28.11: Signaling by Steroid Hormones

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Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Steroid Hormone Receptors: Structure and Mechanism of Nuclear Action

• Describe the four conserved domain architecture of steroid receptors (NTD/AF1, DBD with two Zn fingers, hinge region, LBD/AF2), explain the function of each domain, and trace the lifecycle of a cytoplasmic steroid receptor from synthesis through Hsp70/Hsp90 chaperone association, ligand-induced Hsp dissociation, homodimerization, nuclear translocation, DNA binding, coregulator recruitment, and proteasomal degradation.
• Distinguish between the direct (classical) and indirect mechanisms of steroid-regulated gene transcription: in the direct pathway, the receptor dimer's Zn finger DBD binds palindromic hormone response elements (HREs/SREs) in gene promoters, while in the indirect pathway, the receptor tethers other DNA-bound transcription factors without directly contacting DNA itself.
• Explain how coactivators (which recruit histone acetyltransferases to promote chromatin remodeling and transcription) and corepressors (which recruit histone deacetylases to inhibit transcription) are recruited to the DNA-bound receptor dimer in a ligand- and conformation-dependent manner, and describe how the specific conformation induced by a selective estrogen receptor modulator (SERM) determines whether coactivators or corepressors are preferentially recruited.

Estrogen Receptors, SERMs, and Breast Cancer

• Compare the transcriptional activities of ERα and ERβ homodimers and ERα/ERβ heterodimers—explaining why ERα homodimers promote estrogen-dependent proliferation, ERβ homodimers inhibit it, and heterodimers reduce ERα proliferative signaling—and describe the structural basis by which tamoxifen acts as an antagonist by occupying the ERα ligand-binding domain and inducing a conformation that favors corepressor over coactivator binding in breast tissue.
• Explain how MAPK-mediated phosphorylation of ERα at Ser118 (downstream of EGF/RTK signaling) can activate transcription in a ligand-independent manner, and describe why this cross-talk between growth factor receptor pathways and the estrogen receptor contributes to tamoxifen resistance and hormone-independent proliferation in breast cancer.

Membrane Steroid Signaling and Integration with Other Pathways

• Describe how steroid hormones can elicit rapid signaling responses by binding to membrane receptors—including GPCRs (e.g., GPER for estrogen), ion channels (e.g., TRPM8, NaV1.2, CAV1.3), and zinc transporters (ZIP9)—and explain how these membrane-initiated effects activate downstream pathways (RTK transactivation, MAPK cascade, PI3K/AKT) that are distinct from the slower genomic effects mediated by nuclear receptors.
• Explain how some nuclear steroid receptors (particularly ERα) can be palmitoylated and targeted to the plasma membrane, where they activate signaling pathways independently of their nuclear transcriptional activity, and describe why this duality of membrane and nuclear signaling complicates therapeutic targeting of steroid receptors in hormone-sensitive cancers.

Introduction

We will now consider signaling by steroid hormones, cholesterol derivatives. They are mostly nonpolar. Steroid hormones can affect signaling in two major ways:

• through binding to membrane receptors, which, when occupied, affect signaling through the myriad ways we will discuss throughout this chapter. These effects would be rapid.
• through binding to cytoplasmic receptors after they diffuse into the cell passively or actively. This signaling is similar to retrograde signaling by nitric oxide, which can passively diffuse out of a cell and enter an adjacent cell to affect signaling. If the steroid's primary messenger is in the cell, it often enters the nucleus and regulates gene transcription. Binding to cytoplasmic receptors accounts for most of the biological effects of steroid hormones. Since transcription is involved, the pathways elicit a slower response.

We will briefly discuss the first type of signaling (binding to membrane receptors) by presenting figures describing their signaling pathways. Then, we will focus on steroid hormone activation of gene transcription.

There are many classes of steroid hormones. These are illustrated in Figure \(\PageIndex{1}\) along with their overall synthetic pathway.

Figure \(\PageIndex{1}\): Diagram of the pathways of human steroidogenesis. WikiJournal of Medicine 1 (1). DOI:10.15347/wjm/2014.005. ISSN 20018762.

The families of hormones include progestogens and estrogens (female sex hormones), androgens (male sex hormones), glucocorticoids (such as cortisol, which affects the immune and metabolic systems), and mineralocorticoids (which regulate salt/water balance).

