THE JOLIRNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 20, Issue of May 20, pp. 14715-14722, 1994 Printed in U.S.A.

Insertional Mutagenesis as a Probe of Rhodopsin’s Topography, Stability, and Activity*

(Received for publication, January 4, 1994, and in revised form, March 14, 1994)

Jim0 BorjiginS and Jeremy NathansSOn From the Department of $Neuroscience, Department of $Molecular Biology and Genetics and the Department of §Ophthalmology, §Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

This paper reports a study of rhodopsin’s structure and function using insertional mutagenesis with a flex- ible epitope. Sixteen rhodopsin derivatives were con- structed, each of which carried a 12-amino acid epitope derived from the c-Myc protein flanked by penta-glycine linkers. For eight of the insertion mutants, the mem- brane sidedness of the epitope insert was determined by immunostaining of intact or permeabilized cells. The re- sults confirm the sidedness of each of the six helix con- necting loops and the amino and carboxyl termini as postulated by the current seven-helix models of G- protein-coupled receptors and provide the first experi- mental evidence for the existence of the third extracel- lular loop. In general, inserts that were either closer to the amino terminus or on the extracellular face were more likely to disrupt folding and/or stability than were inserts near the carboxyl terminus or on the cytosolic face. Epitope insertion at positions 139 or 239, in the second and third cytosolic loops, respectively, failed to activate transducin, whereas an insertion at position 333 in the carboxyl-terminal tail was fully functional. The experimental approach described here should prove generally useful for elucidating structural and functional properties of both membrane and globular proteins.

Rhodopsin is the light absorbing protein that mediates vision in dim light. I t consists of an integral membrane apoprotein, opsin, and a covalently bound chromophore, 11-cis-retinal. Light absorption induces retinal to isomerize from ll-cis to all-trans, which triggers a series of conformational changes in the attached protein. One of the conformational intermediates, metarhodopsin 11, interacts with the photoreceptor-specific G- protein, transducin, thereby activating the phototransduction cascade. Because of its role as a receptor, its amenability to spectroscopic methods, and its availability in large quantities, rhodopsin has become one of the most intensively studied in- tegral membrane proteins.

Rhodopsin’s polypeptide chain is 348 amino acids in length, and consists of seven clusters of hydrophobic amino acids in- terrupted by clusters of hydrophilic amino acids (Ovchinnikov et al., 1983; Hargrave et al., 1983; Nathans and Hogness, 1983). ‘lbpographic mapping using lectin binding, limited proteolysis,

and the National Eye Institute (National Institutes of Health). The * This work was supported by the Howard Hughes Medical Institute

costs of publication of this article were defrayed in part by the payment

tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate of page charges. This article must therefore be hereby marked “adver-

this fact.

North Wolfe St., Johns Hopkins University School of Medicine, Balti- ll To whom correspondence should be addressed: 805 PCTB, 725

more, MD 21205. Tel.: 410-955-4679; Fax: 410-614-0827.

and chemical derivatization (Rohlich, 1976; Martynov et a l . , 1983; Barclay and Findlay, 1984) shows that the amino termi- nus faces the lumen of the rod outer segment disc membrane (topologically equivalent to the outside of the cell), the carboxyl terminus faces the cytosol, and there exist three cytosolic and at least two extracellular loops. A third extracellular loop, con- necting the sixth and seventh membrane spanning a-helices, is predicted by the hydropathy profile. This general model, in which seven membrane spanning a-helices are linked by hy- drophilic loops, is presumed to apply to all G-protein coupled receptors. The recently determined two-dimensional projection map of rhodopsin at 9-L% resolution reveals seven major densi- ties that are likely to represent the predicted membrane-span- ning a-helices (Schertler et al., 1993).

During the past several years, experiments with mutant opsins produced in tissue culture cells have begun to address the question of how different regions of rhodopsin contribute to its folding and stability. A large number of one and two codon deletions constructed in the extracellular domains of bovine rhodopsin were found to be unstable in a COS cell expression system (Doi et aZ., 1990), whereas many larger deletions in the second and third cytoplasmic loops were stable (Franke et al . , 1992). Among 31 single amino acid substitution mutations in human rhodopsin that cause autosomal dominant retinitis pig- mentosa, 85% interfere with the folding and/or stability of the protein (Sung et al . , 1991,1993) and this subset of mutations is found exclusively in the transmembrane domains and extracel- lular loops. Taken together, these data suggest that the prin- cipal determinants of the native state reside in the transmem- brane domains and extracellular loops.

Functional mapping experiments have also begun to define the regions that interact with the chromophore, transducin, rhodopsin kinase, and arrestin (reviewed in Khorana (1992) and Nathans (1992)). Residues that interact with the chro- mophore have been mapped to transmembrane segments 3, 4, 6, and 7 in rhodopsin and the highly homologous red and green cone pigments (Bownds, 1967; Wang et al., 1980; Neitz et al . , 1991; Zhukovsky and Oprian, 1989; Sakmar et al., 1989; Nathans, 1990; Chan et al . , 1992; Merbs and Nathans, 1993). Transducin appears to interact with residues in the second and third intracellular loops and in the proximal region of the car- boxyl-terminal tail (Konig et al . , 1989; Franke et al . , 1990, 1992).

