Functional expression of receptors in microorganisms

TiPS - Xnrch i992 [Vol. 131 ation may act on key cytoskeletal proteins and thus affect the organization and functions of the cytoskeleton. Alternatively, cytoskeletal elements may be necessary for the enhanced tyrosine phosphorylation observed in cells consequent to integrin clustering. Future investigations wilI surely sort out these possibilities. In the meantime we do at least have a glimpse of a novel and potentially vital signal transduction pathway, one that may have many ramifications for the control of cell growth and differentiation. L. KORNBERG

AND R. L. JULIAN0

Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA.

References 1 Sanes, J. R. (1989) Annu. Rev. Neurosci. 12,491-516 2 McCIay, D. R. and Ettensohn, C. A. (1987) Annu. Rev. Cell BioJ. 3,319-345 3 Juliano, R. L. (1987) Biochim. Biophys. Actu 907,261-268 4 Ruoslahti, E. and Pierschbacher, M. D. (1987) Science 238,491497

95 5 Akiyama, S. K., Nagata, K. and Yamada, K. (1990) Biochim. Biophys. Acta 1031, 91-110 6 Ruoslahti, E. (1991) J. Clin. Invest. 87, l-5 7 HemIer, M. E. (1990) Annu. Reo. Immunol. 8,365-400 8 Brown, I’. J. and Juliano, R. L. (1985) Science 228, l-1451 9 Takada, Y. et al. (1991) J. Cell Biol. 115, 257-266 10 PbiIIips, D. R., Charo, I. F., Parise, L. V. and Fitzgerald, L. A. (1988) Blood 71, 831-843 11 Obara, M., Kang, M. S. and Yamada, K. M. (1988) Cell 53,64%657 12 tillrich, A. and Schlessinger, J. (1990) Cell 61,203-212 13 Fischer, E. H., Charbonneau, H. and To&s, N. K. (1991) Science 253,401-406 14 Gilman, A. G. (1987) Annu. Rev. Biothem. 56,615-649 15 Ingber, D. E. et al. (1990) J. Cell Biol. 110, 1803-1811 16 Ng-Sikorski, J., Andersson, R., Patarroyo, M. and Andersson, T. (1991) Exp. Cell Res. 195,504508 17 Nojima, Y. et al. (1990) J. Exp. Med. 172, 1185-1192 18 Schreiner, C. L., Hussein, S., Fisher, M. and Juliano, R. L. (1991) Cancer Res. 51, 1738-1740 19 Giancotti, F. and Ruoslahti, E. (1990) Cell 60, 849-859 20 Werb, Z., Tremble, P. M., Behrendtsen, O., Crowley, E. and Damsky, C. H.

Fbnctional expression of receptors in microorgahns Recent advances in the isolation and characterization of genes encoding receptor and effector molecules have revealed the existence of a large number of hitherto unknown subtypes of receptor with common structural properties. Membrane-bound G proteincoupled receptors, each of which has seven putative transmembrane domains, constitute one of the largest receptor families. Hundreds of different receptors in this family have been identified that can couple to tens of different transducer G proteins”. Drug screening G protein-coupled receptors are of particular interest to the pharmacologist since they are the target of scores of drugs, many of which were originally identified using

cumbersome and time-consuming Cloned methods. screening human receptors expressed in separate cell lines are now being exploited by the pharmaceutical industry to provide automated, high-throughput screening procedures for the identification of new ligands selective for one particular receptor subtype. Such screening procedures could be used to replace, or at least supplement, traditional methods that use animal tissues rich in one particulafi receptor. For large-scale screening, the host cell line should be inexpensive and convenient to grow in bulk. Ideally, it should provide a system where, in addition to ligand binding, a coupled biological response can also be measured. Binding of agonists to

