MISCELLANEA

Didier Picard is at the Department of

Cell Biology, University of

Geneva, Sciences III,

1211 GenEve 4, Switzerland.

Steroid-binding domains for regulating the

functions of heterologous proteins in cis

Didier Picard

Many questions in molecular, cellular and developmental biology can only be addressed using technical ap- proaches that allow the introduction or activation of a test protein at a g!yen time-point. Temperature-sensi- tive protein mutants, microinjection, and inducible expression systems have been used extensively in eukaryotic cells. However, these techniques suffer from a number of specific disadvantages. For example, temperature-sensitive mutants are rarely available and protein function can usually only be switched off. The microinjection technique requires a source of protein, and biochemical analysis of microinjected proteins is technically very demanding. While inducible expression systems are well suited for biochemical studies, they are subject to a lag phase dur- ing which rnRNA and protein have to accumulate, and the induction of endogenous genes cannot always be avoided.

A novel inducible system, which largely circumvents these problems, has been developed during the last few years1: the activity of many intra. cellular proteins can be hormonally regulated post-translationally when they are fused to the hormone-bind- ing domain (HBD) of steroid recep- tors. In the absence of hormone, the HBD maintains the heterologous proteins i n an inactive state. On treating cells with ligand, this re- pression is reversed. Induction is very specific, requires no new protein synthesis and happens within sec- onds to minutes. This approach was initially important for analysing the mechanism of signal transduction by steroids 1,2, but has now become a very powerful tool for studying a variety of questions (see Box 1 for technical details).

Several recent publications illus- trate applications of this approach: hormone-regulated Myc 3, Fos4-6, Myb 7, Rel 8 and Abl 9 allowed the characterization of reversible trans- formation or changes in the differen-

tiation state of fibroblast, haemo- poietic and neuronal cells; a Myc- HBD fusion protein was instrumental in showing that Myc can promote progression through the cell cycle lo; both reversible and irreversible changes in the state of cellular differ- entiation of epithelial cells have been studied with a Fos-HBD fusion pro- tein11; hormone-regulated Fos 4-6, Myb 7 and Myc 10 fusion proteins were used to identify Fos-, Myb- and Myc-responsive genes, respectively; and the hormone-reversible inacti- vation of a fusion protein of the CCAAT-enhancer-binding protein (C/EBP) made it possible to generate

cell lines expressing this growth- inhibitory protein 12.

HBDs regulate a var iety of functions

As part of a steroid receptor, the 'inactivation function' of unliganded HBDs represses one or several func- tions including some that map, at least in part, outside the HBD itself: these include dimerization, nuclear localization, DNA binding and tran- scriptional regulation. A variety of heterologous functions can now be added to this list (Box 2). The fact that the activity of several cytoplasmic kinases can be regulated by HBDs definitively demonstrates that func- tions other than nuclear localization or DNA binding can be controlled.

The HBD has a number of other activities in addition to its inacti- vation function. Depending on the specific application, these activities can be either a complication or use- ful in themselves. When fused with transcription factors the hormone- dependent transactivation function of the HBD has been a matter of concern, but this problem might be

BOX 1 - DESIGNING AND USING FUSION PROTEINS WITH STEROID-BINDING DOMAINS

Choice of HBD Avoid the HBD of a steroid receptor present in your biological system. Note that GR, in contrast to other steroid receptors, is present in almost all tissues and cell lines.

Design of fusion Place HBD 'close' to an essential domain or try various fusion points (see text for dis- cussion). Since HBDs form the C-terminus of steroid receptors, HBDs have usually been fused to the C-terminus of heterologous proteins, provided the latter tolerate such additions. While this is technically easier, it is not a necessity. The ER HBD has been shown to work at the N-terminus of Myc 3 and between the two moieties of GAL4-VP16 (Refs 20,21). The former case, however, il!ustrates one of the potential problems of such an arrangement: ER-Myc was found to have a higher activity in the absence of hormone than Myc-ER because of translation initiation at an internal AUG (M. Eilers, pets. commun.), resulting in the deletion of the regulatory HBD from some Myc molecules.

Expression Express the fusion protein constitutively in vivo. To avoid activation of the ER and MR HBDs by spurious steroids in normal tissue culture medium, use medium with- out phenol red, supplemented with charcoal-treated serum. Alternatively, use a mutant HBD with reduced ligand-binding affinity.

Activation For activation of a protein fused to the ER or MR HBDs, add 0.1 IJM 13-estradiol or aldosterone, respectively; with the GR HBD, add 10 I~M dexamethasone. Hormones can be stored as 1000-fold concentrated stock solutions in ethanol at -20°C.

Deinduction Remove the steroid by washing tissue culture cells several times with medium, Tris- buffered saline or phosphate-buffered saline.

278 © 1993 Elsevier Science Publishers Ltd (UK) 0962-8924/93/$06.00 TRENDS IN CELL BIOLOGY VOL. 3 AUGUST 1993

MISCELLANEA

avoided altogether in the future by using transactivation mutants of the HBD 13. By contrast, the hormone- dependent nuclear localization signal (NLS) of the HBD of the glucocorticoid receptor (GR) may be a key regulated function in certain REV fusion proteins TM and the HBD dimerization function may play a role in the regulation of an Abl fusion protein 9.

