purification of the g-protein-coupled receptor rhodopsin for structural ??2008-08-05purification of...
Post on 07-May-2018
Embed Size (px)
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
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: firstname.lastname@example.org
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: email@example.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,
2. RHODOPSIN AND OTHER GPCRs
3. WORKING WITH RHODOPSIN
4. ROD OUTER SEGMENT ISOLATION
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
6. NON-CHROMATOGRAPHIC PURIFICATION OF RHODOPSIN
A. Stock solutions
B. Precipitation of contaminant proteins
C. DEAE-Sepharose chromatography
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.
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
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