(1989) J. Cell Biol. 109, 877-889 21 Spom, S. A. et al. (1990) 1. Immunol. 144, 4434-4441 22 Otey, C. A., Pavalko, F. M. and Burridge, K. (1990) J. Cell Biol. 111, 721-729 23 Bunidge, K., Fath, K, KeIIey, T., NuckoIIs, G. and Turner, C. (1988) Annu. Rev. Cell Biol. 4.487-525 24 Ben-Ze-ev, A., Robinson, G. S., Bucher, N. L. R. and Farmer, S. R (1988) Proc. NutI Acad. Sci. USA 85,2161-2X5 25 StreuIi, C. H. and B&d, M. J_ (1990) J. Cell Biol. 110, 1405-1415 26 Komberg, L., Earp, H. S., Turner, C., Prokop, C. and Juliano, R. L. (1991) Proc. Nat1 Acad. Sci. USA 88,8392-8396 27 Kanner, S. B., Reynolds, A. B., Vines, R. R. and Parsons, J. T. (1990) Proc. N&f Acad. Sci. USA 87,3328-3332 28 Guan, J. L., Trevethick, J. E. and Hynes, R. 0. Cell Regul. (in press) 29 Golden, A., Brugge, J. S. and ShattiI, S. (1990) 1. Cell Biol. 111,3117-3127 30 FerreII, J. E. and Martin, S. (1989) Proc. N&Z Acad. Sci. USA 86,2234-2238 31 VeiIIette, A., Bookman, M. A., Horak, E. M. and Bolen, J. B. (1988) Cell 55, 301-308 32 SameIson, L. E., Philips, A. F., Luong, E. T. and KIausner, R. D. (1990) Proc. NatJ Acad. Sci. USA 87.4358-4362 33 Yamanashi, Y., Kakiuchi, T., Mizuguchi, J., Yamamoto, T. and Toyoshima, K (1991) Science 251, 192-195

G protein-linked receptors leads to dissociation of the coupled G protein into its (Yand (3~subunits. The (Ysubunit activates or inhibits one or several of a variety of effector systems. The same receptor may couple to different G proteins and thus regulate different effecters; the same G protein may similarly associate with many different receptors. The ideal screen should provide the relevant ar subunit that can couple to an effector system adaptable to automated measurement. A wide range of mammalian celI lines is now available, each expressing a different human receptor subtype’. Mammalian cells have the advantage that they often contain the required G protein and corresponding effector sys.tem. For example, the human (3~ adrenoceptor, when expressed in Chinese hamster ovary cells, couples to an endogenous G, protein to stimulate adenylyl cyclase. However, the same cells do not provide the appropriate G protein to couple the dopamine 0 1992. Elsevier Science Publishers

Ltd (UK)

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Fig. 1. Cunwt uses of G pmW=Wedreceplwsexpressed in micmoganims. tea?mbinant Mammalian receptors expressed alone in r+rWganisms (top) famsbm= amvenient re* krogates in fatge &m&and&noMg screenii7g programs for the kfentiffcation of new agonists or antagon&ts. Binding propertiesofmutantsornewfyi.93 fated wild-type receptols may afsQ be detemlined witi these exptes&n systems. h#icWlgm -lessing receptors with recombinant mammafian G pmteins (centre) may serve as a setf-mpficatingWs@&a$wiWPviWP~uW~f hw3en these two families of signat&hg proteins. Discrimination between ago&tic or antagonHc pmpertiis of lW?Wl@ilKfS~yalsok &temnmd in such systems becauseagonistadivatW may be moNored by the hydmf@s by G pmteih of radfoM#led GTP, or by the bfnd@ of the nonhydmtys-

able ladfowbsd anabgue GTpvS to G protein.Altema-

receptor + G protein

receptor + G protein + reporter f

tkeenqmatksystemsare in Wt.mf&;zg eWessing maMSnant receptors and G pmteins, agonist-induced signaIlingmay resutt in the activation of endogenous effectom that are normally finked to the response phemmones. Reporter systems @@torn) could induce enzymatrc acmy w response to agonist binding, by placing the gene that encodes &afactosidase under the controi of a phemmone-responsive promoter.