In this paper we describe the development of an insertional mutagenesis approach to examine the transmembrane topog- raphy, stability, and activity of rhodopsin. In these experi- ments, a unique restriction site was introduced by site-directed mutagenesis at various positions in an expressible cDNA, and a DNA segment encoding a foreign peptide epitope flanked by glycine linkers was cloned into these positions. Both the paren- tal restriction site mutants and the epitope insertion mutants were compared to the wild type with respect to yield and ability to bind ll-cis-retinal. The membrane sidedness of a subset of

14715

14716 Insertional Mutagenesis as a Probe for Rhodopsin

FIG. 1. Diagram of bovine rhodopsin. The central seven helical segments represent the membrane-embedded regions. N , amino terminus; C,

respectively. Adjacent pairs of filled circles represent codon pairs that were mutated to produce BamHI sites. The wild type amino acid sequence carboxyl terminus; zigzag lines, palmityl groups. The amino and carboxyl termini face the extra- and intracellular faces of the membrane,

is shown adjacent to each pair in the single letter code. Each BamHI site (GGATCC) encodes Gly-Ser. Heavy rectangles indicate the locations of the eight c-Myc epitope insertions for which the transmembrane topography was determined.

the epitope insertion mutants was determined by immunocy- tochemical techniques, and three epitope insertion mutants were used to partially delimit the regions on the cytosolic face that contact transducin. The approach described here should prove generally useful for elucidating structural and functional properties of both membrane and globular proteins.

EXPERIMENTAL PROCEDURES DNA and Cells-The mammalian expression vector pCIS (Gorman e.?

al., 1990), bovine opsin cDNA (Nathans and Hogness, 1983), and the c-Myc epitope (MEQKLISEEDLN recognized by mAb’ Mycl-9ElO; Evan e.? al. (1985), Kolodziej andYoung (1991), as modified by Wong and Cleveland (1990)) have been described. For epitope insertion, a unique BamHI site was introduced by site-directed mutagenesis at various locations in the bovine opsin cDNA by replacing two codons with GGATCC (encoding Gly-Ser). The coding region of each BamHI-contain- ing plasmid was cloned into a copy of the expression vector that had not undergone the mutagenesis procedure, and was sequenced on one strand both to confirm the predicted mutations and to rule out spurious mutations. Double-stranded synthetic DNA encoding the c-Myc epitope and flanking penta-glycine linkers (GGGGGMEQKLISEEDLNGGG- GG) was cloned in-frame into the unique BamHI site of the target DNA, resulting in a duplication of the BamHI site. The correct orientation and sequence of each insert was confirmed by DNA sequencing. All con- structs were expressed by transient transfection of human embryonic kidney cells (2938) as described (Gorman et al., 1990).

Spectroscopic Assay of Opsin and Opsin-Myc Hybrids-Two days af- ter transfection the cells were harvested, and total cell membranes were purified on a sucrose step gradient, solubilized in 2% CHAPS (Sigma), and reconstituted with ll-cis-retinal at room temperature in the dark (Nathans, 1990). Photobleaching difference spectra were recorded in the presence of 50 m~ hydroxylamine using a Kontron Instruments Uvikon 860 spectrophotometer (Nathans, 1990).

Protein Blots-Membrane proteins were separated by SDS-polyac- rylamide gel electrophoresis, electrophoretically transferred to nitrocel-

l The abbreviations used are: mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-l-propanesulfonic acid; BP- TI, bovine pancreatic trypsin inhibitor; GPTyS, guanosine 5’-3-0-(thio)- triphosphate; amino acid substitutions are referred to by the identity of the wild type residue, abbreviated using the single letter amino acid designation, followed by the codon number, followed by the introduced residue, e.g. alanine 26 to glycine is A26G.

lulose, and detected immunochemically using an enhanced chemilumi- nescence detection system (Amersham) as previously described (Borjigin and Nathans, 1993).

Immunocytochemistry-For immunostaining, 2939 cells were grown on poly-D-lysine-treated coverslips. Eighteen hours after transfection, the cells were rinsed with phosphate-buffered saline, and fixed and permeabilized with methanol. Coverslips were incubated with the ap- propriate antibody probes (mouse mAb 1D4 (Hodges et al., 1988) and mAb B6-30 (Adamus, et al., 1988) for opsin, and mouse mAb Mycl- 9E10 for c-Myc epitopes (Evan et al., 1985)), and subsequently incu- bated with fluorescein isothiocyanate-conjugated goat anti-mouse im- munoglobulin or biotinylated goat anti-mouse immunoglobulin followed by avidin-biotin complex (ABC) peroxidase staining (Vector Laborato- ries). For staining of intact cells, transfected cells were rinsed twice with ice-cold Dulbecco’s modified Eagle’s medium-Fl2 (Life Technolo- gies Inc.), incubated on ice for 1 h with appropriate antibodies diluted in Dulbecco’s modified Eagle’s m e d i d - 1 2 , 10% calf serum, and visual- ized as described above with anti-mouse secondary antibody.