We will mostly use the estrogens in the section as a prototypical example because of their involvement in breast cancer and epidermal growth factor receptors.

Steroid hormone signaling through binding to membrane receptors

Most of this subsection is taken from and adapted from the following source:  Masi et al. Cells 2021, 10(11), 2999; https://doi.org/10.3390/cells10112999. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Most people think of steroid signaling mediated by their nuclear effects on gene transcription (the predominant signaling effect). Signaling at the cell membrane is an emerging area of interest. We will present steroid signaling in four figures, each with its associated caption, at the cell nucleus. We first present these lesser-known effects of steroid signaling, as they involve many pathways we have already studied. The figures will also briefly review some canonical pathways, which are always good in fields as complicated as signal transduction. The figures focus on the signaling effects in cancer.

Figure \(\PageIndex{2}\) shows membrane signaling through androgen receptors.

Figure \(\PageIndex{2}\): Membrane androgen receptor-associated pathways and their effect on hormone-sensitive cancer progression (breast cancer as BC, prostate cancer as PC). Receptors are ZIP9 (Zinc transporter), OXER1 (Oxoeicosanoid receptor 1), TRPM8 (Transient receptor potential cation channel subfamily M member 8), CAV1.3 (Voltage-dependent L-type calcium channel subunit alpha-1C). Masi et al. ibid

Figure \(\PageIndex{3}\) shows membrane signaling through estrogen receptors

Figure \(\PageIndex{3}\): membrane estrogen receptor-associated pathways and their effect on hormone-sensitive cancer progression (breast cancer as BC, prostate cancer as PC, endometrial cancer as EC, ovarian cancer as OC, and testicular germ cell cancer as TGCC). Other abbreviations: multi-drug resistance (MDR), anchorage-independent growth (AIG). Receptors are GPER (G-protein coupled estrogen receptor), NaV1.2 (Sodium channel protein type 2 subunit alpha). Masi et al. Ibid

A binding site for tamoxifen (a selective estrogen receptor modulator used in breast cancer therapy) has been found in NavMs voltage-gated sodium channels.

Figure \(\PageIndex{4}\) show membrane signaling through membrane progesterone receptors

Figure \(\PageIndex{4}\): Membrane progesterone receptor-associated pathways and their effect on hormone-sensitive cancer progression (breast cancer as BC, prostate cancer as PC, ovarian cancer as OC, and basal phenotype breast cancer as BPBC). Receptors are mPR (Membrane progestin receptor). Masi et al. ibid

Figure \(\PageIndex{5}\) shows membrane-associated progesterone receptor effects from progesterone signaling.

Figure \(\PageIndex{5}\): MAPK-associated pathways breast cancer as BC, endometrial cancer as EC, ovarian cancer as OC, Leydig cell tumor as LCT, and cervical cancer as CC). Other abbreviations: anchorage-independent growth (AIG). Receptors are PGRMV (Membrane-associated progesterone receptor component 1) and GIG47 (Neudesin). Masi et al. ibid.

Steroid hormone signaling through binding to cytoplasmic receptors and activation of gene transcription

Now we will explore the major effects of steroid hormones on intracellular signaling: steroids enter the cell, bind to cytoplasmic receptors, and then translocate to the nucleus, where they regulate gene expression. The naming of steroid receptors can be confusing, as it is essential to differentiate between steroid receptors that reside in the cell membrane and those that move to the nucleus. An added complexity arises because some nuclear receptors can be covalently modified with a fatty acid (palmitoylated) and targeted to the cell membrane. Estrogen receptors targeted to the membrane can then act independently of their nuclear transcriptional activity.

We will primarily focus on estrogen effects in breast cancer (the most diagnosed cancer) that are mediated through the nuclear steroid receptors, which belong to the nuclear receptor superfamily. Tamoxifen, a commonly used drug in the treatment of breast cancer, is an estrogen receptor antagonist (also called an estrogen receptor modulator—SERM).

