Steroid Hormone Receptors and Nuclear Receptors

Now, in his forthcoming book Scally continues his first-rate investigative and research abilities into producing an opus on steroid science. In the tradition of supplying the well-educated and intelligent Meso readers with the best available information available, Dr. Scally is providing Meso with summaries of the forthcoming book. The article following is the first in a series that highlight selected excerpts from this book. The androgen receptor is a member of the nuclear receptor (NR) family. Research and investigations on the androgen receptor (AR) are creating a revolution for ligands, steroidal and nonsteroidal, that produce anabolic changes in muscle. To fully appreciate and understand these new drugs requires knowledge for the nuclear receptor. The first of these articles introduces the nuclear receptor. Following this will be an article on the androgen receptor, introducing the all-important ligand-binding pocket (LBP). The third in the series will document the molecular interactions of various ligands, steroidal and nonsteroidal, for amino acid residues within the LBP.

THE NUCLEAR RECEPTOR

Multicellular organisms require specific intercellular communication to orchestrate properly the complex body plan during embryogenesis and maintain the physiological properties and functions during the entire lifespan. The binding of extracellular signal molecules to either cell-surface receptors or intracellular receptors differs. Most signal molecules are hydrophilic and are therefore unable to cross the plasma membrane directly; instead, they bind to cell-surface receptors, which in turn generate one or more signals inside the target cell. A number of small hydrophobic signal molecules diffuse directly across the plasma membrane of target cells and bind to intracellular receptor proteins. These signal molecules include steroid hormones, thyroid hormones, retinoids, and vitamin D. Although they differ greatly from one another in both chemical structure and function, they all act by a similar mechanism.

Initiation of classical signal transduction cascades are by ligand binding to membrane-anchored receptors, eventually changing the activity of specific nuclear transcription factors. While growth factors, neurotransmitters, and peptide hormones bind to membrane receptors thereby inducing the activity of intracellular signaling pathways, other small hydrophobic signaling molecules such as steroid hormones, certain vitamins, and metabolic intermediates enter the target cells and bind to cognate members of a large family of nuclear receptors (NR).[1] In contrast, members of the nuclear receptor superfamily transduce their signals directly. The receptors have evolved to combine the functions of signal responsiveness, DNA-binding, and transcriptional activation into one protein composed of functionally separated modules/domains.[2]

Nuclear receptors (NR) are members of a large superfamily of evolutionarily related DNA-binding transcription factors that regulate programs involved in a broad spectrum of physiological phenomena.[3] Nuclear receptors regulate diverse functions, such as homeostasis, reproduction, development, and metabolism. Nuclear receptors function as ligand-activated transcription factors, and thus provide a direct link between signaling molecules that control these processes and transcriptional responses.[4]

The NR is of major importance for intercellular signaling, converging intracellular and extracellular signals on the regulation of genetic programs. They are transcription factors that: (1) respond through association with a variety of hormonal and metabolic signals, (2) integrate diverse signaling pathways as they correspond themselves to targets of post-translational modifications, and (3) regulate the activities of other major signaling cascades. The genetic programs that they establish or modify affect virtually all aspects of the life, covering such diverse aspects as embryogenesis, homeostasis, reproduction, cell growth, or death.[5]

NR emerged in the earliest of metazoan evolution, long before the divergence of vertebrates and invertebrates.[6] The NR superfamily consists of a large group of transcription factors that are essential for growth, differentiation, metabolism, reproduction, and morphogenesis. More than fifty members of the NR superfamily all derive from a common ancestor.[7] Complementary DNA (cDNA)[8] for many intracellular receptors have been isolated and characterized, making it possible to deduce the amino acid sequences of various nuclear receptors and related members of the superfamily of nuclear receptors.[9] The phylogenetic transparency of these receptors makes classification by sequence relatively straightforward. Sequence alignment and phylogenetic tree construction resulted in a classification of the human NR family into six evolutionary groups, subfamilies, of unequal size.[10]

The steroid hormone receptors (SHR) subgroup of the nuclear receptor superfamily is nuclear receptor subfamily 3. Subfamily 3A includes the estrogen receptor, which comes in two forms, ERα [NR3A1] and ERβ [NR3A2]. Subfamily 3C includes the cortisol binding glucocorticoid receptor (GR) [NR3C1], the aldosterone binding mineralocorticoid receptor (MR) [NR3C2], the progesterone receptor (PR) [NR3C3], and the dihydrotestosterone (DHT) binding androgen receptor (AR) [NR3C4]. In addition, the SHR subgroup contains three orphan receptors closely related to ER, subfamily 3B: the estrogen related receptors α (ERRα) [NR3B1], β (ERRβ) [NR3B2], and γ (ERRγ) [NR3B3], for which a natural ligand remains to be identified.[11]

To date, no experimentally determined three-dimensional structure is available for a complete receptor. It has so far not been possible to determine the tertiary structure of intact full-length NR. Nevertheless, conformational studies of NR parts yield valuable structural information. There is a consistent structural and functional organization of the nuclear receptor superfamily.

Cloning and sequencing of the genes encoding nuclear receptors permit comparison of their amino acid sequences. Such studies revealed a remarkable conservation in both the amino acid sequences and different functional regions of various nuclear receptors. Nuclear receptors share a common structural organization. NR is a modular protein with distinct functional and structural domains. The members of the steroid hormone receptor family share a similar, modular architecture, consisting of a number of independent functional domains.

