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The mineralocorticoid receptor (MR) mediates the action of two adrenal steroid hormones, aldosterone, and cortisol, in many tissues in response to normal physiological change and in response to stress, adaption, or disease states. Aldosterone is a key regulator of cardiovascular homeostasis, and cortisol mediates adaptive responses to stress, both centrally and in the periphery. The MR is a large multidomain intracellular receptor that once activated triggers multiple cytoplasmic signaling pathways and in the nucleus directly regulates target gene expression to change cellular responses. Human MR gene mutations cause forms of pseudohypoaldosteronism-type-1 with prominent renal dysfunction, abnormalities in HPA axis activity, and cognitive behavior and have recently been associated with autism. MR mutations modeled in the mouse and fish are defining tissue-specific functions and molecular mechanisms related to normal function and disease. Future research will define new roles for the MR in the body and provide further detail of MR-activated cellular responses.

It is well known that many of the actions of 17β-estradiol in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there now exists compelling evidence for membrane steroid receptors for estradiol in hypothalamic and other brain neurons. But it is not well understood how estradiol signals via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for some time that estradiol can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane-delimited events. In addition, estradiol can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review will consider our current knowledge of rapid membrane-initiated and intracellular signaling by estradiol in the brain, the nature of receptors involved, and how they contribute to homeostatic functions.

Progesterone is a pleiotropic hormone that regulates a wide range of physiological and pathological processes in the central nervous system. The actions of progesterone are mediated by classical and nonclassical mechanisms, and many of them depend on its intracellular receptor (PR). PR expresses two main isoforms (PR-B and PR-A), whose actions are driven by the participation of specific coregulators, posttranslational modifications, and epigenetic mechanisms among other events, that in turn are influenced by the cellular context and developmental stage. In the present chapter, we address in depth about the regulation and molecular actions of PR in the brain.

Juvenile hormone (JH) is a key regulator of development and reproduction in mosquitoes. JH delays metamorphosis until larvae have attained an appropriate stage and size. At that point, a drop in JH titre permits a metamorphic moult. As the antimetamorphic role of JH comes to an end, the late pupa becomes again “competent” to synthesize JH, which plays an essential role orchestrating reproductive maturation. JH synthesis is controlled by the rate of flux of isoprenoid precursors, with a complex interplay of changes in precursor pools, enzyme levels and nutritional and developmental modulators, such as 20-hydroxyecdysone, ecdysis-triggering hormone, insulin and allatostatin-C. JH uses multiple molecular mechanisms to exert its pleiotropic functions at different stages of the mosquito life cycle. JH acts via an unidentified membrane receptor and an intracellular receptor, methoprene-tolerant protein. The latter functions as a transcription factor and directly regulates the expression of JH target genes.

β-Adrenergic receptors (βARs), prototypical G-protein-coupled receptors, play a pivotal role in regulating neuronal and cardiovascular responses to catecholamines during stress. Agonist-induced receptor endocytosis is traditionally considered as a primary mechanism to turn off the receptor signaling (or receptor desensitization). However, recent progress suggests that intracellular trafficking of βAR presents a mean to translocate receptor signaling machinery to intracellular organelles/compartments while terminating the signaling at the cell surface. Moreover, the apparent multidimensionality of ligand efficacy in space and time in a cell has forecasted exciting pathophysiological implications, which are just beginning to be explored. As we begin to understand how these pathways impact downstream cellular programs, this will have significant implications for a number of pathophysiological conditions in heart and other systems, that in turn open up new therapeutic opportunities.

The multireceptor multiligand system of neuropeptide Y receptors and their ligands is involved in the regulation of a multitude of physiological and pathophysiological processes. Specific expression patterns, ligand-binding modes, and signaling properties contribute to the complex network regulating distinct cellular responses. Intracellular trafficking processes are important key steps that are regulated in context with accessory proteins. These proteins exert their influence by interacting directly or indirectly with the receptors, causing modification of the receptors, or operating as scaffolds for the assembly of larger signaling complexes. On the intracellular receptor faces, sequence-specific motifs have been identified that play an important role in this process. Interestingly, it is also possible to influence the receptor internalization by modification of the peptide ligand.

The invention relates to the discovery and purification of novel vitamin D response element binding proteins. Vitamin D response element binding proteins are distinct from the vitamin D receptor. Vitamin D response element binding proteins can interfere with the biological activity of the vitamin D receptor and other related intracellular receptor proteins. One aspect of the invention is to provide purified vitamin D response element binding proteins. Another aspect of the invention is to provide antibodies capable of binding to the vitamin D response element binding proteins of the invention. Another aspect of the invention is to provide assays for the detection or screening of therapeutic compounds that interfere with the interaction between vitamin D response element binding protein and vitamin D response elements.