Ds receptor response; for this, another cell type must be used. There are also major practical difficulties with mammalian cells. They are difficult and costly to cultivate, highly susceptible to infection by mycoplasma and relatively unstable, thus requiring frequent subcloning. For these reasons, microbial cells such as bacteria or yeast have been considered as a convenient and inexpensive alternative in ‘which to functionally express mammalian receptors for ligand screening. Figure 1 summarizes the available or potential uses of genetically engineered microorganisms. Several G protein-coupled receptors have already been expressed in Escherichia coli bacteria or in Saccharomyces cerevisiae yeasts. These include the three subtypes

of B-adrenoceptor (Refs 3-7 and V. Ravet, unpublished), the 5-HTIA receptors and the MI muscarinic acetylcholine receptorg. The pharmacological properties (ligand selectivity, stereospecificity and order of binding affinities) of these receptors in microbial membranes are identical to the properties displayed in mammalian tissues or cells transfected with cloned receptor genes. Microbial cells are cheap and easy to grow but have the disadvantage that they may require the introduction of additional genes encoding G proteins in order to activate resident effector systems. Bacterial systems Binding studies may be performed directly on intact bacterial cells instead of on membrane

preparations because there is very low background activity. A few hours of culture in solution provide sufficient material for about ten thousand assays3”,10, favouring high throughput. Cells or membranes may be stored for months without loss of binding activityrl. The guaranteed absence of endogenous receptors in E. coli expressing single human receptors, combined with ease of use, reproducibility and adaptability to automation*, is clearly advantageous compared with the use of membrane preparations derived from mammalian tissues. However, the absence of endogenous transducer proteins means that functional responses cannot be measured following agonist binding (but see below). The availability of microbial expression systems for human receptors has prompted the pharmaceutical industry to adapt these systems for their often promassive drug-screening grams. At Janssen, for example, when the affinities of over 50 B-blockers for rabbit lung Br- and rat lung Bz-adrenoceptors were compared with their affinities for the equivalent human receptor subtypes expressed in bacteria, the correlation was very good”. For hydrophobic ligands, the affinity estimates were more accurate in bacteria, where nonspecific adsorption to the membranes occurs less. One compound was actually metabolized in the lung preparations, thus yielding an apparent affinity loo-fold less than the probably correct value obtained for the human B1-adrenoceptor expressed in bacteria. The advantages of bacterial expression of cloned receptors have also been exploited in basic research, for example in the study of mutant receptors. The molecular basis of ligand binding selectivity to B-adrenoceptor subtypes was investigated by designing chime& Br/Bz-adrenoceptors from a set of reciprocal constructs obtained by exchange of one to three transmembrane regions between the two wild-type receptor genes. These chimeras were expressed in E. coli and probed with selective ligands5. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on an analysis of free-energy change values cal-

TiPS - March 1992 [Vol. 231 culated from the equilibrium binding constants, as a function of the number of substituted transmembrane domains of B2 origin. The data showed that ligand selectivity for Bi- or B2-adrenoceptors results from a particular combination of interactions between each ligand and each transmembrane region of the recepto?. This study, requiring the expression of ten different genes and several thousand binding assays, was considerably eased by the use of the bacterial expression system. An in situ screening procedure has also been developed to characterize mutations in the human B2-adrenoceptor (Fig. 2). Receptorexpressing bacteria were plated out on agar, and binding of radiolabelled ligand was monitored directly by autoradiography of replica filters. nitrocellulose Receptors mutated in the second transmembrane helix by saturation mutagenesis were similarly screened for ligand binding by this procedure. Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid sidechains essential for either ligand binding or structural integrity of the BzadrenoceptorlO. Although bacteria lack effecters that may be activated by G proteins, B-adrenoceptors can be coexpressed with, and can functionally reassociate with, the cr subunit of G, (Ref. 7) in the presence of purified By subunits. This system may be useful in evaluating coupling preferences between defined molecular species of receptor and G protein subunits. For instance, it has recently been shown that the 5-HTiA receptor may be conveniently expressed in E. coli and reconstituted with G; or G, protein’. Indeed, the absence of endogenous G proteins in E. coli allowed Bertin et al.’ to demonstrate that the 5-I-&, receptor does not associate with the G,, subunit, at least in bacteria, suggesting that the reported 5-HT-mediated adenylyl cyclase observed in mamactivation malian tissues may be due to receptor promiscuity or indirect effects. Yeast systems Unlike bacterial systems, expression of receptors in the yeast S. cerevisiae can be coupled to a