Molecular Function of Steroid Receptors—Common Features

Steroid receptors (SR) consist of four main domains: the C-terminal ligand-binding domain (LBD), the DNA-binding domain (DBD), the hinge region, and the amino-terminal domain (NTD). Each SR also contains two motifs, called activation functions 1 and 2 (AF1 and AF2), within the NTD and LBD, respectively, and they are crucial for regulating gene transcription. Two zinc fingers are located in the DBD. Figure \(\PageIndex{6}\):

Figure \(\PageIndex{6}\): Schematic illustration of steroid receptor structure. NTD—amino-terminal domain, DBD—DNA-binding domain, H—hinge region, LBD—ligand-binding domain, AF1—activation function 1, AF2—activation function 2. The diagram does not show the exact length proportion of the domains because it differs between distinct SRs. Kowalczyk, W.; Waliszczak, G.; Jach, R.; Dulińska-Litewka, J. Steroid Receptors in Breast Cancer: Understanding of Molecular Function as a Basis for Effective Therapy Development. Cancers 2021, 13, 4779. https://doi.org/10.3390/cancers13194779. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

The domains are often labeled A-F. The N-terminal domain (NTD) is also called the A/B domain. It can also bind DNA and can weakly activate transcription in the absence of hormones. The C domain is the DNA-binding domain (DBD) containing zinc fingers that bind to the steroid response element in the promoters of key genes. The D domain is the hinge domain, and the E domain binds hormones (like estrogen) and protein regulators, and when bound, can activate gene transcription. The last domain (F) varies in length, and its function is unclear.

Figure \(\PageIndex{7}\) shows the specific DNA structure for the estrogen receptor from Pfam.

Figure \(\PageIndex{7}\): Estrogen receptor domain structure.

The green is the N-terminal Oest_recep domain (NTD, A/B). The red zf-C4 is the DNA-binding domain with two Zn fingers that bind DNA (DBD, C). The blue Hormone_Receptor is the ligand (estrogen) binding domain (LBD, domain E). The yellow is the C-terminal domain (F).

These two genes encode the estrogen receptor, ERα and ERβ. The transcriptionally active form is a dimer that forms on the binding of estrogens. The dimer then translocates to the nucleus and activates transcription at ERE sites. The ERα dimer promotes estrogen-dependent growth while the ERβ dimer inhibits it. Heterodimers can form, which seem to reduce the proliferative effects of ERα. Both ERα and ERβ can be expressed in

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the human estrogen receptor computational model (P03372, AlphaFold)

Figure \(\PageIndex{8}\): Full human estrogen receptor computational model (P03372, AlphaFold) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...XAk1jUHred87c9

• red spacefill: N-terminal Met
• green backbone trace: N-terminal domain disordered, which can bind DNA
• magenta backbone trace: DNA binding domain with Zn fingers. The Cys side chains of the Zn fingers are shown in sticks, CPK colors, and labeled.
• blue: Hormone_Receptor is the ligand (estrogen) binding domain (LBD, domain E)
• yellow: C-terminal tail (domain)
• Cyan spacefill: C terminal Val

There are no full-length crystal structures of the ER dimer. Most of the available structures are of the estrogen-binding domain. It's really useful to see the full predicted structure to see how all the domains are connected, but perhaps more interestingly, the extended regions of disordered structures, which you should imagine adopting specific conformations upon interacting with key signaling partners.

Figure \(\PageIndex{9}\) shows an interactive iCn3D model of the ligand binding domain of human estrogen receptor ERα bound to the antagonist tamoxifen (3ERT).

Figure \(\PageIndex{9}\): Ligand binding domain of human estrogen receptor ERα bound to the antagonist tamoxifen (3ERT) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?hhKXF19sVHS5RPys9

The ligand binding domains (one for each ERα monomer) are shown in magenta and cyan. The antagonist Tamoxifen, bound in each ligand binding domain, is shown as sticks in CPK color. The amino acids comprising the binding site for tamoxifen are shown in stick and CPK colors and labeled in the magenta subunit. The CPK-colored spheres indicate the binding site on the ligand-binding domain for other proteins called corepressors or coactivators (not shown; discussed below).

Figure \(\PageIndex{10}\) shows the structures of estrogens and selective estrogen receptor modulators (SERMs).