This chapter provides detailed description on intracellular receptor binding growth factors. Intracellular receptor binding ligands are identifiable as nuclear receptor binding growth factors such as estrogen, progesterone, VD3, and retinoids and cytoplasmic receptor binding ligands such as the glucocorticoids. The physiological effects of several hormones, androgen, vitamin D3, thyroid hormones, retinoids, and estrogen and progesterone are mediated by the activation of a large family of nuclear receptors. Activated receptors dimerize and bind to responsive elements in the regulatory regions of target genes and induce or repress their transcription of responsive genes, resulting in appropriate physiological function. Thyroid hormone receptors (TRs) repress transcription of target gene in the absence of the ligand but activate transcription when the ligand is present. TRs associate and form complexes with co-repressors such as N-CoR and SMRT. The androgens regulate the growth and differentiation of the normal prostate and are closely implicated in the pathogenesis of prostate cancer. Androgens enter into interactive regulation with growth factors, often influencing mutual expression and so contribute to the progression of prostate cancer. Steroid hormones and glucocorticoids are known to modulate the biological function of IGFs. Estrogens can harness insulin and IGF signaling, and estrogen and progesterone receptors are known to engage with signaling through EGFR, and indeed not insignificantly with IGFR and EGFR interaction in tamoxifen resistance. They then translocate into the nucleus to activate genetic transcription.

Publisher Summary The first steroid hormone to be isolated, purified, and characterized was the estrous-inducing substance, estrin (C18H22O2) obtained in gram quantities from the urine of pregnant women. The steroid and thyroid hormones, and other lipophilic messengers are carried by specific binding proteins in the circulation. It has been thought that they penetrate cell membranes and enter cells passively, but this is not always the case. In the cytosol, most of them bind to intracellular receptor molecules that then migrate to the nucleus, attach to particular regulatory DNA sequences (response elements), and activate or inhibit transcription (transactivation or transrepression). The receptors for steroid hormones are members of a very much larger class of ligand-activated transcription factors. It includes the receptors for thyroid hormones, for retinoids (i.e., all-trans-retinoic acid or vitamin A, and its isomer 9–cis-retinoic acid) and for vitamin D (cholecalciferol). This family is called the nuclear receptor superfamily. Its members have a wide range of biological functions including the regulation of growth and embryonic development, the maintenance of phenotype, and the regulation of metabolic processes, such as cholesterol and bile acid metabolism. Disorders of these systems can lead to infertility, obesity, diabetes, and cancer. Over the years, the study and classification of nuclear receptors has suffered from the problem of multiple names for the same gene. A systematic nomenclature has therefore been established. The general structure of the nuclear receptors is highly conserved, consisting of a set of common functional domains. This chapter also discusses activation, interaction, and repression of transcription pathways by nuclear receptors and their other nontranscriptional actions.

Estrogen has multifaceted effects on the hypothalamus that regulate a number of homeostatic functions including reproduction, temperature, energy balance, stress, and motivated behaviors. Estrogen targets all of the major hypothalamic neuroendocrine and autonomic cellular groups to activate multiple signaling pathways. Originally it was thought that all of these actions of estrogen could be ascribed to its binding to its "classical" intracellular receptor and to alterations in gene transcription. However, we now know that this steroid hormone activates multiple signaling pathways to affect neuronal excitability and gene transcription. Although the "classical" genomic signaling pathway has been recognized for almost half a century, until recently little attention has been paid to the rapid membrane-initiated signaling by estrogen in neurons. It has been known since the 1970s that estrogen can rapidly alter neuronal firing within seconds, indicating that some cellular effects of estrogen could occur via rapid, non-transcriptional mechanisms. Therefore, this chapter reviews the current status of estrogen signaling in the hypothalamus via membrane-initiated and nuclear-mediated events that affect the excitability of hypothalamic neurons and, ultimately, neuroendocrine and autonomic functions.

Drug discovery using intracellular receptors (IRs) as targets presents its own set of unique complications and advantages. The natural ligands for these receptors are, in many cases, already used as drugs. To effectively exploit these targets, newer molecules must have either increased receptor selectivity or increased tissue or gene selectivity to reduce side effects. The search for these molecules will yield new therapeutics as well as new insights into the mechanism of action of these receptors and their ligands.

Publisher Summary This chapter discusses the serum transport of steroid hormones. Steroid hormones are extensively bound to plasma proteins including albumin, corticosteroid binding globulin (CBG), and sex hormone binding globulin (SHBG). Because of its high concentration, albumin binding is important in determining the magnitude of the nonprotein bound or free fraction of a steroid in plasma. The generally accepted model of steroid hormone action suggests that free steroid (in equilibrium with circulating binding proteins) diffuses passively through target cell membranes and binds to a soluble intracellular receptor. The steroid-receptor complex apparently moves into the nucleus where it modifies the chromatin transcriptional activity which results in, among other things, altered levels of protein synthesis. CBG has been differentiated from the intracellular glucocorticoid and progesterone receptors by its inability to bind synthetic glucocorticoids and progestins.

