- Email: [email protected]
Analysis of structure-function from expression of G protein-coupled receptor fragments
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The combination of RASSL technology with the tet system provides a reversible molecular switch that allows researchers to control the timing, location, and specificity of G protein signaling in viva. With the completion of the Human Genome Project, all GPCRs will soon be identified. We can then focus on the significant challenge of understanding how this diverse family of proteins modulates physiological processes. Recent findings revealing the significance of dimerization,24 protein-protein interactions,25 and alternative splicing of GPCRs2(j suggest that the signaling and modulation of these proteins are even more complex than previously believed. RASSLs provide a new tool to help study these processes in viva and provide specific functional data for a variety of physiologically important signaling pathways.
24H. Mijhler and J.-M. Fritschy, Trends Pharmacol. Sci. 20,87 (1999). 25Y. Tang, L. A. Hu, W. E. Miller, N. Ringstad, R. A. Hall, J. A. Pitcher, P. DeCamilli, and R. J. Leikowitz, Proc. Natl. Acad. Sci. U.S.A. 96, 12559 (1999). 26G. J. Kilpatrick, F. M. Dautzenberg,G. R. Martin, and R. M. Eglen, Trends Pharmacol. Sci. 20,294 (1999).
[ 171 Analysis of Structure-Function from Expression of G Protein-Coupled Receptor Fragments By SADASHIVA S. KARNIK The family of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins (G proteins) consists of transmembrane receptors that transduce signals in response to light, odorants, hormones, neurotransmitters, and a variety of intracellular signals of unknown nature. 1,2G protein-coupled receptors (GPCRs) share a common structural motif consisting of seven transmembrane (7TM) a-helical segments separated by four extracellular segments and four cytoplasmic segments. The extracellular segments predominantly play a role in the folding and the assembly of the receptor. In a variety of peptide hormone receptors the extracellular domain is also involved in hormone binding. As a consequence of this role, an N-terminal hormone binding domain distinct from the 7TM structure is evolved in some receptors, which is structurally distinct and functionally autonomous. In the majority of GPCRs, the ligand pocket is formed by the TM domain. This structure is fundamental to signal generation in GPCRs. The intracellular regions interact ’ T. H. Ji, M. Grossmann,and I. Ji, J. Biol. Chem. 273, 17299 (1998). ’ U. Gether and B. K. Kobilka, J. Biol. Chem. 273,17979 (1998).
METHODS
IN ENZYMOLOGY,
VOL. 343
Copyright 0 2002 by Academic Press All rights of reproduction in any form reserved. 0376.6879/02 $35.00
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with G proteins and other cytoplasmic proteins in mediating the signals. Mutagenesis studies have played a major role in defining the structure and the molecular mechanisms that govern GPCR function. Structure-function analysis from expression of GPCR fragments has become a powerful tool in the study of GPCRs and other membrane proteins. The approach opens up new insights into tertiary structure analysis and molecular mechanisms governing GPCR functions. Promise of developing new tools for regulating specific GPCR functions has emerged in the study of several GPCRs. Cytoplasmic
Domain
Fragments
Mutagenesis of the cytoplasmic loops (CL) in GPCRs suggests a prominent role for the CL-3 in G protein activation; however, other cytoplasmic loops also play a role. Peptides derived from CL-2, CL-3, and CL-4 in a variety of GPCRs have been shown to block the receptor-specific interaction of G proteins.3-7 However, the exact role of each cytoplasmic loop in the specificity of G protein binding, stimulation of GDP release and GTP uptake in the receptor-bound G protein, or dissociation of G protein subunits from the receptor is not very well understood. Advances in protein engineering have demonstrated that it is possible to separate individual domains of multidomain proteins preserving the domain function. The G protein activating function in GPCRs is accomplished by the cytoplasic domain consisting of discontinuous loops connecting TM helices. Therefore, engineering a soluble G protein-activating domain of a GPCR would require extensive reengineering of the loops. This article describes the construction of TAPER (transducin activating protein engineered from rhodopsin) through a series of minigenes for expressing in Escherchia coli. Design of TAPER Constructs TAPER I contains four cytoplasmic segments of bovine rhodopsin (see Fig. 1)-loop I (Thr58-Asn73), loop II (Lys141-His’52), loop III (Lys231-Arg252), and loop IV (Asn310-Cys322)-and the octapeptide epitope (Thr340-A1a348) for the monoclonal antibody lD4. Because the conserved ERY sequence located at the cytoplasmic end of TM3 appears to play an important role in rhodopsin function, the segment Ile133-Cys’40 was designed to be a part of loop II in all TAPER constructs that contain this loop. Two of the cysteines, CYS’~’and CYSTICwere retained, 3 B. Ktinig, A. Arendt, J. H. McDowell, M. Kahlert, P. A. Hargrave, and K. P. Hofmann, Proc. Narl. Acad. Sci. U.S.A. 86,6878 (1989). 4 A. H. Cheung, C. H. Ruey-Ruey, M. P. Granziano, and C. D. Strader, FEES Lett. 279,277 (1991). 5 T. Okamoto, Y. Murayama, Y. Hayashi, M. Inagaki, E. Ogata, and I. Nishimoto, Cell 67,723 (1991). 6 T. Okamoto and I. Nishimoto, J. Viol. Chem. 267,8342 (1991). ’ H. M. Dalman and R. R. Neubig, J. Biol. Chem. 266,11025 (1991).
250
G PROTEIN-COUPLED RECEPTORS
iI71 lD4SpItopa
,
TM1
V
Y~USOA
TMa
TM3
TM4
lERVVWC~hplI
TM6
AO8M
TM6
I”“pIvIOlW4
,
TM7
-1
FIG. 1. Schematic representationof the cytoplasmic domain of bovine rhodopsin and TAPER polypeptides constructed.The residue numbering is according to bovine opsin. The boundaries of transmembranehelices and loops are chosen arbitrarily based on recent structure-function studies. The ERY sequencewas retained as an extension of transmembranehelix 3, which may be important for activating transducin. Palmitoylated Cys-322 and Cys-323 are indicated. The lD4 epitope was placed at the C-terminal end of all TAPER constructs.The tetrapeptidelinker sequenceis indicated.
whereasCys3t6was replacedwith an Ala. A tetrapeptidelinker consistingof amino acids Met, Ser, Gly, and Ala was used in different sequencebecausetheseamino acids occur with nearly equal preferencein different protein secondarystructural elements.Thereforethe linker sequenceis lesslikely to influencethe conformation assumedby the loops. Becausethe functional contribution of different loops to various stepsof the GTPasecycle of transducinis not clearly established,a series of TAPER constructsthat differ in the loop composition were also constructed. They are shown in Fig. 1.
FIG. 2. The synthetic gene encoding TAPER I. The gene was assembled from synthetic oligonucleotides and characterized as described in Ferretti et al8 Theremaining TAPER constructs contain the identical sequence except for the segment that is missing. Segments encoding tetrapeptide linkers are shown in boxes and the ribosome binding site is underlined.