Figure \(\PageIndex{10}\): Structures of estrogens and selective estrogen receptor modulators (SERMs). Smith et al. Breast Cancer Research. 2014, 16:212. http://breast-cancer-research.com/content/16/3/212. CC BY 2.0

The iCn3D model for the human estrogen receptor ERα bound to the antagonist tamoxifen (3ERT) showed binding sites for other proteins called coactivators or corepressors. After binding to DNA, the ER-estrogen complex can also bind a protein coactivator, which activates transcription. Likewise, binding of a corepressor to the DNA-bound complex inhibits transcriptional activity. Tamoxifen binding to the ERα monomer leads to dimerization and DNA binding. The DNA-bound dimer can then bind either a corepressor (the usual case for tamoxifen binding to ER in the breast), leading to inhibition of DNA transcription (i.e., tamoxifen antagonizes ER transcriptional function), or a coactivator, which stimulates gene transcription.

A cartoon diagram illustrating the role of ER coactivators and corepressors is shown in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\): Molecular activity of selective estrogen receptor modulators (SERMs) at estrogen receptors. Smith et al., ibid.

When a SERM binds to the estrogen receptor, the receptor adopts a unique conformation that allows dimerization and interaction with the target genes' estrogen response elements (EREs). The unique conformational change induced by SERM binding may result in a distinct pattern of cofactor recruitment.

Before steroid binding, most steroid receptors are found in the cytoplasm, bound to heat shock proteins like Hsp90. Phosphorylation of the Hsp:SR complex leads to dissociation of the Hsp, followed by dimerization and translocation into the nucleus. In some cases, hormone binding occurs in the nucleus.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of the estrogen receptor DNA-binding domain bound to DNA (1HCQ).

Figure \(\PageIndex{12}\): Estrogen receptor DNA-binding domain bound to DNA (1HCQ) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...rUUhnx8J8ezh8A

The backbones of the dsDNA are shown in spacefill cyan and magenta. The DNA bases are shown in CPK colors. The two chains of the DNA-binding domain of the estrogen receptor are shown in gray and gold. Zn²⁺ ions are shown as brown spheres. The coordinating Cys side chains in the gray DNA binding domain are shown as sticks, in CPK colors, and labeled "C". The amino acid side chains of the gold DNA-binding domain that interact with DNA are shown as sticks in CPK colors.

There are two ways steroid hormones activate gene transcription: direct and indirect.

Direct (classical): The DNA-binding domain (containing the Zn fingers) of the dimer binds to the target hormone response element (HRE) or, for steroids, the steroid response element (SRE) sequences in the promoter site of specific genes under steroid hormone control. As seen in the iCn3D above, one of the two Zn fingers on each hormone receptor binds to the target site in the major groove of DNA. The other Zn finger is involved in hormone receptor dimerization. The SRE contains two 6-base pair repeats separated by three base pairs. The DNA sequence shown in the iCn3D above is CCAPGGTCA. The consensus sequence for steroid hormones is 5′-GGTACAnnnTGTTCT-3′. The ER binds to 5′-GGTCAnnnTGACC-3′. Note that the complementary strand sequence is 5'-GGTCAnnTGACC, so the sequence is a palindrome (the complementary strand has the same sequence going in the opposite direction. After binding, additional coregulators bind. These modify histones and remodel the DNA to facilitate or inhibit transcription.

Indirect: In this method, the steroid receptors bridge other DNA-bound transcription factors without the steroid hormone binding to its response element.

The direct and indirect methods for steroid hormone effects on transcription are shown in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): SR synthesis, genomic signaling, and degradation.

(1) Translation of a SR and binding of Hsp70. (2) Hsp70 to Hsp90 transition. (3) Ligand binding, Hsp90 dissociation, and dimerization. (4) Nuclear translocation. (5) Transcriptional action: induction (5a, 5c) or inhibition (5b, 5d) of target gene expression, performed either in the classical mechanism involving SRE-binding (5a, 5b) or by tethering other TFs (5c, 5d). (6) Ligand dissociation followed by disassembly of the transcriptional complex and SR binding to a molecular chaperone. (7) Rebinding of the ligand. (8) Ubiquitination. (9) Proteasomal degradation. SR—steroid receptor, SH—steroid hormone, Hsp 70—heat shock protein 70, Hsp90—heat shock protein 90, SRE—steroid response element, CoA—coactivators, CoR—corepressors, HAT—histone acetyltransferase, HDAC—histone deacetylase, TF—transcription factor, TFRE—transcription factor response element, Ub—ubiquitin. Although histone acetyltransferases (HATs) and histone deacetylases (HDACs) are classified as coregulators, they are shown separately here to emphasize their roles. Illustration created using elements from Servier Medical Art https://smart.servier.com/, reproduced under Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0/.