Adrenal medullary tissue contains a soluble protein of M.W. 47,000 which specifically binds Ca2+ and, in the presence of Ca2+, induces the fusion of the outer leaflets of chromaffin granule membranes. We have named this protein synexin, from the Greek word “synexis” which means “a meeting”. We suggest synexin may be an intracellular receptor for Ca2+ in the process of exocytosis, acting to promote fusion between secretory vesicles and the plasma membrane, or, in the case of compound exocytosis, between vesicles.

Publisher Summary This chapter describes characterization, assay, and purification of steroid receptors. Steroid hormones achieve their effects on target tissues through intracellular receptor proteins. Estrogen and progestin receptors are localized in the nuclear compartment of the cells, whereas glucocorticoid receptors may reside in both the cytoplasm and the nucleus. Steroid receptors should have (1) a high affinity towards the ligand, (2) a limited binding capacity, |(3) a high-ligand specificity, and (4) a high degree of tissue specificity. Application of these criteria is useful for the identification of steroid-binding macromolecules in tissues previously not identified as steroid-target tissues. The first two properties of steroid receptors, i.e., the binding affinity and capacity are derived from binding curves. The interaction between a steroid and its receptor obeys the law of mass action. The third property of steroid receptors, i.e., their ligand specificity, is inferred from competition studies. In such studies, receptors are labeled with radioactive ligands and competitors are added at different concentrations. Receptor assays are based on binding of a radioactive form of the ligand and such assays are relatively simple to perform. A suitable receptor preparation is incubated with either a single saturating concentration or a number of different nonsaturating concentrations of the radioactive ligand.

Publisher Summary This chapter discusses recent results with respect to steroid-binding serum globins. Several proteins in the blood serum of humans and other mammalian species form stoichiometric complexes with steroid hormones, namely, albumin, corticosteroid-binding globulin (CBG), testosterone–estradiol-binding globulin (TeBG), and α 1 -acid glycoprotein (AAG). Whereas human serum albumin (HSA) consists of a polypeptide chain free of carbohydrate, AAG, CBG, and TeBG are glycoproteins with relatively high sugar content. The binding of the steroid hormones to the serum proteins is mediated essentially by hydrophobic forces and hydrogen bonds. Noncovalent complexes that are dissociable are formed. The dissociation constant increases with rising temperature. The steroids are biologically inactive as long as they are associated with the serum proteins. They can be activated by dissociation to the free hormone. In this manner, a relatively large amount of steroid can be carried in an indifferent storage form and can be made available immediately at the target tissue. The dissociation rate of the serum–protein complex determines the availability of the steroid hormone to the target cell. Binding of the steroid to an intracellular receptor protein serves a different function—the hormone remains attached to the receptor while conformational changes and nuclear transfer take place.

Publisher Summary This chapter discusses that steroid, retinoid, and thyroid hormones regulate development and homeostasis in complex eukaryotes. The diverse physiological actions of these hormones on metabolism and differentiation are predominantly mediated by intracellular receptor proteins that directly regulate patterns of gene expression in target cells. The chapter reviews that the primary structures of all of the known steroid hormone receptors and two thyroid hormone receptors and two retinoic acid receptors, have been elucidated by the cloning and sequencing of their complimentary DNAs (cDNAs). These studies indicate that the receptor gene products comprise a superfamily of regulatory proteins that are capable of modulating gene expression in a ligand-dependent fashion. As transcription factors, these receptors are of great interest as molecular machines through which mechanisms of transcriptional control is studied. These receptors reflect considerable specificity and selectivity in the genetic programs which they ultimately influence. This modulation of gene expression leads to profound changes in protein synthesis within cells and consequential changes in cell function.

Publisher Summary This chapter discusses the crystallographic studies of calmodulin. Calmodulin has been extensively studied, and it is now recognized as a principal intracellular receptor for calcium. Binding of calcium induces a conformational change that allows calmodulin to interact with enzymes, peptides, and pharmacological agents such as the phenothiazines. There have been a number of chemical and physicochemical studies to understand the nature of conformational changes that calmodulin undergoes in response to calcium binding, and also to determine its structure. At present, X-ray crystallography is the only method for determining the three-dimensional structure of proteins at the atomic level. The first step in any structural analysis by X-ray diffraction is the crystallization of the protein. The crystals must be well formed, have a minimum of mosaic spread, and be large enough to cause significant X-ray diffraction. The X-ray analysis at 3.0-A o resolution provides the general structural features of calmodulin; refinement of the model at higher resolution is required for a detailed analysis of the structure. A number of small polypeptides are shown to bind tightly to calmodulin. They provide information on the mechanisms of interaction among calmodulin and calmodulin-dependent proteins.