Minigene Expression
A synthetic gene encodingTAPER I is shown in F ig. 2. M inigenesencoding different TAPER polypeptideswere constructedby the methodsdevelopedby Ferrettiandcolleagues,*which is describedin detail elsewherein this volume[ 191. To facilitate efficient expressionin E. co& a syntheticribosome-bindingsite was introducedupstreamof the initiator Met codon in eachm inigene.Combinations of the cytoplasmicloops in eachTAPER are illustrated in F ig. 1. For example, TAPER I containsall four cytoplasmicloops, whereasTAPER II containsonly loops II, III, and IV, which are thought to be important for G protein coupling, and TAPER III containsloops III and IV. An octapeptideepitopefor the monoclonal antibody, lD4 is presentat the C-terminal end of all TAPER constructs. This facilitated easydetectionof the TAPER by immunoblotting,as well as pu9,‘o Expressionof thesem inigenes rification by immunoaffinity chromatography. utilizes the bacteriophage T7 RNA polymeraseto drive transcriptionof the TAPER m inigene.”The E. coli strain,BL21(DE3), is a lysogenbearingthe bacteriophage 8 L. Ferretti, S. S. Kamik, H. G. Khorana, M. Nassal, and D. D. Oprian, Pmt. Nutl. Acad. Sci. U.S.A. 83,599 (1986). 9 R. S. Molday and D. McKenzie, Biochemistry 22,653 (1983). lo S. S. Kamik and H. G. Khorana, J. Bid. Chem. 265, 17520 (1990).
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T7 RNA polymerase gene under the control of the lac UV5 promoter. The second component of this system is the T7 promoter, which is located upstream of the TAPER minigene in the pT7-5 expression vector. Expression is initiated by the addition of 5 mM nonmetabolizable inducer isopropylthio+D-galactoside (IPTG). Addition of IPTG derepresses T7 RNA polymerase expression in turn, which will activate expression of the TAPER gene. Induction of Minigene Expression A single colony of the BL21(DE3)/pT7-5-TAPER transformant is used to inoculate 5 ml LB medium containing 50 pg/ml ampicillin. The overnight culture (37”) is then diluted lOOO-fold in 10 ml of enriched medium (2% tryptone, 1% yeast extract, 0.5% NaCl, 0.2% glycerol, and 50 mM KH2PO4, pH 7.2) containing ampicillin and grown overnight at 30” with shaking at 150-200 ‘pm. This culture is used to inoculate 1 liter of rich medium containing ampicillin in 2-liter Erlenmeyer flasks, maintaining shaking at 150-200 rpm at 30”. When the OD 650 nm reaches 0.3, IPTG (30 pA4) and chloramphenicol (1 pg/ml) are added. Chlorophenicol addition improves the expression of TAPER sometimes, but does not inhibit expression anytime. About 5 hr after the addition of the inducer, bacteria are collected by centrifugation at 4”. The cells are washed with ice-cold TES buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 2 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mg/ml benzamidine]. Cell pellets are frozen by immersion in liquid nitrogen and kept frozen at -80”. Lysis of E. coli About 5 g (wet weight) of a fully thawed cell pellet is suspended in 20 ml of TES buffer containing 10% sucrose and 10 mg/ml lysozyme (Sigma). Mix well and place on ice for 1 hr. Add 150 ml 50 mM Tris (pH 8.0) buffer, containing 10 mM MgC12 and 10 mM 2-mercaptoethanol. Mix well on ice, and sonicate using a probe sonicator until the viscosity is reduced. Add 500 ~1 DNase I (1 mg/ml stock) and incubate for 1 hr at 37”. Place on ice, add NaCl to a final concentration of 200 mM, mix for 30 min, and spin at 10,OOOgfor 10 min. The supematant is spun again at 50,000 g for 1 hr. Add CHAPS [ 10% (w/v) stock in water] to a final concentration of 0.5% (w/v), and spin at 50,000 g for 1 hr. TAPER is recovered in the supematant as indicated by the immunoblot analysis shown in Fig. 3A. PuriJcation of TAPER Peptides Further purification is carried out by immunoaffinity chromatography on lD4-Sepharose. The 50K supematant is mixed with 5 ml of lD4-Sepharose (50% I1 S Tabor 1 in “Current Protocols in Molecular Biology” (K. Janssen, series ed.), p. 16.2.1. Current Protocols; Green Publishing Associates, Inc., and John Wiley & Sons, Inc., 1987.
253
GPCR FRAGMENTS
[171 A. lW-
Sepharose
Flow
chromatography
Acid
Through
of TAPER
Eluata
-I IIIIIIlvVvIIIIIIIIvvvI
+---
B. Purified
TAPER
TAPER I
II III
Iv
v VI
FIG. 3. (A) Immunoaffinity purification of TAPER polypeptides from E. coli lysates. The cleared lysate prepared from lysed E. coli BL2 1(DE3) cells expressing various TAPER constructs was subjected to immunoafhnity chromatography on lD4-Sepharose as described in the text. Sample fractions (10 ~1) were resolved by SDS-PAGE on a 14% polyacrylamide gel. The resolved proteins were transferred to nitrocellulose for Western blot analysis with the lD4 monoclonal antibody as the primary antibody and the alkaline phosphatase-conjugated antimouse IgG as the secondary antibody. Protein bands were visualized by reaction with NBT and BClP according to directions from the manufacturer @omega). All sample buffers contained B-mercaptoethanol. (B) Purified TAPER samples (10 ~1) after dialysis were resolved on a 14% polyacrylamide gel and immunobloted as described earlier. Note that the resolution obtained on this gel does not distinguish differences in the size of TAPER polypeptides.