So far, few experiments have ad- dressed which function(s) is directly inactivated by the HBD. In the case of transcription factors, NLSs present in the heterologous moiety may 1,8 or may not 1 be inactivated. The estrogen receptor (ER) and the pro- gesterone receptor (PR) are localized to the nucleus in the absence of hor- mone, suggesting that their un- liganded HBDs may not repress NLS activity in their natural context. In the case of REV-HBD fusion proteins, however, nuclear localization may well be the only regulated function. In the absence of hormone Rev fused to the GR HBD is maintained in the cytoplasm and is inactive, whereas when hormone is added Rev-HBD moves to the nucleus and transactivates target genes TM. How- ever, dominant negative mutants of Rev, fused to the GR HBD, escape hormonal control despite their cyto- plasmic localization in the absence of hormone 15. They presumably exert their negative effect by oligomer- izing with wild-type Rev.

BOX 2 - A VARIETY OF HETEROLOGOUS PROTEINS CAN BE REGULATED BY FUSION TO A STEROID-BINDING DOMAIN

inac,~vel Protein X J ~ § b ' ~

H Q " ~ _

t - . . ~ HSP90

Heterologous proteins (Protein X) can be regulated by a hormone-binding domain (HBD). In the absence of hor- mone the hybrid protein may be maintained in an inactive state due to formation of a complex including HsPg0, which results in steric hin- drance of other activities (see main text). Hormone (H) binding results in the release of the HSP90 complex and hence derepression. The HBDs of GR 1, ER 3 and MR (C. Fankhauser,

J-F. Louvion, P-A. Briand and D. Picard, unpublished) have been shown to contain such a hormone-regulable inactivation function. This list of heterologous proteins is necessarily incomplete although it includes all the published examples.

Protein X Regulated as

E1A (adenovirus) c-Myc c-Fos v-Myb C/EBP v-Rel GATA-1, -2, -3

GAL4-VP16 Rev (HIV) c-Abl Raft STE11

Transcription factod, oncoprotein a Oncoprotein 3 Oncoprotein, transcription factor 4 Transcription factor 7 Transcription factor 12 Oncoprotein, transcription factor 8 Transcription factor, promoter of proliferation (GATA-2) 22

Transcription factor (yeast and tissue culture cells)20, 21 Transactivator TM

Oncoprotein, tyrosine kinase 9 Oncoprotein, serine/threonine kinase b Serine/threonine kinase (yeast) c

ap. Jansen-D~rr et aL, unpublished. bM. Samuels, I. M. Bishop, M. J. Weber and M. McMahon, pers. commun. cJ-F. Louvion and D. Picard, unpublished.

A model for the mechanism of pro te in inac t iva t ion

How can the apparent lack of specificity of the HBD in regulating a variety of functions be explained? It has been proposed that inactivation by unliganded HBDs is mediated by HSP90 (Refs 1,2). This highly abun- dant and conserved protein forms a complex with each of the five ver- tebrate steroid receptors in the absence of hormone. This complex, which may include several other proteins 16, is disrupted upon hor- mone addition. The HBD is sufficient for hormone-reversible complex for- mation even when fused to heter- ologous proteins 17. The proposed dependence of regulation by the HBD on the HSP90 complex sug- gests that such regulation might be restricted to protein domains re- maining in the cytosolic or nuclco plasmic compartments; translocat!on into a vesicular compartment such as mitochondria or the endoplasmic reticulum would either not allow the

formation of the regulatory complex or result in its disruption.

I favour a model in which the HSP90 complex interferes with func- tions of the fusion protein by steric hindrance. Thus, inactivation would be effected by blocking access to other proteins (for example, other subunits, coactivators, substrates and components of the nuclear localization machinery), or to DNA and RNA target sites. This model predicts that the tightness of regu- lation depends on how components of the HsPg0 complex become pos- itioned relative to a critical functional domain in the heterologous moiety; domains that are facing away from the HSPg0 complex or are physically too far away would not be regulated. Extensive studies of E1A fusion pro- tpins support the notion that physi- cai proximity may indeed be im- portant for tight regulation. The regulation of the transactivation function, which requires E1A con-

served region 3 (CR3), was gradually lost when the HBD was moved fur- ther and further away on the linear protein map 1, which presumably reflects spacing in three dimensions to some extent. The transformation function of E1A, requiring CR1 and CR2, could be brought under hor- monal control only by moving the HBD even closer (D. Picard, unpub- lished; see also Box 2). In the light of this model, it is to be expected that certain functions remain active even in the absence of hormone. This is indeed the case for the nuclear local- ization function of ER, PR and certain fusion proteins 1, the oligomerization capacity of REV fusions 15 and the growth-inhibitory function of Abl fusions 9. Preliminary findings with enzymes such as ~-galactosidase, galactokinase, dihydrofolate reduc- tase and URA3 are also compatible with this view. HBDs fail to inacti- vate these functions (D. Picard, unpublished), presumably because

TRENDS IN CELL BIOLOGY VOL. 3 AUGUST 1993 279

MISCELLANEA

Acknowledgements

I am grateful to a large number of

colleagues for sharing unpub-

lished results, many of which I

have not been able to include. I thank Katharina

Strub and Tiziana Mattioni for

critical reading. This work was

supported by the Swiss National

Science Foundation and

the Canton de Geni~ve.

access of small substrates to the active sites is not sterically blocked.