All steroid hormone receptors are composed of a variable N-terminal domain (NTD, A/B); a highly conserved DNA Binding Domain (DBD, C); a flexible hinge region (D); and a C-terminal Ligand Binding Domain (LBD, E). The estrogen receptor α is unique in that it contains an additional C-terminal (F) domain with unknown function.[12]

Nuclear receptors contain a variable N-terminal amino acid sequence, (NTD, A/B), which contains an autonomous transcriptional activation function known as AF-1. The AF-1 shows weak conservation across the nuclear receptor superfamily and may mediate differential promoter regulation in vivo. The AF-1 sequence functions as a ligand-independent transcriptional activator, but can also functionally synergize with AF-2. The NTD is unique to each SHR and has variable sequence and length. The highly conserved C region harbors the DNA-binding domain that confers sequence-specific DNA recognition (DBD, C). Situated between the DBD and the LBD is a linker region, domain D. This region functions as a flexible hinge and contains the nuclear localization signal (NLS). The LBD (E) is responsible for the binding of cognate ligand or hormone. This domain also contains a ligand-dependent transcriptional activation function (AF-2) necessary for recruiting transcriptional coactivators, which interact with chromatin remodeling proteins and the general transcriptional activation machinery. Nuclear receptors may or may not contain a final domain in the C-terminus, the F region, whose sequence is extremely variable and whose structure and function are unknown.[15]

A/B REGION (N-TERMINAL DOMAIN, NTD)

Thus far, there is no elucidation of a crystal structure of an A/B domain. The A/B region in the different NR is highly variable, revealing a very weak evolutionary conservation. The N-terminal region is the least conserved region among NR, both in size and sequence.[16] All the nuclear receptors have a unique N-terminal region (NTD) of variable length (100–500 amino acids) whose 3D structure is unknown.

NR contains two transactivation functions. One maps to the structurally flexible N-terminal domain (NTD) and is termed AF-1. The poorly defined N-terminal A/B region contains a transcriptional activation function, referred to as activation function 1 (AF-1) that can operate autonomously. AF-1 can act in a ligand-independent manner when placed outside of the receptor. Besides the one constitutionally active transactivation region (AF-1), NTD includes several autonomous transactivation domains (AD). The activation domains (AD) contain transcriptional activation functions that can activate transcription when fused to a heterologous DNA-binding domain.

The main determinants for transactivation map to NTD of both the androgen (AR) and glucocorticoid (GR) receptors and while generally there is little sequence conservation between the different NTD, in contrast to the DBD and LBD, short regions of similarity have been observed for the AR and GR.[17] The NTD is potentially involved in multiple protein-protein interactions and the length of this domain has a positive correlation with the activity of AF-1 for different members of the nuclear receptor superfamily.[18]

C REGION (DNA BINDING DOMAIN, DBD)

The DBD consists of a highly conserved residue core located between the N-terminal domain and the C-terminal ligand-binding domain. The DNA binding domain lies toward the center of the molecule. The amino acid sequence of this domain is similar among different steroid receptors (56–79% identity). The 3D structure of the DBD has been resolved for a number of nuclear receptors. Nuclear magnetic resonance and crystallographic studies for different NR DBD in their DNA uncomplexed and complexed forms with the GR and ER homodimers on their cognate DNA sequence were the first 3D crystal structure reported.[19]

D REGION (HINGE REGION)

The D region, which is a poorly conserved domain, serves as a hinge between the DBD and the LBD, allowing rotation of the DBD. The hinge region allows the DBD and LBD to adopt different conformations without creating a steric hindrance. This domain also harbors a nuclear localization signal (NLS) or at least some elements of a functional nuclear localization signal.

E REGION (LIGAND BINDING DOMAIN, LBD)

The largest domain is the moderately conserved ligand-binding domain (LBD, E region). The hallmark of a nuclear receptor is its ligand-binding domain (LBD). This domain is highly structured, and encodes a wealth of distinct functions most of which operate in a ligand-dependent manner. The highly conserved region of the nuclear receptor proteins lies near the carboxyl terminus. The AR C-terminal ligand-binding domain contains about 290 amino acids and represents about 30% of the receptor. The ligand-binding domains of AR from humans, rats, and mice are identical, and sequence homology with other steroid receptors ranges between 15% and 54%.

Members of the NR superfamily (GR, MR, PR, AR, and ER) interact with heat-shock proteins via their LBD.[20] The LBD is a region where chaperone proteins, such as Hsp90, bind NR when they are present in the cytoplasm. Upon ligand binding, exposure of a NLS in the LBD induces the nuclear translocation of the NR. NR-LBD together with NR-DBD also consists of a sequence involved in NR dimerization with the NR partner.[21] After dissociation of chaperones, the liganded NR–complexes can bind to chromatin organized DNA sequences near target genes, termed hormone response elements (HRE). The HRE-recruited hormone-receptor-complexes are then able to initiate chromatin remodeling and to relay activating or repressing signals to the target genes transcription machinery.

Nuclear receptor pharmacology has led the way in the appreciation that ligands may exert very diverse pharmacology, based on their individual chemical structure and the allosteric changes induced in the receptor/accessory protein complex.[22] Binding of agonistic or antagonistic ligands leads to different allosteric changes of NR making them competent to exert positive or negative effects on the expression of target genes by different mechanisms. In the upcoming articles, discussion of these interactions is with a focus on the androgen receptor. The past decade has seen a revolution in the study and investigation of androgen receptor ligands. This brief introduction of the nuclear receptor serves as background for a more complete understanding of new and revolutionary drugs currently under study for clinical use.

References