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G PROTEIN-COUPLED
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slurry prepared as described earlier) with an end-to-end mixing at 4” overnight. The Sepharose beads are recovered by a gentle spin at 3000 rpm on a refrigerated tabletop centrifuge. The resin is washed extensively (3 x 20 bed volumes each) with 50 mMTris-Cl (pH 6.5%), 200 mMNaC1, and 0.1% CHAPS. BoundTAPER is eluted from the column in 0.1 M acetic acid in 0.1% CHAPS. The eluate is mixed with Tris base to adjust the pH to 7.0, dialyzed against 10 mM Tris buffer (pH 7.5) containing 20% glycerol and 5 mM DTT, and stored at -20”. Protein concentration is determined, and the samples are analyzed by immunoblotting shown in Fig. 3B. Functional Assay for TAPER Two different functional assays are employed. In the first assay, the ability of TAPER to interfere with rhodopsin-transducin interaction is measured by following the light-dependent binding of [35S]GTPyS by transducin. In the second assay, the ability of TAPER to stimulate GTPase activity of transducin is measured by following [y-32P]GTP hydrolysis by transducin. Transducin is purified from bovine retinae according to the procedure of Fung et al. ‘* and is subjected to ion-exchange chromatography on a DE-52 column. The eluate from the DE-52 column is subjected to dialysis against 10 r&4 Tris buffer (pH 7.5) containing 50% glycerol, 2 mM MgC12, and 1 n-&f DTT and is stored at -20”. The assay mixture for [35S]GTPyS binding to transducin consists of 2 nM spectroscopically pure rhodopsin solubilized in 0.1% dodecyl maltoside, 2 p1J4 transducin, 4 @4 [35S]GTPyS in the assay buffer [lo mM Tris buffer (pH 7.5), 100 m&f NaCl, 5 mM MgC12, 2 n&f DTT, 0.01% dodecyl maltoside]. Purified TAPER is added to 100 nM final concentration. The reaction is initiated in the dark by the addition of [35S]GTPyS; after 1 min of incubation (20”), the assay mixture is illuminated at >495-nm light. Samples are withdrawn at 1-min intervals and filtered through nitrocellulose. Filters are washed five times with 5 ml of 10 mM Tris buffer (pH 7.5) 100 nut4 NaCl, 5 mM MgC12, and 2 m&Z DTT. Radioactivity retained on the filter is measured by scintillation counting. The assay mix for the measurement of GTPase activity of transducin consists of either 100 nM purified TAPER or 0.01 t-J4 light-activated pure rhodopsin in 0.1% dodecyl maltoside, 1.2 r&Y transducin, 1 pM [Y-~*P]GTP in the assay buffer [lo mM Tris buffer (pH 7.5), 100 mM NaCl, 5 mM MgC12, 2 rniW DTT, 0.01% dodecyl maltoside]. The assay is initiated by the addition of [Y-~*P]GTP and is followed for 20 min at 20”. The ym3*Pi released during the assay is extracted into a molybdic acid complex and estimated as described earlier. lo Table I shows the activity of various TAPER constructs in the two assays. All TAPER constructs, except TAPER IV, were effective in inhibiting transducin activation by the activated rhodopsin. However, none of the TAPER constructs were l2 B. K. K. Fung, J. B. Hurley, and L. Stryer, Proc. Nurl. Acd. Sci. U.S.A. 78, 152 (1981).
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TABLE I FUNCTIONAL ACTIVITY OF PUREED TAPER F’EFTUIES Relative transducin activation
Control TAPER TAPER TAPER TAPER TAPER TAPER
I II III IV V VI
Rhodopsin-dependent [35S]GTPyS binding
[Y-~‘P]GTP hydrolysis
l.oa 0.32 0.38 0.55 1.0 0.68 0.51
1.06
a Light-activatedrhodopsinactivated270 k 15 moltransducin/mol rhodopsin. This value was used as 1.0 to calculate the relative activity of each of the TAPERS (50-fold molar excess in the reaction). b Light-activated rhodopsin stimulated 40 f 5 mol/mol GTP hydrolysis. This value was used as 1.0 to calculate the GTPaseactivating potential of each TAPER construct (100 n&f final GTP concentration). Basal GTPase activity of transducin in our assay system was 4 * 2mol/mol.
very effective in the direct activation of transducin. Successful design and construction of a more effective transducin-activating protein domain, starting from the cytoplasmic loops of rhodopsin, could be useful for undertaking structural studies. However, the observation from TAPER studies is consistent with the hypothesis that critical contacts between GPCR and G proteins may involve residues within TM domain. The movement of TM helices may actually be necessary to bring critical residues to the receptor-G protein interface to initiate the biochemical events involved in nucleotide exchange. Previous studies employing purified, reconstituted G proteins and receptorderived peptides or toxins have demonstrated that specific peptides are capable of stimulating G proteins. 4-6 For example, peptides derived from the carboxyl region of the third cytoplasmic loop of /?-adrenergic receptor, MCMuscatine receptor, and az,-adrenergic receptor mimic the receptor, can directly activate G-proteins in vitro. However, most GPCR-derived peptides are effective in blocking receptor G protein interaction through competition. 7*13*14It has been proposed that this form of antagonism may provide a model for development of a class of receptor
t3 L. M. Luttrell, S. Cotecchia, .I. Ostrowski, H. Kendall, andR. .I. Letkowitz, Science 259,1453 (1993). l4 B. E. Hawes, L. M. Lutterell, S. T. Exum, and R. J. Leftkoitz, J. Viol. Chem. 269,15776 (1994).
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antagonists that specifically blocks the receptor G protein interaction. Studies of Luttrell et al. l3 suggest that the cellular expression of cytoplasmic loops of both a!m-adrenergic receptor and D~A dopamine receptor results in specific antagonism of the receptor G protein interaction in intact cells. Several possible mechanisms could be responsible for the observed antagonism. Competition between the loop peptides and the activated agonist-receptor complex is most likely, as the inhibition is overcome by an increase in receptor density. Receptor specificity of inhibition, i.e. am-loop peptide, is more effective in blocking the am-adrenergic receptor signal than any other receptor that uses Gdll suggesting that the mechanism of antagonism may be more complicated than simple receptor antagonism. The heterogeneity of G protein heterodynes and intermolecular interactions of CL-3 with other intracellular regions of the receptor may have significant impact on the specificity observed. Transmembrane
Domain
Fragments
Many different membrane proteins have been shown to be capable of assembly in functional form from two or more protein fragments.‘5-25 Examples include bacteriorhodopsin,‘5-‘7 &adrenergic receptor,ls muscarinic acetylcholine receptor,‘9v20 lactose permease?’ rhodopsin?2-24 and the yeast a-factor receptor.25 The assembled functional unit casually referred to as a “split receptor” could be a valuable research tool in many studies. The approach of using polypeptide fragments to study the mechanism of integral membrane protein folding and assembly was pioneered during investigations on bacteriorhodopsin, a seven transmembrane helical light-transducing proton pump. l5 Proteolysed polyp ep tide fragments containing one or more transmembrane segments were found to be able to insert into lipid vesicles, assemble with their complementary partners, and form a chromophore with all-truns retinal and a light-driven proton pump. A two-step model for folding of this family of proteins envisaged is depicted in Fig. 4, in which each of the transmembrane a! helices
I5 K S Huang, H. Bayley, M. J. Liao, E. London, and H. G. Khorana, .I. Viol. Chem. 256,3802 (1981). l6 T.‘i. Kahn and D. M. Engehnan, Biochemistry 31,6144 (1992). ” 0. K. Hansen, M. Pompejus, and H. J. Fritz, Biol. Chem. Hoppe-Seyler 375,715 (1994). l8 B. K. Kobilka, T. S. Kobila, K. Daniel, J. W. Regan, M. G. Caron, and R. J. Lefiowitz, Science 240, 1310 (1988). l9 R. Maggio, Z. Vogel, and J. Wess, FEBS I&t. 319, 195 (1993). *’ T. Schoneberg, J. Liu, and J. Wess, .I. Biol. Chem. 270,180oO (1995). *’ E. Bibi and H. R. Kaback, Proc. Natl. Acad. Sci. U.S.A. 87,432s (1990). 22 K. D. Ridge, S. S. J. Lee, and L. L. Yao, Proc. Natl. Acad. Sci. U.S.A. 92,3204 (1995). 23 H. Yu, M. Kono, T. D. Melcee, and D. D. Oprian, Biochemistry 34,14963 (1995). 24 K. D. Ridge, S. S. J. Lee, and N. G. Abdulaev, J. Viol. Chem. 271,786O (1996). 25 N. P. Martin, L. M. Lea&t, C. M. Sommers, and M. E. Dumont, Biochemistry 38,682 (1999).