Dansducin Assay-Transducin was purified to apparent homogene- ity by GTP elution from bovine rod outer segments as described by Kuhn (1982). To assay transducin activation, membranes from tran- siently transfected cells expressing either wild type or mutant opsins were purified on a sucrose step gradient, incubated with 114s-retinal for several hours in the dark, sonicated for 10 s in 5 M urea to remove peripheral membrane proteins (Shichi and Somers, 19781, and recov- ered by ultracentrifugation. After the addition of ll-cis-retinal, all ma- nipulations were carried out in the dark or under dim red light. The membrane pellets were solubilized in 70 mM KPO,, pH 7.5,5 mM MgCl,, 2 mM dithiothreitol (RK buffer) containing 0.3% dodecylmaltoside to give a final rhodopsin concentration of 0.75 p~ as determined spectro- photometrically. ’ICventy microliters of recombinant rhodopsin was com- bined with 5 pl of 5 mM Tris, pH 7.5, 0.5 mM MgCl,, 1 mM dithiothreitol, 0.1 m~ phenylmethylsulfonyl fluoride, and 0.1 mM GTP containing 0.5 p~ transducin. The reaction was initiated by the addition of 20 pl of RK buffer containing 5 pCi of [35SIGTPyS and was maintained at room temperature throughout the experiment. At intervals of 1 min, 5-1.11 aliquots were removed and assayed for [35SlGTPyS binding to transdu- cin by nitrocellulose filter binding using a slot-blot format. The 35S retained by the filter was measured using a Molecular Dynamics Phos- phorImager. Two reactions were prepared for each rhodopsin sample: one was maintained in the dark, and the other was exposed for 1 min to a broad band fiber optic light of sufficient intensity to photoactivate >95% of the rhodopsin.

Insertional Mutagenesis as a Probe for Rhodopsin 14717

TABLE I Properties of BamHI substitution mutants and the corresponding

c-Myc epitope insertion mutants The pair of numbers in each name refers to the pair of codons that

were mutated to create the BamHI site. Amino acid substitutions are indicated by the identity of the wild type (wt) residue (in the single letter code), followed by the codon number, followed by the identity of the substituted residue, i.e. A26G refers to the substitution of glycine for alanine at codon 26. Protein yield on Western blot was scored as if undetectable or barely detectable, "+" if consistently lower than 10- 20% of wild type, and "++" if within severalfold of the intensity of wild type. The pigment yield following reconstitution with 114s-retinal (measured by the optical density at 500 nm in a photobleaching differ- ence spectrum) is expressed as percent of wild type recombinant rho- dopsin prepared in the same experiment. EC, extracellular; IC, intra- cellular. For three c-Myc insertion mutants the sidedness determination is shown in parentheses to indicate uncertainty as to whether the im- munostained protein is in its native structure due to the low yield observed upon reconstitution with ll-cis-retinal.

Names Protein yield

blot

Sidedness Mutations on western :%in",' izvkon

sites

26,27-BamHI

32,33-BarnHI 32,33-Myc 64,65-BamHJ

69,70-BamHI

97,98-BamHI

106,107-BamHI

139,140-BamHI

147,148-BamHI

194,195-BamHI

197,198-BamHI 197,198"~~ 239,240-BamHI

250,251-BamHI 250,251-Myc 280,281-BamHI 280,281"~~

26,27-My~

64,65-Myc

69,7O-My~

97,98-My~

106,107-MYC

139,140-MYC

147,148"~~

194,195"~~

239,240"~~

283,284-BamHI 283,284"~~ 318,319-BamHI 318,319"~~ 333,334-BamHI 333,334"~~

A26G c-Myc insertion A32G, E33S c-Myc insertion Q64G, H65S

R69T, T70S c-Myc insertion

c-Myc insertion T97G c-Myc insertion P107S

V139G, C140S c-Myc insertion

c-Myc insertion R147G, F148S c-Myc insertion P194G, H195S c-Myc insertion E197G, T198S

E239G c-Myc insertion

V250G, T251S c-Myc insertion

c-Myc insertion wt c-Myc insertion F283G, G284S c-Myc insertion V318G, T319S

A333G c-Myc insertion

c-Myc insertion

++ + ++ - ++ +

++ + ++ +

++ -

++ ++ ++

++ ++

++ ++ +

++ ++ ++ ++ ++ + ++ + ++ ++ ++ ++

% of wt 25 0

60 0 80

0-5 50

0 45 0

50 0

100 85

105 50

100 30 80

5 105 80 80 75

100 10 25

0-5 95 5

85 160

RESULTS

Choice of Insertion Sites and Epitopes--Two sites in each of the eight hydrophilic domains were chosen as positions for insertion of foreign epitopes. These positions were chosen to increase the likelihood that the inserted epitope would pro- trude from the protein surface, thereby minimizing structural perturbations and maximizing accessibility to binding proteins. Additional criteria used in choosing the points of insertion in- cluded avoiding highly conserved amino acids or amino acids known to be important for the stability of rhodopsin (e.g. Cysiio and Cysis7, which form an important disulfide bond (Karnik and Khorana, 1990)). At each point of insertion, site-directed mutagenesis was first used to replace the wild type sequence with GGATCC, thus introducing a unique BamHI site. In each case the BamHI site was introduced in-frame and therefore encodes Gly-Ser. Among the 16 BamHI mutants, one preserved the wild type pair of amino acids, five produced single amino acid substitutions, and the remainder produced double substi- tutions (Fig. 1 and Table I). The BamHI mutants and their

insertion derivativeb) were assayed by transient transfection of a human embryonic kidney cell line (2938) followed by re- constitution in vitro with 11-cis-retinal. The yield of native opsin was determined by the efficiency of binding to ll-cis- retinal as measured from a photobleaching difference spec- trum.

