Trafficking of nuclear receptors in living cells

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Abstract

Most of the steroid receptor family, with the exception of the estrogen receptor, are classically viewed as ‘translocating receptors’. That is, they move from an exclusively, or principally, cytoplasmic distribution in the absence of hormone to a predominately nuclear localization in hormone stimulated cells. The estrogen receptor and the nuclear receptor family are found exclusively in the nucleus, both in hormone stimulated and hormone free cells. This behavior has now been studied with GFP-fusions in living cells, and has in general been confirmed. However, there are important exceptions, and new findings, particularly with regard to sub-nuclear localization. We propose that the intracellular distribution of both receptor classes is dependent not only on subcellular localization signals directly encoded in the receptors, but also on the nature and composition of the large, macromolecular complexes formed by each receptor. Furthermore, we find that most members of the receptor superfamily form focal accumulations within the nucleus in response to ligand, and suggest that these structures may participate in the biological life cycle of the receptors. Finally, we propose that receptor movement in the nucleus is highly dynamic, with the receptors undergoing constant exchange between genomic regulatory elements, multi-protein complexes with other transcription factor partners, and subnuclear structures that are as yet poorly defined.

Introduction

The distribution of steroid/nuclear receptors within the major subcellular compartments is an important component of their biological activity. For members of the steroid receptor family, the mechanisms governing the cytoplasmic/nuclear distribution have been modeled primarily in terms of the conditional interaction of nuclear localization signals with the import/export apparatus present in nuclear pores [1]. In the absence of hormone, these receptors are found in association with a complex set of chaperones in a large complex [2] interaction of the cognate ligand with these receptors induces a conformational rearrangement that results in dissociation of the complex and loss of many of the associated factors. This reorganization is thought in some cases to expose previously masked translocation signals, and the receptors are then recognized by the transport machinery.

In contrast, members of the ‘nuclear’ subgroup are found constitutively in the nucleus, and are believed not to interact with the heat shock protein/chaperon complex. Some of these receptors, the thyroid hormone receptor in particular, have been shown to interact with specific DNA regulatory elements in the absence of ligand, and also repress selective genes in the hormone-free cells [3], [4]. These observations have led to the general hypothesis that the ‘nuclear’ receptors are nuclear localized because they are constantly present on chromatin. The dominant paradigm for subcellular localization of the receptors thus focuses on ligand-independent interaction with chaperone complexes for the steroid receptors, and ligand-independent binding to DNA elements for the nuclear receptors. Despite the wide-spread acceptance of this model, there are significant disparities in the literature. For example, the estrogen receptor clearly moves from a chaperone complex (8s) form to a dissociated form (4s) in a ligand dependent fashion, but it is constantly present in the nucleus. Furthermore, with the exception of the nuclear matrix, which is a controversial structure, current models of receptor trafficking view the nucleus essentially as a soluble compartment with little internal structure.

With the advent of the green fluorescent protein (GFP) [5], and the exciting potential this development provides for real time monitoring of protein trafficking [6], we initiated a program to utilize this reagent to study intracellular distribution and movement for members of the steroid/nuclear receptor family. The reagent has proven to be quite useful. Receptors labeled with GFP in general retain their normal transcriptional activity and ligand dependence [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. We now have approximately five years of experience using these chimeras, and several unexpected findings have emerged regarding subcellular trafficking for members of the receptor superfamily.

Section snippets

Progesterone receptor

The progesterone receptor (PR) is found in mammalian cells as two variants, the A and B forms. The A form is an expression variant, utilizing an alternate initiation signal, with the N-terminal 165 a.a. of the B form deleted (Fig. 1). These receptors are identical within the remaining 768 a.a. identical. PR-A can exert a dominant negative effect on PRB in some systems [17] and variation in dimerization efficiencies have been reported [18]. The formation of AA dimers appears more likely than AB

Estrogen receptor-alpha

While the alpha form of the estrogen receptor (ER) is classically a member of the steroid receptor family, and was the first to be identified in a molecular chaperone complex, it is unique among this group in its strictly nuclear localization. We recently confirmed this distribution in living cells with GFP derivatives of the human estrogen receptor [12]. Human ER labeled at the N-terminus with GFP was found to be expressed as a stable protein, and retains transactivational properties very

Thyroid hormone receptor

The thyroid hormone receptor (TR) has been described since its first identification as a nuclear receptor, that is, it is nuclear localized both in the absence and presence of ligand [28], [29], [30]. Several observations suggest that hormone-independent association with DNA is responsible for this localization. Hormone-free TR can bind to specific DNA elements in vitro [31], and is found in chromatin in association with a set of corepressors. Furthermore, unliganded TR can repress some

Dynamics of receptor movement

A classic view of receptor trafficking has often invoked a static location for a given receptor in a continuous ligand state. The steroid receptors are seen as continuously bound to a hormone response element in the constant presence of ligand, leaving the regulatory elements only when hormone is withdrawn. Conversely, a nuclear receptor, with TR as the prototype, is envisaged in a chromatin bound state in the absence of T3, in association with corepressors and chromatin silencing activities

Conclusions

These observations on receptor trafficking in living cells suggest a more complex and dynamic view of receptor movement than entertained in current models. We suggest that the distribution of nuclear/steroid receptors between the nucleoplasmic and cytoplasmic compartments is dependent not only on the intrinsic NLS signals present on the receptors, but equally subject to constraints imposed by the many partners found in the large macromolecular complexes with which the receptors associate (Fig. 5

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    Present address: Department of Pharmaceutics and Pharmaceutical Chemistry, 421 Wakara Way #318, University of Utah, Salt Lake City, UT 84108, USA.

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