GPCR FRAGMENTS 1. Cotransfection
of minigenes r, -
2. Fragments
3. Assembly
Kozak Flngment
encoding
sequence
b AM
1 coding
fold independently
of functional
each fragment
codon
,+
3‘ UTR
Kozak
sequence
Fragment 2 Coding
cmrihItron
b AM
co&n
3‘OTn intron
in membrane
receptor
A
B
c
D
E
F
0
FIG. 4. Schematic representation of assembly of the functional receptor from coexpressed complementary polypeptide fragments. The highly conserved disulfide linkage in the extracellular domain shown is expected to he formed after the assembly of fragments to form the functional receptor.
fold independently of each other, insert into the membrane, and following which assemble functional transmembrane bundle without major rearrangement. Complementary fragment assembly into functional entities has been demonstrated unequivocally in a number of polytopic transmembrane proteins since then. These studies have demonstrated that each of the polypeptide fragments appears capable of endoplasmic reticulum translocation and topogenesis. Singly expressed fragments are not degraded readily by cellular proteases suggesting that each helical segment is an independent folding domain. The experiments also suggest that complementary segments associate through a side-to-side interaction within the membrane. General Principles
The approach of using fragments of the receptor polypeptide needs demonstration that the cotransfection of gene fragments generates a functional split receptor that folds into a native conformation as judged by ligand selectivity. In rhodopsin, fragments generated by proteolytic cleavage remain associated in the membrane
258
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and after solubilization in detergent solution.14-22Recombinant expressionstudies indicate that coexpressionof two or three fragments allows the formation of noncovalently assembledGPCRs,which exhibit propertiessimilar to the wild-type receptor.For a detailed treatmentof this topic, the readeris referred to Refs. 2 l-25. Choosing Fragments for Expression
Split receptors exhibit functional activity comparableto that of the wild type when the split site did not disrupt segmentsknown to be important for function. For instance,a split in the C-terminal segmentof EF loop led to a -75% decrease in G protein coupling in both rhodopsin and /I-adrenergic receptor.A normal precaution that must be exercisedin selecting a split site is to carefully restrict to those regions of the polypeptide chain shown to be tolerant of mutagenic changes.For example,a split that resultsin a discontinuousTM helix or loop region that is important for helical alignment may not be assembledeasily into a stable split receptor. Expression of Fragments
For expression,in each case the fragments are encoded on separateexpression vectors so that the fragments can be expressedindividually or they can be coexpressed.Each fragment is cloned into the vector as a cassettecontaining a Kozak consensusribosome-binding sequence,CCACC, immediately 5’of the initiator methionine codon, ATG, followed by the coding sequenceof the fragment placed in frame with the ATG codon and ending with one or two nonsensecodons. Semiconfluent COS 1 cells (-2-3 x 106/60-mm dish) grown in Dulbeco’s minimal essentialmedium (DMEM) containing 10% bovine calf serum are transfected by the lipofectamine method per the manufacturer’srecommendation.In situ functional studies are carried out 48 hr after transfection. Transfectedcells are grown for 72 hr for the purposeof maximizing the protein level for ligand binding or functional reconstitution studies.Purification of the split receptor constructscan follow solubilization with either 1% (w/v) CHAPS or p-D-dodecyl maltoside. The details of solubilization and purification stepsneed careful standardizationfor each system. The cellular expressionof polypeptide fragments was examined by protein immunoblotting of whole cell detergent extracts with fragment-specific monoclonal antibodies.In general, the yield of the split receptor is -30% comparedto the yield of the wild-type receptor in the sameexpressionsystem. Application
The split receptor approach could be a general, rapid, and easy assay for the routine determination of tertiary contacts in single chain, polytopic, integral membrane proteins. Split receptors used in combination with techniques such as Cys cross linking and site-directed reporter attachment studies would prove
1171
GPCR FRAGMENTS
259
extremely useful. The approach is to use disulfide cross-linking of site-directed Cys mutations to demonstrate the proximal location of residue side chains within the protein. Cys residues can be engineered into split receptor constructs. Crosslinking can be detected readily by a mobility shift on SDS-PAGE. This approach has been used successfully in rhodopsin. 22-24 Traditionally, the disulfide crosslinking method has found limited application because it is restricted to proteins composed of multiple subunits. With split receptors, however, the limitation can be overcome. Cys cross-linking demonstrates that the two candidate residues are capable of coming close together enough to form a covalent bond in the protein.26 It is important to consider other interpretations of the observation in terms of protein structure and dynamics of protein conformations. A major factor for consideration is the trapping of random conformational fluctuation in the protein. For this reason, it is very important to functionally characterize the cross-linked proteins. In addition, analysis of the global data set obtained from a systematic Cys scan of the domain for a pattern of self-consistency can thwart such anomalies. Studies on functional heterodimerization and protein complementation in GPCR function elude to an entirely novel application of the split receptor approach.27,28 It should now be possible to design GPCR mutants and fragments that are able to interfere with the specific aspects of wild-type receptor function or folding. The experimental design and general principles will be identical to those described earlier for split receptors. Thus, the construction of minigenes to express carefully designed GPCR fragments could be extremely valuable in analysis of the tertiary structure of GPCR, elucidation of complex GPCR signaling networks, and development of novel in viva modifiers of GPCR functions. Therefore, this approach should be very valuable. Acknowledgments This work was supported in part by National Institute of Health Grants EY9704, HL57470 and an Established Investigator Award from the American Heart Association. I am grateful to Thomas Boyle, Shreeta Acharya, and Yasser Saad for the experimental work; and John Boros, Jingli Zhang, and Robin Lewis for assistance in the preparation of the manuscript.
26 J. J. Falke and D. E. Koshland, Jr., Science 237,1596 (1987). 27 Z. Zhu and J. Wess, Biochemistry 37,15773 (1998). 28 B. A. Jordan and L. A. Devi, Nature 399,697 (1999).