In summary, in the absence of hormones, steroid receptors are inactive when complexed with chaperones such as Hsp70 and Hsp90.  Hormone binding to the receptor complex changes the conformation of the receptors, leading to chaperone dissociation.  The hormone-bound receptor is now free to bind DNA and regulate transcription.  In the nucleus, the receptor is active as a dimer.

The steroid hormone receptor can be targeted for proteasomal degradation via ubiquitination in either the nucleus or the cytoplasm.

Modulation of ER function by phosphorylation

No pathways stand in isolation, so it should be no surprise that the estrogen receptor (again used as an example) is regulated by post-translational modification, especially phosphorylation. Phosphorylation can be ligand-dependent or independent. Multiple kinases are involved in the phosphorylation of the N-terminal region. An especially important one occurs at Ser 118 at a cyclin-dependent kinase (involved in cell cycling) when the receptor is bound to estradiol. Ser 118 is also phosphorylated by epidermal growth factor signaling via MAPK. This may increase cell proliferation in breast cancer even in the absence of estrogen. Figure \(\PageIndex{14}\) shows ER activation by phosphorylation through growth factor and cytokine signaling pathways.

Figure \(\PageIndex{14}\): Activation of ER by phosphorylation induced by growth factor and cytokine signaling pathways. Siersbæk et al, Genes Dev. 2018 Sep 1; 32(17-18): 1141–1154. doi:10.1101/gad.316646.118. Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

Steroid hormones are cholesterol-derived, predominantly nonpolar signaling molecules that influence cell behavior through two mechanistically distinct modes: rapid, membrane-initiated signaling and slower, genomic signaling via nuclear receptors. In the membrane pathway, steroids bind to GPCRs (such as GPER for estrogen), ion channels (TRPM8, NaV1.2, CAV1.3), and other surface receptors, activating familiar downstream cascades including MAPK, PI3K/AKT, and intracellular Ca²⁺ signaling within seconds to minutes. These rapid effects are particularly relevant in hormone-sensitive cancers, including breast, prostate, endometrial, and ovarian cancers, and represent an emerging area of pharmacological interest.

The predominant and more extensively characterized steroid signaling mode involves passive diffusion of the hormone into the cell and binding to cytoplasmic receptor proteins. In the resting state, steroid receptors (SRs) are held in an inactive conformation by Hsp70 and Hsp90 chaperone complexes. Hormone binding triggers Hsp dissociation, receptor homodimerization, and translocation to the nucleus. All SRs share a conserved four-domain architecture: an N-terminal domain (NTD) containing the ligand-independent activation function AF1; a DNA-binding domain (DBD) with two Zn finger motifs, one of which contacts the palindromic hormone response element (HRE) in the major groove of DNA and the other of which mediates receptor dimerization; a flexible hinge region; and a C-terminal ligand-binding domain (LBD) containing the ligand-dependent activation function AF2. Once the DNA-bound dimer is established, the specific conformation of the LBD determines whether coactivators (which recruit histone acetyltransferases to open chromatin and promote transcription) or corepressors (which recruit histone deacetylases to compact chromatin and repress transcription) are recruited. An indirect genomic pathway also exists in which the receptor tethers other DNA-bound transcription factors without itself contacting a hormone response element.

The estrogen receptor (ER) system illustrates the therapeutic and biological complexity of steroid signaling. ERα and ERβ homodimers have opposing proliferative effects in breast tissue, and their relative expression levels and heterodimerization state determine the net transcriptional output. Tamoxifen and other selective estrogen receptor modulators (SERMs) exploit the conformational plasticity of the LBD to act as tissue-selective agonists or antagonists depending on the coregulator complement present in each tissue. Critically, the ER does not operate in isolation: MAPK-mediated phosphorylation of ERα at Ser118, downstream of EGF receptor signaling, can activate receptor-driven transcription in a ligand-independent manner, and palmitoylated ERα at the plasma membrane activates RTK-associated signaling pathways independently of nuclear function—both mechanisms contributing to resistance to endocrine therapies in breast cancer.