In a preliminary series of experiments we evaluated the rela- tive efficacy of 3 sequences as insertional mutagens: bovine pancreatic trypsin inhibitor (BPTI), an influenza hemaglutinin epitope (YPYDVPDYA; Wilson et al. (1984) and Kolodziej and Young (1991)), and a c-Myc epitope (MEQKLISEEDLN; Evan et al. (1985), Kolodziej and Young (1991), and Wong and Cleve- land (1990)). In previous work, we observed that an in-frame insertion of BPTI into bovine opsin following codon 194 re- sulted in a hybrid protein in which both BPTI and opsin appear to be correctly folded as judged by the binding of 11-cis-retinal to opsin and trypsin to BPTI (Borjigin and Nathans, 1993). BPTI was similarly inserted into opsin at seven other engi- neered BamHI sites: after codons 21, 69, 101, 147, 239, 280, and 333. In four of these positions (69, 147, 239, and 333), the native rhodopsin structure was able to accommodate BPTI in- sertion, although, like the BPTI insertion after codon 194, each insertion mutant accumulated to a level lower than either the parental BamHI mutant or the wild type (data not shown).

The failure of the native rhodopsin structure to accommodate BPTI insertion at some sites could reflect steric constraints imposed by the highly structured insert. To explore the feasi- bility of using more flexible inserts, we compared the influenza hemaglutinin epitope, the c-Myc epitope, and BPTI insertions using two constructs for each insert following codon 280, one of the positions where BPTI insertion interfered with the folding and/or stability of opsin. In one set of constructs the inserted sequence was flanked directly by the BamHI cloning sites, and in the second set the inserted sequence was flanked on each side by five glycines. The properties of these six constructs can be summarized as follows: 1) BPTI insertions failed to produce native opsin with or without glycine linkers; 2) influenza hema- glutinin epitope insertions failed to produce native opsin with- out glycine linkers, but did produce native opsin with glycine linkers; and 3) c-Myc epitope insertions produced native opsin with or without glycine linkers. In those cases in which native opsin was observed, the yield was approximately one-tenth that of wild type (Table I and data not shown). The c-Myc epitope was further tested with or without glycine linkers fol- lowing codons 194 and 333. The yield of native opsin with the c-Myc epitope with glycine linkers was comparable (at position 333) or better (at position 194) than that obtained in the ab- sence of glycine linkers (data not shown). Based on these re- sults, we decided to use a c-Myc epitope with glycine linkers (GGGGGMEQKLISEEDLNGGGGG) for the remainder of the study.

Effects of c-Myc Insertion on Protein Stability-For the 16 BamHI substitution mutants the yield of visual pigment fol- lowing reconstitution with 11-cis-retinal ranged from 25 to 100% relative to the wild type (Fig. 2 and Table I). In each case, the characteristic shape and wavelength of maximal absorption were indistinguishable from that of wild type bovine rhodopsin. The apoproteins were further analyzed by Western blotting of total membrane proteins using mAb 1D4, which recognizes an epitope near the carboxyl terminus (Hodges et al., 1988). In each case, the yield and relative distribution of electrophoretic species observed by Western blotting closely resembled that of the wild type (Table I and Fig. 3). The Western blot reveals both heterogeneous glycosylation, giving rise to monomers with ap- parent molecular mass between 35 and 50 kDa, and aggrega- tion of opsin into dimers and higher order multimers.

The c-Myc epitope with glycine linkers (hereafter referred to

14718 Insertional Mutagenesis as a Probe for Rhodopsin

I I I I

300 500 100 300 500 700

Wavelength (nm) FIG. 2. Photobleaching difference absorption spectra of 3

BamHI substitution mutants ( lef t ) and the corresponding c-Myc epitope insertion mutants (right). Each of the BamHI mutants produces a photolabile pigment indistinguishable from the wild type. Insertion of the c-Myc epitope after codon 194 (top) or after codon 239 (middle) results in pigments with yields that are within a factor of 2 of the wild type. Insertion of the myc epitope after codon 32 abolishes pigment formation in vitro.

as “c-Myc”) was then inserted into the unique BamHI site in each of the 16 BamHI-opsin substitution mutants, and the resulting fusion proteins were analyzed as described above. Eleven of the c-Myc insertion mutants were capable of gener- ating visual pigments, although with widely varying yields’ (Fig. 2 and Table I). The remaining five c-Myc insertion mu- tants produced no detectable visual pigment upon incubation with ll-cis-retinal. The c-Myc insertion mutants were analyzed by Western blotting using mAb 1D4 and a mAb directed against c-Myc (Mycl-9El0, Evan et al. (1985); hereafter referred to as “anti-myc”). The 1D4 Western blots showed the expected lower mobility of the c-Myc insertion mutants relative to the BamHI substitution mutants and wild type rhodopsin (Fig. 3). As sum- marized in Table I, the c-Myc mutants that were capable of binding ll-cis-retinal migrated predominantly as monomers in SDS-polyacrylamide gel electrophoresis, whereas those that failed to bind ll-cis-retinal migrated predominantly as multi- mers (for example, c-Myc insertion following codons 32,64, and 106), a correlation that had been observed in previous studies (Doi et al., 1990; Sung et al., 1991, 1993). The yield of immu- noreactive protein was also observed to be lower among those mutants that failed to bind 114s-retinal.