G PROTEIN-COUPLED RECEPTORS
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The combination of RASSL technology with the tet system provides a reversible molecular switch that allows researchers to control the timing, location, and specificity of G protein signaling in viva. With the completion of the Human Genome Project, all GPCRs will soon be identified. We can then focus on the significant challenge of understanding how this diverse family of proteins modulates physiological processes. Recent findings revealing the significance of dimerization,24 protein-protein interactions,25 and alternative splicing of GPCRs2(j suggest that the signaling and modulation of these proteins are even more complex than previously believed. RASSLs provide a new tool to help study these processes in viva and provide specific functional data for a variety of physiologically important signaling pathways.
24H. Mijhler and J.-M. Fritschy, Trends Pharmacol. Sci. 20,87 (1999). 25Y. Tang, L. A. Hu, W. E. Miller, N. Ringstad, R. A. Hall, J. A. Pitcher, P. DeCamilli, and R. J. Leikowitz, Proc. Natl. Acad. Sci. U.S.A. 96, 12559 (1999). 26G. J. Kilpatrick, F. M. Dautzenberg,G. R. Martin, and R. M. Eglen, Trends Pharmacol. Sci. 20,294 (1999).
[ 171 Analysis of Structure-Function from Expression of G Protein-Coupled Receptor Fragments By SADASHIVA S. KARNIK The family of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins (G proteins) consists of transmembrane receptors that transduce signals in response to light, odorants, hormones, neurotransmitters, and a variety of intracellular signals of unknown nature. 1,2G protein-coupled receptors (GPCRs) share a common structural motif consisting of seven transmembrane (7TM) a-helical segments separated by four extracellular segments and four cytoplasmic segments. The extracellular segments predominantly play a role in the folding and the assembly of the receptor. In a variety of peptide hormone receptors the extracellular domain is also involved in hormone binding. As a consequence of this role, an N-terminal hormone binding domain distinct from the 7TM structure is evolved in some receptors, which is structurally distinct and functionally autonomous. In the majority of GPCRs, the ligand pocket is formed by the TM domain. This structure is fundamental to signal generation in GPCRs. The intracellular regions interact ’ T. H. Ji, M. Grossmann,and I. Ji, J. Biol. Chem. 273, 17299 (1998). ’ U. Gether and B. K. Kobilka, J. Biol. Chem. 273,17979 (1998).
METHODS
IN ENZYMOLOGY,
VOL. 343
Copyright 0 2002 by Academic Press All rights of reproduction in any form reserved. 0376.6879/02 $35.00
GPCR FRAGMENTS
1171
249
with G proteins and other cytoplasmic proteins in mediating the signals. Mutagenesis studies have played a major role in defining the structure and the molecular mechanisms that govern GPCR function. Structure-function analysis from expression of GPCR fragments has become a powerful tool in the study of GPCRs and other membrane proteins. The approach opens up new insights into tertiary structure analysis and molecular mechanisms governing GPCR functions. Promise of developing new tools for regulating specific GPCR functions has emerged in the study of several GPCRs. Cytoplasmic
Domain
Fragments
Mutagenesis of the cytoplasmic loops (CL) in GPCRs suggests a prominent role for the CL-3 in G protein activation; however, other cytoplasmic loops also play a role. Peptides derived from CL-2, CL-3, and CL-4 in a variety of GPCRs have been shown to block the receptor-specific interaction of G proteins.3-7 However, the exact role of each cytoplasmic loop in the specificity of G protein binding, stimulation of GDP release and GTP uptake in the receptor-bound G protein, or dissociation of G protein subunits from the receptor is not very well understood. Advances in protein engineering have demonstrated that it is possible to separate individual domains of multidomain proteins preserving the domain function. The G protein activating function in GPCRs is accomplished by the cytoplasic domain consisting of discontinuous loops connecting TM helices. Therefore, engineering a soluble G protein-activating domain of a GPCR would require extensive reengineering of the loops. This article describes the construction of TAPER (transducin activating protein engineered from rhodopsin) through a series of minigenes for expressing in Escherchia coli. Design of TAPER Constructs TAPER I contains four cytoplasmic segments of bovine rhodopsin (see Fig. 1)-loop I (Thr58-Asn73), loop II (Lys141-His’52), loop III (Lys231-Arg252), and loop IV (Asn310-Cys322)-and the octapeptide epitope (Thr340-A1a348) for the monoclonal antibody lD4. Because the conserved ERY sequence located at the cytoplasmic end of TM3 appears to play an important role in rhodopsin function, the segment Ile133-Cys’40 was designed to be a part of loop II in all TAPER constructs that contain this loop. Two of the cysteines, CYS’~’and CYSTICwere retained, 3 B. Ktinig, A. Arendt, J. H. McDowell, M. Kahlert, P. A. Hargrave, and K. P. Hofmann, Proc. Narl. Acad. Sci. U.S.A. 86,6878 (1989). 4 A. H. Cheung, C. H. Ruey-Ruey, M. P. Granziano, and C. D. Strader, FEES Lett. 279,277 (1991). 5 T. Okamoto, Y. Murayama, Y. Hayashi, M. Inagaki, E. Ogata, and I. Nishimoto, Cell 67,723 (1991). 6 T. Okamoto and I. Nishimoto, J. Viol. Chem. 267,8342 (1991). ’ H. M. Dalman and R. R. Neubig, J. Biol. Chem. 266,11025 (1991).
250
G PROTEIN-COUPLED RECEPTORS
iI71 lD4SpItopa
,
TM1
V
Y~USOA
TMa
TM3
TM4
lERVVWC~hplI
TM6
AO8M
TM6
I”“pIvIOlW4
,
TM7
-1
FIG. 1. Schematic representationof the cytoplasmic domain of bovine rhodopsin and TAPER polypeptides constructed.The residue numbering is according to bovine opsin. The boundaries of transmembranehelices and loops are chosen arbitrarily based on recent structure-function studies. The ERY sequencewas retained as an extension of transmembranehelix 3, which may be important for activating transducin. Palmitoylated Cys-322 and Cys-323 are indicated. The lD4 epitope was placed at the C-terminal end of all TAPER constructs.The tetrapeptidelinker sequenceis indicated.
whereasCys3t6was replacedwith an Ala. A tetrapeptidelinker consistingof amino acids Met, Ser, Gly, and Ala was used in different sequencebecausetheseamino acids occur with nearly equal preferencein different protein secondarystructural elements.Thereforethe linker sequenceis lesslikely to influencethe conformation assumedby the loops. Becausethe functional contribution of different loops to various stepsof the GTPasecycle of transducinis not clearly established,a series of TAPER constructsthat differ in the loop composition were also constructed. They are shown in Fig. 1.
FIG. 2. The synthetic gene encoding TAPER I. The gene was assembled from synthetic oligonucleotides and characterized as described in Ferretti et al8 Theremaining TAPER constructs contain the identical sequence except for the segment that is missing. Segments encoding tetrapeptide linkers are shown in boxes and the ribosome binding site is underlined.
