular ligands to rhodopsin-like

    receptors and activation of G proteins by ligand-occupied receptor, termed G protein-

    coupled receptors (GPCRs), is a common mechanism in biology of signal transduction

    across the plasma membranes (Gether 2000; Ballesteros et al. 2001). More than a

    thousand GPCR genes, including sensory receptors, were identified in human genome,

    and today ~50% of all pharmaceutical targets are directed toward modulating the

    physiological responses of these receptors (Drews 2000; Ballesteros and Palczewski

    2001; Klabunde and Hessler 2002). Among this signaling superfamily of transmembrane

    receptors, rhodopsin is the best-studied prototypical member (Menon et al. 2001). Many

    useful methods to study rhodopsin have been developed that could be adapted to other

    GPCRs due to their structural, biochemical and functional similarities (Hargrave and

    McDowell 1992; Filipek et al. 2003a). Here, we describe several protocols for the

    purification of rhodopsin that will enable structural studies. These methods that have

    been adapted and refined in this study are based on previously described procedures first

    developed in the 70s (Hargrave 1976; Papermaster 1982; Oprian et al. 1987; Okada et al.

    1998).

    3

  • 2. RHODOPSIN AND OTHER GPCRs

    Vertebrate rhodopsin is one of the best-studied GPCRs because of its important role

    in vision, its high expression in the eye, the technical accessibility of its visual system for

    electrophysiological manipulations, and the fast and quantitative conversion of the

    chromophore ligand from inverse agonist to agonist by light (Ebrey and Koutalos 2001;

    Hargrave 2001; Okada et al. 2001; Okada and Palczewski 2001; Filipek et al. 2003b).

    Many of the methods used to study GPCRs structure and function were first developed

    to investigate rhodopsin. Structural studies on rhodopsin have also led the GPCR field,

    beginning with the understanding of the general topology of the receptor by electron

    microscopy of two-dimensional crystals of bovine and frog rhodopsin (Schertler et al.

    1993; Unger and Schertler 1995). The recent elucidation of the high-resolution three-

    dimensional structure of bovine rhodopsin further paved the way for our current

    understanding of the organization of the polypeptide chain of rhodopsin and its interplay

    with the chromophore (Palczewski et al. 2000; Okada et al. 2001; Okada and Palczewski

    2001; Teller et al. 2001). The structural core of GPCRs is characterized by a heptahelical

    bundle of transmembrane segments (TM) with an additional helix 8 running parallel to

    the membrane (reviewed in (Filipek et al. 2003b)).

    GPCRs contain an extracellular N-terminus, three extracellular loops (ECL), seven

    transmembrane helices (TMH), three intracellular loops (ICL), and an intracellular C-

    terminus. On the basis of the conservation pattern of the primary amino acid sequence,

    GPCRs have been grouped into three subfamilies (for review see Ref. (Ghanouni et al.

    2000)) (Table I). The majority of GPCRs belong to Family 1, which is defined by

    receptors whose sequences are related to rhodopsin (Mirzadegan et al. 2003). The overall

    sequence identity within GPCR Family 1 is relatively low (in many cases,

  • between these three families, although it is believed that these receptors share similar

    polypeptide fold (Filipek et al. 2003a; Malherbe et al. 2003).

    In general, GPCRs are activated as a result of the binding of extracellular, receptor-

    specific ligands to extracellular and/or transmembrane domains. Conformational changes

    of the receptor induced by the ligand are relayed to the cytoplasmic surface, allowing

    productive coupling of the receptor with a heterotrimeric G protein. In this context,

    rhodopsin, as well as other visual pigments, is unique in that its intrinsic ligand, 11-cis-

    retinal, is coupled via a protonated Schiff base (PSB) with the side-chain of

    transmembrane Lys296 residue. The 11-cis-retinylidene moiety acts as an inverse agonist,

    suppressing the activity of the receptor to an undetectable level. This feature is especially

    important for the function of rod cells, which contain ~108 molecules of rhodopsin per

    cell and specialize in detecting light at low intensities. Any spontaneous activation of

    rhodopsin present at such a high concentration would trigger phototransduction, lowering

    the overall light sensitivity (Ebrey and Koutalos 2001). The ground state of rhodopsin

    with its intrinsic ligand and its rigid extracellular structure appears to be specifically

    suited for maintaining the receptor in the inactive state.

    Rhodopsin has been described as a prototypical GPCR and the structural similarities

    between all GPCRs may suggest a common mechanism by which GPCRs activate G

    proteins (Mirzadegan et al. 2003) and how these receptors may self-organize within

    native membranes (Fotiadis et al. 2003; Liang et al. 2003).

    3. WORKING WITH RHODOPSIN

    Rhodopsin is a photosensitive membrane protein. Therefore, to avoid denaturing

    and/or photobleaching, there are several precautions that need to be taken when working

    with this receptor (see Fig. 5B). The following recommendations apply for any

    manipulation of rhodopsin (sections 4 through 6).

    Experiments with rhodopsin must be performed in a dark room with dim red light,

    using a Kodak 2-way Safelamp with Kodak 1A Safelight Filter (or a Kodak Adjustable

    Safelight Lamp with Kodak Safelight Filter No 2) and 15 W bulb. Although rhodopsin is

    stable for a short exposure up to ~55oC; experiments that take several hours should be

    carried out in a cold room to prevent bleaching and denaturation. To measure rhodopsin

    5

  • concentration in a sample, an aliquot of suspended membranes is solubilized in the

    detergent-containing buffer (1-10 mM n-dodecyl--D-maltoside (DM) or other

    alkylglucoside, 20 mM 1,3-bis[tris(hydroxymethyl)methylamino]-propane (BTP), 5-50

    mM

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