In these experiments, two general trends were observed with respect to the location of the c-Myc insertion and the stability of the resulting hybrid protein. First, positions within the amino- terminal one-third of the opsin polypeptide (e.g. codons 26, 32, 64, 69, 97, and 107) were less likely to tolerate the c-Myc in- sertion than was the rest of the polypeptide. Second, the extra- cellular face of the protein was in general less tolerant of the c-Myc insertion than was the cytoplasmic face. This latter ob- servation is in keeping with the asymmetry of destabilizing

1 D4

anti-myc

FIG. 3. Immunoblota of expressed opsins. Expression plasmids

BamHI substitution mutants were transfected into 2939 cells. Five encoding eight c-Myc epitope insertions and the corresponding parental

micrograms of membrane protein was loaded on each lane. The Western blot was reacted sequentially with mAb 1D4 (top) and anti-Myc (bot- tom). Molecular mass standards (right) are from top to bottom: 106,80, 49.5, and 32.5 m a . ROS, rod outer segments. Only those opsins used for mapping of transmembrane topography are shown in this figure.

deletion mutations observed in earlier studies of bovine (Doi et al. 1990; Franke et al., 1992) and human (Sung et al., 1991, 1993) rhodopsins. We note that some of the extracellular inser- tions, in particular those near the sites of N-linked glycosyla- tion at residues 2 and 15, could destabilize the protein indi- rectly by perturbing glycosylation.

Dansmembrane Topography-Eight of the 16 c-Myc inser- tion mutants, one in the amino-terminal domain, one in each of the six putative helix connecting loops, and one in the carboxyl- terminal region, were used to study the topography of opsin in the plasma membrane (Fig. 4). In each experiment, transiently transfected cells were stained under two conditions: as intact cells in culture medium at 4 “C, or following fixation and per- meabilization with methanol. Three antibodies were used for staining: mAb B6-30 which binds an amino-terminal epitope on the extracellular face of the bilayer (Adamus et al., 19881, mAb 1D4 which binds a carboxyl-terminal epitope on the in- tracellular face of the bilayer (Hodges et al., 19881, and anti- myc. As expected, 2939 cells transfected with wild-type opsin reacted with both B6-30 and 1D4 when fixed and permeabi- lized with methanol, whereas intact cells reacted only with B6-30. The dependence of 1D4 staining on methanol treatment is likely to reflect permeabilization of the cell rather than de- naturation of opsin because 1D4 is known to bind native rho- dopsin with high affinity (Oprian et al., 1987). No immuno- staining of cells transfected with wild type rhodopsin was ob- served with anti-Myc in either condition, confirming the speci- ficity of this reagent.

Immunofluorescent staining of cells expressing each of the eight c-Myc insertion mutants revealed immunoreactive mate- rial with all three antibodies following methanol permeabiliza- tion (Fig. 4). Those three insertion mutants that accumulated to lower levels, as determined by Western blotting and recon-

Insertional Mutagenesis as a Probe for Rhodopsin 14719

FIG. 4. Immunofluorescent staining of 2935 cells expressing c-Myc epi- tope insertion mutants. Cells were grown on glass coverslips and immunos- tained either in culture medium a t 4 "C ("intact cells," left) or following methanol treatment at room temperature ("perme- abilized cells," right). For each set of transfected cells, staining was performed with three mAbs: B6-30, which recog- nizes rhodopsin's extracellular amino-ter- minal domain (left); 1D4, which recog- nizes rhodopsin's intracellular carboxyl- terminal domain (center); and anti-Myc. Several brightly stained cells expressing the 32,33-Myc construct are shown in the intact cell experiment; these cells are pre- sumed to be leaky.

wt opsin

32,33-myc

64,65-myc

106,107-myc

139,140-myc

194,195-myc

239,240-myc

280,281 -myc

333,334-myc

i ntact cells 86-30

stitution with ll-cis-retinal, (32,33-Myc, 64,65-Myc, and 106,107-Myc) showed a lower level of immunostaining with each of the antibodies. In all cases the plasma membrane of the transfected cells appeared to contain correctly oriented opsin as judged by the ability of B6-30 and the inability of 1D4 to bind intact cells. Immunostaining of intact cells with the anti-Myc mAb revealed surface labeling of insertion mutants 32,33-Myc, 106,107-Myc, 194,195-Myc, and 280,281-Myc. Surface labeling was not detectable with 64,65-Myc, 139,140-Myc, 239,240-Myc, and 333,334"~~. Identical results were obtained when the immunostaining was performed using an immunoperoxidase detection system (data not shown). Intense uniformly labeled cells were occasionally seen in the absence of methanol treat- ment; examples are shown in two of the 32,33-Myc panels. These are presumed to be leaky or ruptured cells. Interestingly, their frequency was consistently higher following transfection with mutants that were poorly expressed, suggesting a delete- rious effect of the unstable opsin.