Minigene Expression
A synthetic gene encodingTAPER I is shown in F ig. 2. M inigenesencoding different TAPER polypeptideswere constructedby the methodsdevelopedby Ferrettiandcolleagues,*which is describedin detail elsewherein this volume[ 191. To facilitate efficient expressionin E. co& a syntheticribosome-bindingsite was introducedupstreamof the initiator Met codon in eachm inigene.Combinations of the cytoplasmicloops in eachTAPER are illustrated in F ig. 1. For example, TAPER I containsall four cytoplasmicloops, whereasTAPER II containsonly loops II, III, and IV, which are thought to be important for G protein coupling, and TAPER III containsloops III and IV. An octapeptideepitopefor the monoclonal antibody, lD4 is presentat the C-terminal end of all TAPER constructs. This facilitated easydetectionof the TAPER by immunoblotting,as well as pu9,‘o Expressionof thesem inigenes rification by immunoaffinity chromatography. utilizes the bacteriophage T7 RNA polymeraseto drive transcriptionof the TAPER m inigene.”The E. coli strain,BL21(DE3), is a lysogenbearingthe bacteriophage 8 L. Ferretti, S. S. Kamik, H. G. Khorana, M. Nassal, and D. D. Oprian, Pmt. Nutl. Acad. Sci. U.S.A. 83,599 (1986). 9 R. S. Molday and D. McKenzie, Biochemistry 22,653 (1983). lo S. S. Kamik and H. G. Khorana, J. Bid. Chem. 265, 17520 (1990).
252
G PROTEIN-COUPLED RECEPTORS
1171
T7 RNA polymerase gene under the control of the lac UV5 promoter. The second component of this system is the T7 promoter, which is located upstream of the TAPER minigene in the pT7-5 expression vector. Expression is initiated by the addition of 5 mM nonmetabolizable inducer isopropylthio+D-galactoside (IPTG). Addition of IPTG derepresses T7 RNA polymerase expression in turn, which will activate expression of the TAPER gene. Induction of Minigene Expression A single colony of the BL21(DE3)/pT7-5-TAPER transformant is used to inoculate 5 ml LB medium containing 50 pg/ml ampicillin. The overnight culture (37”) is then diluted lOOO-fold in 10 ml of enriched medium (2% tryptone, 1% yeast extract, 0.5% NaCl, 0.2% glycerol, and 50 mM KH2PO4, pH 7.2) containing ampicillin and grown overnight at 30” with shaking at 150-200 ‘pm. This culture is used to inoculate 1 liter of rich medium containing ampicillin in 2-liter Erlenmeyer flasks, maintaining shaking at 150-200 rpm at 30”. When the OD 650 nm reaches 0.3, IPTG (30 pA4) and chloramphenicol (1 pg/ml) are added. Chlorophenicol addition improves the expression of TAPER sometimes, but does not inhibit expression anytime. About 5 hr after the addition of the inducer, bacteria are collected by centrifugation at 4”. The cells are washed with ice-cold TES buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 2 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mg/ml benzamidine]. Cell pellets are frozen by immersion in liquid nitrogen and kept frozen at -80”. Lysis of E. coli About 5 g (wet weight) of a fully thawed cell pellet is suspended in 20 ml of TES buffer containing 10% sucrose and 10 mg/ml lysozyme (Sigma). Mix well and place on ice for 1 hr. Add 150 ml 50 mM Tris (pH 8.0) buffer, containing 10 mM MgC12 and 10 mM 2-mercaptoethanol. Mix well on ice, and sonicate using a probe sonicator until the viscosity is reduced. Add 500 ~1 DNase I (1 mg/ml stock) and incubate for 1 hr at 37”. Place on ice, add NaCl to a final concentration of 200 mM, mix for 30 min, and spin at 10,OOOgfor 10 min. The supematant is spun again at 50,000 g for 1 hr. Add CHAPS [ 10% (w/v) stock in water] to a final concentration of 0.5% (w/v), and spin at 50,000 g for 1 hr. TAPER is recovered in the supematant as indicated by the immunoblot analysis shown in Fig. 3A. PuriJcation of TAPER Peptides Further purification is carried out by immunoaffinity chromatography on lD4-Sepharose. The 50K supematant is mixed with 5 ml of lD4-Sepharose (50% I1 S Tabor 1 in “Current Protocols in Molecular Biology” (K. Janssen, series ed.), p. 16.2.1. Current Protocols; Green Publishing Associates, Inc., and John Wiley & Sons, Inc., 1987.
253
GPCR FRAGMENTS
[171 A. lW-
Sepharose
Flow
chromatography
Acid
Through
of TAPER
Eluata
-I IIIIIIlvVvIIIIIIIIvvvI
+---
B. Purified
TAPER
TAPER I
II III
Iv
v VI
FIG. 3. (A) Immunoaffinity purification of TAPER polypeptides from E. coli lysates. The cleared lysate prepared from lysed E. coli BL2 1(DE3) cells expressing various TAPER constructs was subjected to immunoafhnity chromatography on lD4-Sepharose as described in the text. Sample fractions (10 ~1) were resolved by SDS-PAGE on a 14% polyacrylamide gel. The resolved proteins were transferred to nitrocellulose for Western blot analysis with the lD4 monoclonal antibody as the primary antibody and the alkaline phosphatase-conjugated antimouse IgG as the secondary antibody. Protein bands were visualized by reaction with NBT and BClP according to directions from the manufacturer @omega). All sample buffers contained B-mercaptoethanol. (B) Purified TAPER samples (10 ~1) after dialysis were resolved on a 14% polyacrylamide gel and immunobloted as described earlier. Note that the resolution obtained on this gel does not distinguish differences in the size of TAPER polypeptides.