97

E. CO/itransformed with plasmids containing mutant or wild-type receptor gene

transformed colonies selected on antibiotic = master plate

SUCCaSSfUlly

replicas grown in presence of inducer for plasmid promotor and: J radiolabelled ligand for encoded receptor

radiolabelled ligand + excess unlabelied ligand added to medium

filter lift + autoradiography

total colonies exhibiting ligand binding

colonies exhibiting nonspecific ligand binding

E. coli bacferia expressing funcrha/ ~ptors. E. coli transfomed with genes coding for receptors may be directly screened for binding of radiolabelled ligand. For example, colonies expressing B-adrenocepfors were fdenMied afler autoradi raphy. On the left, colonies were grown in the presence of the radioligand [’ 2 I]iodocyanopindolol alone, while on fhe right, unlabeled PmPranoloL another #Ladrenoceptor antagonist, was added in the medium.

Fig. 2. Replica filter assay for

functional response. S. cerevisiae has an intrinsic signal-transducing pathway, similar to that found in mammalian cells, that couples pheromone receptors to a trimeric GTP-binding protein homologous to mammalian G proteins. King et a2.l’ achieved the functional expression of human Ba-adrenoceptors in yeast. As in E. coli, the receptor expressed in S. cerevisiae displays characteristic

affinity, specificity and stereoselectivity, measured by directand competition-binding assays on yeast membranes, for agonists and antagonists. Partial activation of the yeast pheromone response was restored in mutant yeast lacking endogenous G protein by coexpressing the human receptor with a rat G, protein OLsubunit, confirming that these components can couple to each other and to the

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downstream effector. In these of p-adrenocells, activation ceptors by agonists induced morphological changes typical of pheromone-dependent activation of the mating signal transduction pathway. This effect was blocked by the specific antagonist alpren0101. When yeasts were transfected with the receptor gene alone, no mating pathway activation by fiadrenoceptor agonists occurred, confirming that this receptor is unable to couple with endogenous yeast G protein. To confirm that the pheromonesignaliing pathway was activated by the human receptor and rat King et al. measured the G ti&tion by agonist of a pheromone-responsive gene promoter linked to the gene coding for figalactosidase. Again, fl-adrenoceptor agonists induced a positive signal, reflected in addition this time by the specific induction of the @-galactosidase reporter gene, as detected by a biochemical assay for enzyme activity. This expressio*reporter system, now composed of a discriminator (the receptor) and a detector (the enzyme), thus offers all the theoretical prerequisites of the pharmaceutical industry, although it is elaborate to design and complex to handle. Shxoid receptor reporting sysSteroid and thyroid hormones interact with specific intracellular receptors; following ligand binding, these receptors bind to welldefined DNA response elements upstream of target genes. Transcription of these genes is thus regulated by activation of steroid receptors. Receptor-responsive transcription units have been reconstituted in heterologous cells by coexpression of both the receptor and a target gene. Receptors for human estrogen, vitamin D, progesterone and glucocorticoids, when expressed in E. coli, retain their steroid-binding and hormone-responsive transcription propertiePls. Wittliff et all3 showed that human estrogen receptor expressed in yeast binds estrogens and associates specifically with DNA estrogen-response elements normally present in bacteria. The advantage of the yeast expression system over transiently

transfected mammalian cells was recently outlined by Pham et al. who, in yeast, were able to study directly the interaction of the estrogen receptor with chromatin and to demonstrate that antiestrogens such as nafoxidine promote DNA binding of the receptor without efficient induction of transcriptian. When a steroid response element was placed in the promoter of a chimeric fusion vector containing the gene that @galactosidase, the encodes receptor transcriptional activity following drug treatments could be monitored by an in vitro figalactosidase enzyme assay”. Current work by D. P. McDonnell and colleagues (pers. commun.) aims at developing an in vivo screening assay, where the structural gene for g-galactosidase is replaced by that of URA3, an enzyme involved in uracil utilization by yeasts. Yeasts deleted for this gene but expressing the new construct will grow in the presence of uracil only when the steroid receptor is activated by steroids and is directing the transcription of the plasmid-borne URA3 gene. This will allow screening for new steroid drugs by assaying growth of yeast.