Each of the three mutants that failed to bind 11-cis-retinal or that bound ll-cis-retinal poorly, accumulated to sufficient lev- els in the plasma membrane to permit topographic analysis by immunostaining (Fig. 4). In these cases, the transmembrane

1 D4 a-mvc

permeablized cells 86-30 1 D4 a-myc

topography of the c-Myc insert is shown in parentheses in Table I to indicate that the correct tertiary structure of these mutant proteins has not been demonstrated by 114-retinal binding. Experiments with a variety of membrane proteins, including rhodopsin, indicate that only correctly folded proteins efi- ciently exit the endoplasmic reticulum (Klausner and Sitia, 1990; Doi et al., 1990; Sung et al., 1991). This mechanism pre- sumably accounts for a low level of accumulation of correctly folded protein at the plasma membrane in those cases in which the mutant protein folds inefficiently or exhibits reduced sta- bility. For these mutants the failure to observe reconstitution with 114s-retinal could reflect denaturation of unstable mu- tant proteins during the in vitro incubation in the presence of detergent.

Based on the pattern of immunostaining described above, we conclude that the regions encompassing codons 32, 106, 194, and 280 reside on rhodopsin's extracellular face, and that the regions encompassing codons 64, 139, 239, and 333 reside on rhodopsin's intracellular face. We cannot rule out the possibil- ity that in mutants with epitope insertions at one or more of the latter positions the c-Myc epitope was located on the extracel- lular face but buried or otherwise rendered inaccessible to an-

Insertional Mutagenesis as a Probe for Rhodopsin

15 I 139,140-myc -hv

rn 139.140-rnyc +hv

0 WT+hv / O WT-hv

0

12

'" I A 239,240-myc -hv

A 239,240-myc +hv

15

10

5

0

o 333,334-myc -hv

+ 333,334-myc +hv

0 WT-hv

2 4 6 8

Time (min)

mutants on the intracellular face of rhodopsin. The extent of FIG. 5. Transducin activation by three c-Myc epitope insertion

GTP-GDP exchange was measured using GTPyS retention on a nitro- cellulose (see "Experimental Procedures"). Similar results were ob- tained in two independent experiments. WT, wild type; hv, light expo- sure.

tibody binding under conditions where the cells remained in- tact. However, the large size of the c-Myc insert and the presence of flanking glycine linkers makes this scenario un- likely. These data provide direct support for the seven-trans- membrane model of rhodopsin and demonstrate for the first time the existence of a third extracellular loop.

Insertional Mapping of Sites on the Cytosolic Face That Do Not Znteract with Dansducin-Most mutational analyses of protein-protein interaction involve substituting or deleting one or more amino acids. Any mutation that produces a decrement in afflnity is presumed to reside in the region of contact be- tween the two proteins. While this method can, in theory, iden- tify each interacting residue and its contribution to the ener- getics of the interaction, it is often difficult to distinguish between afflnity differences due to changes in residues that directly contact the target protein and those due to perturba- tions in protein structure that indirectly affect binding.

A mutational analysis of protein-protein interactions can also be performed using insertional mutagenesis. In this case, the interpretation may be simplified because any insertion in a region of contact is expected for steric reasons to be completely defective in the protein-protein interaction. Conversely, any region that can tolerate insertion and still bind the target pro-

tein is unlikely to be part of the binding site. To explore the utility of this approach we examined the ability of three c-Myc insertion mutants to activate transducin. 139,140-Myc, 239,240-Myc, and 333,334"~~ reside, respectively, in the sec- ond and third cytosolic loops, and in the carboxyl-terminal tail. Each accumulates in 2938 cells to the wild type level, and each can be reconstituted with 11-cis-retinal to form a photolabile pigment that is indistinguishable from the wild type in its yield and absorption maximum. The activity of these three rho- dopsins and expressed wild type rhodopsin in catalyzing the binding of [35S]GTPyS to purified bovine transducin was meas- ured using a filter binding assay. As shown in Fig. 5, both wild type rhodopsin and 333,334"~~ catalyzed the binding of GTPyS with similar efficiences and in a light-dependent man- ner, while 139,140"~~ and 239,240"~~ failed to activate transducin. Similar results were obtained in two independent experiments. This experiment indicates that the region near codons 333 and 334 in the carboxyl-terminal domain of rhodop- sin is not involved in interacting with transducin, whereas the second and the third cytoplasmic loops are important for the activation of transducin, consistent with current models of the rhodopsin-transducin interaction (Franke et al., 1992; Konig et al., 1989). We note that the defects in transducin activation observed in these two mutants could involve alterations in the conformation of photoactivated rhodopsin or in the catalysis of GTP-GDP exchange rather than or in addition to alterations in transducin binding.

DISCUSSION

In this paper, we describe the use of an insertional mutagen- esis approach to analyze rhodopsin's stability, transmembrane topography, and ability to activate transducin. This approach has several experimental attributes that may make it generally useful. First, the use of an epitope with glycine linkers in- creases the likelihood that insertion into the target protein will be tolerated and that the epitope will be accessible to antibody binding. Second, like all genetic methods, flexible epitope in- sertion allows the native protein to be probed at any location where the sequence alteration does not interfere with folding or stability. By contrast, using antibodies directed against differ- ent epitopes from the protein of interest requires the produc- tion of multiple high affhity antibodies, a potentially difficult task due both to the constrained conformation and incomplete accessibility of many protein surface regions and the nonran- domness of the immune response. Third, and also in contrast to the use of antibodies directed against the native protein, epitope insertion permits standardization of the experimental protocol because all of the antibody binding experiments can be performed under identical conditions with a single antibody reagent. Fourth, the use of epitope insertions with glycine link- ers would be expected to insulate the target protein from struc- tural perturbations associated with antibody binding, which could be significant and which are difficult to assess when using antibodies directed against the target protein. And fifth, the use of insertional mutagenesis to map the sites of protein- protein interaction should complement the use of single amino acid substitution mutants by revealing in an all-or-none fash- ion those regions where epitope insertion does not sterically interfere with binding.