254
G PROTEIN-COUPLED
RECEPTORS
[I71
slurry prepared as described earlier) with an end-to-end mixing at 4” overnight. The Sepharose beads are recovered by a gentle spin at 3000 rpm on a refrigerated tabletop centrifuge. The resin is washed extensively (3 x 20 bed volumes each) with 50 mMTris-Cl (pH 6.5%), 200 mMNaC1, and 0.1% CHAPS. BoundTAPER is eluted from the column in 0.1 M acetic acid in 0.1% CHAPS. The eluate is mixed with Tris base to adjust the pH to 7.0, dialyzed against 10 mM Tris buffer (pH 7.5) containing 20% glycerol and 5 mM DTT, and stored at -20”. Protein concentration is determined, and the samples are analyzed by immunoblotting shown in Fig. 3B. Functional Assay for TAPER Two different functional assays are employed. In the first assay, the ability of TAPER to interfere with rhodopsin-transducin interaction is measured by following the light-dependent binding of [35S]GTPyS by transducin. In the second assay, the ability of TAPER to stimulate GTPase activity of transducin is measured by following [y-32P]GTP hydrolysis by transducin. Transducin is purified from bovine retinae according to the procedure of Fung et al. ‘* and is subjected to ion-exchange chromatography on a DE-52 column. The eluate from the DE-52 column is subjected to dialysis against 10 r&4 Tris buffer (pH 7.5) containing 50% glycerol, 2 mM MgC12, and 1 n-&f DTT and is stored at -20”. The assay mixture for [35S]GTPyS binding to transducin consists of 2 nM spectroscopically pure rhodopsin solubilized in 0.1% dodecyl maltoside, 2 p1J4 transducin, 4 @4 [35S]GTPyS in the assay buffer [lo mM Tris buffer (pH 7.5), 100 m&f NaCl, 5 mM MgC12, 2 n&f DTT, 0.01% dodecyl maltoside]. Purified TAPER is added to 100 nM final concentration. The reaction is initiated in the dark by the addition of [35S]GTPyS; after 1 min of incubation (20”), the assay mixture is illuminated at >495-nm light. Samples are withdrawn at 1-min intervals and filtered through nitrocellulose. Filters are washed five times with 5 ml of 10 mM Tris buffer (pH 7.5) 100 nut4 NaCl, 5 mM MgC12, and 2 m&Z DTT. Radioactivity retained on the filter is measured by scintillation counting. The assay mix for the measurement of GTPase activity of transducin consists of either 100 nM purified TAPER or 0.01 t-J4 light-activated pure rhodopsin in 0.1% dodecyl maltoside, 1.2 r&Y transducin, 1 pM [Y-~*P]GTP in the assay buffer [lo mM Tris buffer (pH 7.5), 100 mM NaCl, 5 mM MgC12, 2 rniW DTT, 0.01% dodecyl maltoside]. The assay is initiated by the addition of [Y-~*P]GTP and is followed for 20 min at 20”. The ym3*Pi released during the assay is extracted into a molybdic acid complex and estimated as described earlier. lo Table I shows the activity of various TAPER constructs in the two assays. All TAPER constructs, except TAPER IV, were effective in inhibiting transducin activation by the activated rhodopsin. However, none of the TAPER constructs were l2 B. K. K. Fung, J. B. Hurley, and L. Stryer, Proc. Nurl. Acd. Sci. U.S.A. 78, 152 (1981).
255
GPCR FRAGMENTS
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TABLE I FUNCTIONAL ACTIVITY OF PUREED TAPER F’EFTUIES Relative transducin activation
Control TAPER TAPER TAPER TAPER TAPER TAPER
I II III IV V VI
Rhodopsin-dependent [35S]GTPyS binding
[Y-~‘P]GTP hydrolysis
l.oa 0.32 0.38 0.55 1.0 0.68 0.51
1.06
a Light-activatedrhodopsinactivated270 k 15 moltransducin/mol rhodopsin. This value was used as 1.0 to calculate the relative activity of each of the TAPERS (50-fold molar excess in the reaction). b Light-activated rhodopsin stimulated 40 f 5 mol/mol GTP hydrolysis. This value was used as 1.0 to calculate the GTPaseactivating potential of each TAPER construct (100 n&f final GTP concentration). Basal GTPase activity of transducin in our assay system was 4 * 2mol/mol.
very effective in the direct activation of transducin. Successful design and construction of a more effective transducin-activating protein domain, starting from the cytoplasmic loops of rhodopsin, could be useful for undertaking structural studies. However, the observation from TAPER studies is consistent with the hypothesis that critical contacts between GPCR and G proteins may involve residues within TM domain. The movement of TM helices may actually be necessary to bring critical residues to the receptor-G protein interface to initiate the biochemical events involved in nucleotide exchange. Previous studies employing purified, reconstituted G proteins and receptorderived peptides or toxins have demonstrated that specific peptides are capable of stimulating G proteins. 4-6 For example, peptides derived from the carboxyl region of the third cytoplasmic loop of /?-adrenergic receptor, MCMuscatine receptor, and az,-adrenergic receptor mimic the receptor, can directly activate G-proteins in vitro. However, most GPCR-derived peptides are effective in blocking receptor G protein interaction through competition. 7*13*14It has been proposed that this form of antagonism may provide a model for development of a class of receptor
t3 L. M. Luttrell, S. Cotecchia, .I. Ostrowski, H. Kendall, andR. .I. Letkowitz, Science 259,1453 (1993). l4 B. E. Hawes, L. M. Lutterell, S. T. Exum, and R. J. Leftkoitz, J. Viol. Chem. 269,15776 (1994).
256
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RECEPTORS
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antagonists that specifically blocks the receptor G protein interaction. Studies of Luttrell et al. l3 suggest that the cellular expression of cytoplasmic loops of both a!m-adrenergic receptor and D~A dopamine receptor results in specific antagonism of the receptor G protein interaction in intact cells. Several possible mechanisms could be responsible for the observed antagonism. Competition between the loop peptides and the activated agonist-receptor complex is most likely, as the inhibition is overcome by an increase in receptor density. Receptor specificity of inhibition, i.e. am-loop peptide, is more effective in blocking the am-adrenergic receptor signal than any other receptor that uses Gdll suggesting that the mechanism of antagonism may be more complicated than simple receptor antagonism. The heterogeneity of G protein heterodynes and intermolecular interactions of CL-3 with other intracellular regions of the receptor may have significant impact on the specificity observed. Transmembrane
Domain
Fragments
Many different membrane proteins have been shown to be capable of assembly in functional form from two or more protein fragments.‘5-25 Examples include bacteriorhodopsin,‘5-‘7 &adrenergic receptor,ls muscarinic acetylcholine receptor,‘9v20 lactose permease?’ rhodopsin?2-24 and the yeast a-factor receptor.25 The assembled functional unit casually referred to as a “split receptor” could be a valuable research tool in many studies. The approach of using polypeptide fragments to study the mechanism of integral membrane protein folding and assembly was pioneered during investigations on bacteriorhodopsin, a seven transmembrane helical light-transducing proton pump. l5 Proteolysed polyp ep tide fragments containing one or more transmembrane segments were found to be able to insert into lipid vesicles, assemble with their complementary partners, and form a chromophore with all-truns retinal and a light-driven proton pump. A two-step model for folding of this family of proteins envisaged is depicted in Fig. 4, in which each of the transmembrane a! helices
I5 K S Huang, H. Bayley, M. J. Liao, E. London, and H. G. Khorana, .I. Viol. Chem. 256,3802 (1981). l6 T.‘i. Kahn and D. M. Engehnan, Biochemistry 31,6144 (1992). ” 0. K. Hansen, M. Pompejus, and H. J. Fritz, Biol. Chem. Hoppe-Seyler 375,715 (1994). l8 B. K. Kobilka, T. S. Kobila, K. Daniel, J. W. Regan, M. G. Caron, and R. J. Lefiowitz, Science 240, 1310 (1988). l9 R. Maggio, Z. Vogel, and J. Wess, FEBS I&t. 319, 195 (1993). *’ T. Schoneberg, J. Liu, and J. Wess, .I. Biol. Chem. 270,180oO (1995). *’ E. Bibi and H. R. Kaback, Proc. Natl. Acad. Sci. U.S.A. 87,432s (1990). 22 K. D. Ridge, S. S. J. Lee, and L. L. Yao, Proc. Natl. Acad. Sci. U.S.A. 92,3204 (1995). 23 H. Yu, M. Kono, T. D. Melcee, and D. D. Oprian, Biochemistry 34,14963 (1995). 24 K. D. Ridge, S. S. J. Lee, and N. G. Abdulaev, J. Viol. Chem. 271,786O (1996). 25 N. P. Martin, L. M. Lea&t, C. M. Sommers, and M. E. Dumont, Biochemistry 38,682 (1999).