Experimentally, the inactivation function of unliganded HBDs has so far been demonstrated only for the GR, ER and mineralocorticoid re- ceptor (MR). However, since the PR and androgen receptor (AR) also associate with HSP90 in a hormone- reversible fashion, the model would predict that their HBDs could be used for the regulation of heterolo- gous functions as well.

A tool fo r a var ie ty o f biological systems

If the HSPgO model is correct, the activity of a protein fused to an HBD should be hormone dependent in any cell type that provides the other components of the HSP90 complex. At present, HBD fusion proteins have been successfully used in mam- malian and avian tissue culture cells and in budding yeast (Box 2). Several laboratories are currently testing this tool in transgenic mam- mals. The presence of endogenous steroid receptors and hormones in animals may reduce the specificity and the tightness of this regulatory system. However, it could be inter- esting to take advantage of sex- specific differences in steroid con- centrations to regulate a fusion pro- tein differentially in males and females. It is conceivable that the HBD could also be used as a regu- latory domain in plants and insects

for which vertebrate steroids are gra- tuitous signals. Indeed, wild-type GR has been shown to function in a hor- mone-dependent fashion in tobacco protoplast 18 and in Drosophila tissue culture cells TM.

Thus, HBDs are a novel tool that can be used in a variety of biological systems to regulate activities of het- erologous proteins. In fact, the HBDs of GR, ER, MR and possibly PR and AR represent a whole set of regu- latory domains. With this choice, activation of endogenous steroid receptors in a particular cell line or tissue can be avoided and different HBDs could be used for independent regulation of more than one heterol- ogous protein in the same cell.

References 1 PICARD, D., SALSER, S. J. and

YAMAMOTO, K. R. (1988) Cell 54, 1073-1080

2 YAMAMOTO, K. R., GODOWSKI, P. J. and PICARD, D. (1988) Cold Spring Harbor Syrup. Quant. Biol. 53, 803-811

3 EILERS, M., PICARD, D., YAMAMOTO, K. R. and BISHOP, J. M. (1989) Nature 340, 66-68

4 SUPERTI.FURGA, G., BERGERS, G., PICARD, D. and BUSSLINGER, M. (1991) Proc. Nail Acad. Sci. USA 88, 5114-5118

S BRASELMANN, S., BERGERS, G., WRIGHTON, C., GRANINGER, P., SUPERTI-FURGA, G. and BUSSLINGER, M. (1992) J. Cell Science Suppl. 16, 97-109

6 WRIGHTON, C. and BUSSLINGER, M. M'~I. Cell. Biol. (in press)

7 BURK, O. and KLEMPNAUER, K-H. (1991) EMBOi. 10, 3713-3719

8 BOEHMELT, G. etaL (1992) EMBOJ. 11, 4641-4652

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10 EILERS, M., SCHIRM, S. and BISHOP, J. M. (1991) EMBOJ. 10, 133-141

11 REICHMANN, E. et al. (1992) Cell 71, 1103-1116

12 UMEK, R. M., FRIEDMAN, A. D. and MCKNIGHT, S. L. (1991) Science 251, 288-292

13 DANIELIAN, P. S., WHITE, R., LEES, J. A. and PARKER, M. G. (1992) EMBO J. 11, 1025-1033

14 HOPE, T. I., HUANG, X. J., MCDONALD, D. and PARSLOW, T. G. (1990) Proc. Natl Acad. Sci. USA 87, 7787-7791

15 HOPE, T. J., KLEIN, N. P., ELDER, M. E. and PARSLOW, T. G. (1992)/. Viral. 66, 1849-1855

16 SMITH, D. F. and TOFT, D. O. (1993) Mol. Endocrinol. 7, 4-11

17 SCHERRER, L. C. etal. Biochemistry(in press) 18 SCHENA, M., LLOYD, A. M. and DAVIS,

R. W. (1991) Proc. Natl Acad. Sci. USA 88, 10421-10425

19 YOSHINAGA, S. K. and YAMAMOTO, K. R. (1991) MoL Endocrinol. 5, 844-853

20 BRASELMANN, S., GRANINGER, P. and BUSSLINGER, M. (1993) Proc. NatlAcad. Sci. USA 90, 1657-1661

21 LOUVION, J-F., HAVAUX-COPF, B. and PICARD, D. Cene (in press)

22 BRIEGEL, K., LIM, K.C., BEUG, H., ENGEL, J. D. and ZENKE, M. Genes Dev. (in press)

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