cl

0

q

Members of two families of receptors have now been functionally expressed in microorganisms. G protein-coupled receptors, despite their profound hydrophobic character, when inserted in the inner membrane of E. coli or in S. cerevisiae membranes, display all the ligandbinding and G protein-coupling properties observed in mammalian cells. The soluble steroid receptors also functionally interact with E. coli or S. cerevisiae DNA as they would in mammalian cells. Two challenges now lie ahead for the molecular pharmacologist: to develop convenient and quantitative effector systems that can be automated, and to extend the microbial expression to other receptors, including those that consist of more than one polypeptide chain. Expression of the 55 kDa chain of the interleukin 2 receptorI represents a first step in this direction.

Acknowledgements Support for our work comes mostly from the Centre National de la Recherche Scientifique, the Institut National de la Sant4 et de la Recherche Mkdicale, University Paris VII, the Ministry for Research and the Bristol Myers-Squibb Company. We are also grateful for help from the Ligue National contre le Cancer, the Fondation pour la Recherche MCdicale Franqaise and the Association pour la Recherche sur le Cancer. A. DONNY

STROSBERG

STEFANO

AND

MARULLO

Laboratoire d’lmmuno-Pharmacologic Mol&uJaire, CNRS UPR 0415 and UniversiH Paris GJl, Jnstitut Cochin de Ghitique MoJtfculaire, 22 rue M&chain, 75014 Paris, France.

References 1 Strosberg, A. D. (1991) Eur. 1. Biochem.

196, l-10 2 Strosberg, A. D. and Leysen, J. E. (1991) Curr. Opin. Biotechnol. 2,30-36 3 Mamllo, S., Delavier-Klutchko, C., Eshdat,. Y.,. Strosberg, A. D.. and Emorine, L. (1988) Proc. NatJ Acad. Sci. USA 85,7551-7555 4 Marullo, S. et al. (1989) Bio/TechnoJogy 7, 923-927 5 Marullo, S., Emorine, L. J., Stmsberg, A. D. and Delavier-Klutchko, C. (1990) EMBO 1. 9,1471-1476 6 Galli, G. et al. (1990) Biochem. Biophys. Res. Commun. 173,680-688 7 Freissmuth, M., Seizer, E., MaruUo, S., Schlitz, W. and Strosberg, A. D. (1991) Proc. NatJ Acad. Sci. USA 88,854EX552 8 Bertin, B., Freissmuth, M., Strosberg, A. D. and Marullo, S. J. BioJ. Chem. (in press) 9 Payette, P., Gossard, F., Whiteway, M. and Dennis, M. (1990) FEBS Lett. 266, 21-25 10 Breyer, R. M., Strosberg, A. D. and Guillet, J. G. (1990) EMBO .I. 9, 2679-2684 11 Luyten, W. H. M. L. et al. (1991) Drug Invest. 3. 3-12 12 King, K:, Dohlman, H. G., Thomer, J., Camn, M. G. and LetIowitz, R. J. (1990) Science 250,121-123 13 Wittliff, J. L., Wenz, L. L., Dong, J., Nawaz, Z. and Butt, T. R. (1990) 1. BioJ. Chem. 265,220X-22022 14 Metzger, D., White, J. H. and Chambon, P. (1988) Nature 334, 3136 15 Mak, P., McDonnell, D. P., Wiegel, N. L., Schrader, W. T. and O’Malley, B. W. (1989) J. BioJ. Chem. 264, 21613-21618 16 Schena, M. and Yamamoto, K. R. (1988) Science 241, 965-967 17 Pham, T. A. et al. (1991) Proc. NatJ Acad. Sci. USA 88,312c312< 18 McDonnell, S. P., Pike, J. W., Dritz, D. I.. Butt. T. R. and O’Mallev. 8. W. (1989) MoJ: Cell BioJ. 9, 3517-3&3 19 Hiiskcn, D., Beckers, T. and Engles, J. W. ,199O) Eur. 1. B&hem. 193,387-394