GPCR FRAGMENTS 1. Cotransfection
of minigenes r, -
2. Fragments
3. Assembly
Kozak Flngment
encoding
sequence
b AM
1 coding
fold independently
of functional
each fragment
codon
,+
3‘ UTR
Kozak
sequence
Fragment 2 Coding
cmrihItron
b AM
co&n
3‘OTn intron
in membrane
receptor
A
B
c
D
E
F
0
FIG. 4. Schematic representation of assembly of the functional receptor from coexpressed complementary polypeptide fragments. The highly conserved disulfide linkage in the extracellular domain shown is expected to he formed after the assembly of fragments to form the functional receptor.
fold independently of each other, insert into the membrane, and following which assemble functional transmembrane bundle without major rearrangement. Complementary fragment assembly into functional entities has been demonstrated unequivocally in a number of polytopic transmembrane proteins since then. These studies have demonstrated that each of the polypeptide fragments appears capable of endoplasmic reticulum translocation and topogenesis. Singly expressed fragments are not degraded readily by cellular proteases suggesting that each helical segment is an independent folding domain. The experiments also suggest that complementary segments associate through a side-to-side interaction within the membrane. General Principles
The approach of using fragments of the receptor polypeptide needs demonstration that the cotransfection of gene fragments generates a functional split receptor that folds into a native conformation as judged by ligand selectivity. In rhodopsin, fragments generated by proteolytic cleavage remain associated in the membrane
258
G PROTEIN-COUPLED
RECEPTORS
1171
and after solubilization in detergent solution.14-22Recombinant expressionstudies indicate that coexpressionof two or three fragments allows the formation of noncovalently assembledGPCRs,which exhibit propertiessimilar to the wild-type receptor.For a detailed treatmentof this topic, the readeris referred to Refs. 2 l-25. Choosing Fragments for Expression
Split receptors exhibit functional activity comparableto that of the wild type when the split site did not disrupt segmentsknown to be important for function. For instance,a split in the C-terminal segmentof EF loop led to a -75% decrease in G protein coupling in both rhodopsin and /I-adrenergic receptor.A normal precaution that must be exercisedin selecting a split site is to carefully restrict to those regions of the polypeptide chain shown to be tolerant of mutagenic changes.For example,a split that resultsin a discontinuousTM helix or loop region that is important for helical alignment may not be assembledeasily into a stable split receptor. Expression of Fragments
For expression,in each case the fragments are encoded on separateexpression vectors so that the fragments can be expressedindividually or they can be coexpressed.Each fragment is cloned into the vector as a cassettecontaining a Kozak consensusribosome-binding sequence,CCACC, immediately 5’of the initiator methionine codon, ATG, followed by the coding sequenceof the fragment placed in frame with the ATG codon and ending with one or two nonsensecodons. Semiconfluent COS 1 cells (-2-3 x 106/60-mm dish) grown in Dulbeco’s minimal essentialmedium (DMEM) containing 10% bovine calf serum are transfected by the lipofectamine method per the manufacturer’srecommendation.In situ functional studies are carried out 48 hr after transfection. Transfectedcells are grown for 72 hr for the purposeof maximizing the protein level for ligand binding or functional reconstitution studies.Purification of the split receptor constructscan follow solubilization with either 1% (w/v) CHAPS or p-D-dodecyl maltoside. The details of solubilization and purification stepsneed careful standardizationfor each system. The cellular expressionof polypeptide fragments was examined by protein immunoblotting of whole cell detergent extracts with fragment-specific monoclonal antibodies.In general, the yield of the split receptor is -30% comparedto the yield of the wild-type receptor in the sameexpressionsystem. Application
The split receptor approach could be a general, rapid, and easy assay for the routine determination of tertiary contacts in single chain, polytopic, integral membrane proteins. Split receptors used in combination with techniques such as Cys cross linking and site-directed reporter attachment studies would prove
1171
GPCR FRAGMENTS
259
extremely useful. The approach is to use disulfide cross-linking of site-directed Cys mutations to demonstrate the proximal location of residue side chains within the protein. Cys residues can be engineered into split receptor constructs. Crosslinking can be detected readily by a mobility shift on SDS-PAGE. This approach has been used successfully in rhodopsin. 22-24 Traditionally, the disulfide crosslinking method has found limited application because it is restricted to proteins composed of multiple subunits. With split receptors, however, the limitation can be overcome. Cys cross-linking demonstrates that the two candidate residues are capable of coming close together enough to form a covalent bond in the protein.26 It is important to consider other interpretations of the observation in terms of protein structure and dynamics of protein conformations. A major factor for consideration is the trapping of random conformational fluctuation in the protein. For this reason, it is very important to functionally characterize the cross-linked proteins. In addition, analysis of the global data set obtained from a systematic Cys scan of the domain for a pattern of self-consistency can thwart such anomalies. Studies on functional heterodimerization and protein complementation in GPCR function elude to an entirely novel application of the split receptor approach.27,28 It should now be possible to design GPCR mutants and fragments that are able to interfere with the specific aspects of wild-type receptor function or folding. The experimental design and general principles will be identical to those described earlier for split receptors. Thus, the construction of minigenes to express carefully designed GPCR fragments could be extremely valuable in analysis of the tertiary structure of GPCR, elucidation of complex GPCR signaling networks, and development of novel in viva modifiers of GPCR functions. Therefore, this approach should be very valuable. Acknowledgments This work was supported in part by National Institute of Health Grants EY9704, HL57470 and an Established Investigator Award from the American Heart Association. I am grateful to Thomas Boyle, Shreeta Acharya, and Yasser Saad for the experimental work; and John Boros, Jingli Zhang, and Robin Lewis for assistance in the preparation of the manuscript.
26 J. J. Falke and D. E. Koshland, Jr., Science 237,1596 (1987). 27 Z. Zhu and J. Wess, Biochemistry 37,15773 (1998). 28 B. A. Jordan and L. A. Devi, Nature 399,697 (1999).