A number of studies during the past several years have uti- lized genetic and/or immunological methods to study mem- brane protein topography, stability, or function (Jennings, 1989). These include immunostaining of cells or membrane preparations with antibodies directed against specific epitopes in the native protein (e.g. Pederson et al. (19901, Imajoh-Ohmi et al. (1992), Roitelman et al. (19921, and Lei et al. (1993)); insertion ofAsn-linked glycosylation sites as markers for lume-

Insertional Mutagenesis as a Probe for Rhodopsin 14721

A. stable rhodopsinlmyc insertion mutations

B. unstable rhodopsinlmyc insertion mutations

c. unstable autosomal dominant RP mutations

FIG. 6. Transmembrane models of human and bovine rhodop- sin. A, locations of Myc epitope insertions that produced stable pro- teins. Filled circles, yield upon reconstitution with 114s-retinal greater than 10% of wild type; half-filled circles, yield upon reconstitution be- tween l and 10% ofwild type. B, locations of Myc-epitope insertions that produced unstable proteins, ie. no reconstitution with 11-cis-retinal, and greatly reduced yield by immunoblotting. C, mutations in human rhodopsin that are responsible for autosomal dominant retinitis pig- mentosa and that produce an unstable protein when produced in 2933 cells (Sung et al., 1991, 1993).

nal or extracellular domains (Olender and Simoni, 1992); con- struction of alkaline phosphatase (Escherichia coli; Manoil and Beckwith (1986)) or histidinol dehydrogenase (yeast; Sengstag et al. (1990)) fusions to progressively truncated protein targets; and insertion of foreign epitopes (Charbit et al . , 1986; Freudl et al . , 1986; Freudl, 1989; Campbell et al . , 1992; Anand et al . , 1993; Teufel et al . , 1993). The latter studies include the dem- onstration that four of six insertions into the helix-connecting loops in bacteriorhodopsin are functional (Teufel et al., 1993); the topographic mapping by immunological methods of two insertions in the E. coli LamB protein (Charbit et al . , 19861, two insertions in the a1 subunit of the nicotinic acetylcholine re- ceptor by antibody binding (Anand et al . , 1993), and the car- boxyl termini of two alternative forms of a calcium ATPase (Campbell et al., 1992); and topographic mapping by partial proteolysis of two insertions in the E. coli OmpA protein (Freudl et al . , 1986; Freudl, 1989).

With respect to rhodopsin’s structure and function, the prin- cipal results of this study are: 1) a confirmation of the existence and topography of each of the six helix-connecting loops and the amino- and carboxyl-terminal tails, including the third extra- cellular loop for which experimental evidence had previously been lacking; 2) the identification of positions which do or do not tolerate insertion of a flexible epitope, these are presumed to be either buried or involved in interactions that are impor- tant for stability and/or folding (Fig. 6); and 3) the identifica- tion of two sites (positions 139 and 239) at which epitope in- sertion blocks transducin activation, and one site (position 333) where it does not.

Five of the 16 c-Myc insertion mutants showed a large de- crease in yield (Fig. 6B, filled circles) and four showed a mod- erate decrease in yield (Fig. SA, halffilled circles). In general, the least stable insertion mutants cluster nearer to the amino terminus or on the extracellular face of the protein, a pattern that resembles to some extent the distribution of unstable mu- tations in human rhodopsin responsible for autosomal domi- nant retinitis pigmentosa, most of which are single amino acid substitutions (Fig. 6C; Sung et al . (1991, 1993)). While the positions for epitope insertion were not chosen at random and are therefore unlikely to encompass all important regions, it is likely that the set of naturally occurring mutations in human rhodopsin represents a relatively unbiased sampling of desta- bilizing changes. These data suggest that in rhodopsin the hy- drophilic domains that are critical for folding and/or stability of rhodopsin are asymmetrically distributed throughout the polypeptide.

REFERENCES

Adamus, G., Arendt, A., Zam, Z. S., McDowell, J. H., and Hargrave, P. A. (1988)

Anand, R., Bason, L., Saedi, M. S., Gerzanich, V., Peng, X., and Lindstrom, J.

Barclay, P. L., and Findlay, J. B. C. (1984) Biochem. J. 220, 75-84 Borjigin, J., and Nathans, J. (1993) Proc. Nutl. Acad. Sci. U. 5. A. 90,337-341 Bownds, D. (1967) Nature 216, 1178-1181 Campbell, A. M., Kessler, P. D., and Fambrough, D. M. (1992) J. Biol. Chem. 267,

Chan T., Lee, M., and Sakmar, T. P. (1992) J. Bid. Chem. 267,947S9480 Charbit, A., Boulain, J. C., Ryter, A,, and Hofnung, M. (1986) EMBO J. 5, 3029-

Doi, T., Molday, R. S., and Khorana, H. G. (1990) Proc. Nutl. Acad. Sci. U. S. A. 87,

Evan G. I., Lewis, G. K., Ramsay, G., and Bishop, M. J. (1985) Mol. Cell. Biol. 5,

Franke, R. R., Konig, B., Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990)

Franke, R. R., Sakmar, T. P., Graham, R. M., and Khorana, H. G. (1992) J. Biol

Freudl, R. (1989) Gene (Amst. 82, 229-236 Freudl, R., MacIntyre, S., Degen, M., and Henning, U. (1986) J. Mol. Bid. 188,

Gorman, C., Gies, D. R., and McCray, G. (1990) DNA Protein Eng. rich. 2, 3-9 Hargrave, P. A., McDowell, J. H., Curtis, D. R., Wang, J. K., and Argos, P. (1983)

Peptide Res. 1, 42-47

(1993) Biochemistry 32, 9975-9984

9321-9325

3037

4991-4995

3610-3616

Science 250, 123-125

Chem. 267, 14767-14774

491-494

Biophys. Struct. Mech. 9, 235-244

14722 Insertional Mutagenesis as a Probe for Rhodopsin Hodges, R. S., Heaton, R. J., Parker, J. M., Molday, L., and Molday, R. (1988) J.

Imajoh-Ohmi, S., lbkita, K., Ochiai, H., Nakamura, M., and Kanegasaki, S. (1992)

Jennings, M. L. (1989) Annu. Reu. Biochem. 58,999-1027 Karnik, S. S., and Khorana, H. G. (1990) J. Biol. Chem. 265, 17520-17524 Khorana, H. G. (1992) J. Bid . Chen. 267, 1-4 Klausner, R. D., and Sitia, R. (1990) Cell 62, 611-614 Kolodziej, P. A,, and Young, R. A. (1991) Methods Enzymol. 194,508-519 Konig, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A,, and Hofmann,

Kuhn, H. (1982) Methods Enzymol. 81, 556564 Lei, S., Raftery, M. A,, and Conti-Tronconi, B. M. (1993) Biochemistry 32,91-100

Martynov, V. I., Kostina, M. B., Feigina, M. Y., and Miroshnikov, A. I. (1983) Bioorg. Manoil, C., and Beckwith, J. (1986) Science 233, 1403-1408

Merbs, S. L., and Nathans, J. (1993) Photochern. Photobiol. 58, 706-710

Nathans, J. (1992) Biochemistry 31,4923-4931 Nathans, J. (1990) Biochemistry 29, 97469752

Nathans, J., and Hogness, D. S. (1983) Cell 34,807314 Neitz, M., Neitz, J., and Jacobs, G. H. (1991) Science 252, 971-974 Olender, E. H., and Simoni, R. D. (1992) J. Biol. Chem. 267,42234235 Oprian, D. D., Molday, R. S., Kaufman, R. J., and Khorana, H. G. (1987) Proc. Natl.

Ovchinnikov, Yu. A,, Abdulaev, N. G., Feigina, M. Yu., Artamonov, I. D., Bogachuk,

Biol. Chem. 263, 11768-11775

J. B i d . Chem. 267,180-184

K. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 , 68784882

Khim. 9,735-745

Acad. Sci. U. S. A. 84, 8874-8878

A. S., 2olotarev.A. S., Eganyan, E. R., andKostetskii, P. V. (1983)Bioorg. Khim. 9, 1331-1340

Pederson, S. E., Bridgman, P. C., Sharp, S. D., and Cohen, J. B. (1990) J. Biol. Chem. 265,569-581

Rohlich, P. (1976) Nature 263, 789-791 Roitelman, J., Olender, E. H., Bar-Nun, S., Dunn, W. A,, and Simoni, R. D. (1992)

Sakmar, T. P., Franke, R. R., and Khorana, H. G. (1989) Proc. Natl. Acad. Sci. J. Cell Biol. 117, 959-973

Schertler, G. F. X., Villa, C., and Henderson, R. (1993) Nature 362, 770-772 Sengstag, C., Stirling, C., Schekman, R., and Rine, J. (1990) Mol. Cell. Biol. 10,

Shichi, H., and Somers, R. L. (1978) J. Biol. Chem. 253, 7040-7046

U. S. A. 86,830943313

672480

Sung, C.-H., Schneider, B.,Agarwal, N., Papermaster, D. S., andNathans, J. (1991)

Sung, C-H., Davenport, C. M., and Nathans, J. (1993) J. Biol. Chem. 268,26645- Proc. Natl. Acad. Sci. U. S. A. 88, 88404844

Teufel, M., Pompejus, M., Humbel, B., Friedrich, K., and Fritz, H.4. (1993) EMBO

Wang, J. K., McDowell, J. H., and Hargrave, P. A. (1980) Biochemistry 19, 5111-

Wilson, I. A,, Niman, H. L., Houghten, R. A., Cherenson, A. R., Connolly, M. L., and

Wong, P. C . and Cleveland, D. W. (1990) J. Cell Biol. 111,1987-2003 Zhukovsky, E. A,, and Oprian, D. D. (1989) Science 246,928-930

26649

J. 12,3399-3408

5117

Lerner, R. A. (1984) Cell 37,767-778