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Transcript
BS2550 D R Davies MODE OF ACTION OF STEROID HORMONES (with particular reference to oestradiol – a typical steroid hormone) The commercial availability of [3H]steroids since the early 1970’s has enabled considerable progress to be made to clarify the mode of action of steroid hormones. Most of the effects of steroid hormones are mediated by cell-specific regulation of gene transcription, with multiple genes regulated resulting in a physiological response to a particular hormone. All steroids bind to specific receptors in target cells. Steroids with a high specific radioactivity (high dpm/mol) can be used to study the binding of steroid to receptors and we can use the principles of kinetic analysis to measure the binding of any ligand to its receptor (see later in course) How is a target tissue identified? If you administer a physiological doses of a radiolabelled steroid (e.g. [6,7 3H] oestradiol) to an immature female rat or mouse the level of steroid in the blood reaches a maximum very rapidly and decreases in a matter of minutes because of metabolism to inactive derivatives and excretion. Since the steroid is lipophilic it readily crosses plasma membranes and there is a rapid equilibration so that the concentration in the cells is the same as that in the blood plasma. The level of a steroid in non-target tissues mirrors the change in its plasma concentration. Target tissues on the other hand take up and retain the steroid for several hours before the steroid disappears. The steroid can be recovered from the target tissue unchanged by extracting with organic solvents suggesting that it exerts its effect without being covalently modified. Note: oestradiol is the same as estradiol (USA spelling) and 17 (o)estradiol Fate of [3H]steroid within target cells There are two primary target tissues for (o)estrogens (a collective name given to steroids such as oestradiol and other compounds with oestradiol-like effects), the uterus and the vagina. Oestrogens stimulate growth and development by switching on or off the expression of a number of gene products at various stages of the menstrual cycle (in humans) or oestrus cycle in animals. If we take a cross section of the uterus and use autoradiography to show the fate of the labelled oestradiol it is possible to show that the radioactivity is mainly associated with the endometrium (the inner lining of the uterus) and at a higher level of magnification, at a subcellular level, most of the activity is found associated with the nucleus. This label can be displaced by following the dose of radioactive steroid with a dose of unlabelled steroid, indicating that the steroid binding to the nucleus is a reversible process. During the menstrual (or oestrus) cycle there are reversible changes in the morphology of the endometrium which are regulated by oestradiol and other hormones and growth factors. The purpose of these changes is to prepare the uterus for a possible pregnancy in every cycle. The Binding of [3H] steroid to Steroid Receptor (SR) When oestradiol (or any steroid) enters the target cell it binds to a so-called ‘soluble’ receptor associated with the cytoplasm or nucleoplasm. The SR complex formed undergoes a transformation process and then becomes associated with the nucleus in a tightly- bound form. In the initial studies of SR, labelled steroid was incubated with protein fractions from cells and the bound and unbound steroid were separated by sucrose density gradient (linear gradient 5%-20% sucrose) centrifugation for many hours. The receptor-bound steroid migrates through the gradient while the unbound steroid remains at the top of the gradient. The rate of migration (Sedimentation Coefficient or S value ) depends on a number of factors including the Molecular Wt, shape and density of the receptor and in general the size of the receptor complex is related to the S value but not in a linear fashion. Oestradiol binding in the uterus If you make extracts of uterus with a low salt (50 mM KCl) buffer and examine the binding kinetics by Scatchard analysis it is possible to resolve two classes of binding site Type I: a high affinity site with a KD in the range 2 x 10-10 to 1 x 10-9 M. There is a limited concentration of these receptor sites estimated at about 16,000 receptor molecules per cell (a concentration of about 10 nM) Type II: a low affinity site with a KD of about 1 x 10-7 M but present at a much higher concentration. The function of Type II is not known but there does appear to be a correlation between the occurrence of this receptor and the oestrogen-sensitivity of the tissue. However in these lectures are concerned with the high affinity Type I receptor site. If a cytosolic fraction is incubated with [3H] oestradiol and then the mixture is subjected to sucrose density gradient centrifugation, the receptor sediments with a coefficient of 9S. On the other hand if an extract is made with a high salt (400 mM KCl) buffer the receptor sediments at a slower rate, the 4S form. This indicates that the 9S form is a complex consisting of a number of different sub-units, whilst the 4S form turned out to be the monomeric form of the receptor. There is also a 5S form, which can be extracted with high salt from uterine nuclei which was revealed to be a dimer. In the early work on receptor proteins these forms were referred to by their S values. Oestradiol Receptor Complex (steroid hormone receptor complexes in general) The 9S oestradiol receptor complex consists of a number of different proteins which appear to be essential for its activity and there are number of accessory proteins which appear to be essential for the assembly of this complex. The 9S complex consists of the following: The receptor monomer itself ( 66 Kda) Two molecules of a heat shock protein hsp90 (90 Kda) – a chaperone protein* P23 (23Kda) also a chaperone protein An immunophilin (FKB51) which has peptidyl prolyl isomerase activity and may be involved in protein folding An inactive precursor complex also has the following: One molecule of another heat shock protein (hsp70) (70Kda) –also a chaperone A hsp40 (40Kda) which is a cofactor for hsp70 HOP (60Kda) which is a scaffolding protein – an assembly factor for hsp70 and hsp90 HiP (p48) (48 Kda) –also a hsp70 cofactor The 9S complex therefore has a Molecular Weight of > 300 Kda, and in the absence of hormone, is found in the cytoplasm (or possibly nucleoplasm) of the cell. The receptor protein will not bind DNA in this form. Similar complexes are found associated with other Steroid Receptors. (for Diagram see handout entitled Activation of Steroid Hormone Receptors) Note: {* hsp90 is an ubiquitous, abundant, essential and highly conserved protein which performs a general molecular chaperone function in cells. In this system it maintains the Steroid Receptor in the high affinity, ligand binding conformation and prevents nuclear binding of the receptor} Transformation of the 4S 5S form Nuclear retention of the high affinity receptor is essential for the effects of oestradiol on uterine developmental changes. Since the receptor associated with the DNA in the nucleus is 5S and the monomer extracted from the cytoplasm is 4S it is clear that some event must have occurred which results in strong nuclear binding. The free 4S receptor is also much more labile than either the 9S complex or the 5S form When the steroid binds to the 9S form a conformational change occurs resulting in the release of the 4S monomer which then undergoes dimerization to the 5S form. The 5S homodimer has an increased affinity for oestradiol and is also capable of binding with high affinity to DNA. This transformation is dependent on oestradiol, the cytosolic proteins and the salt concentration and is temperature-sensitive implying a protein catalysed conformational change in the receptor protein: 9S complex 4S monomer 5S dimer the appearance of the 5S form in the nucleus is always accompanied by the loss of the 4S monomer from the cytoplasm. The 5S form can be dissociated into the 66 Kda monomer by dialysis against 0.5 M sodium thiocyanate. Regulation of Gene Transcription The consequence of oestradiol binding to the transformed receptor in the nucleus results in the induction or repression of the transcription of specific genes. This can be shown by the incubation of uterine tissue with [35S] methionine in the presence or absence of oestradiol and then extracting the total protein and subjecting it to separation on 2 Dimensional IEF/SDS gels and then subjecting the gels to autoradiography. It is then clear that a number of proteins are induced or repressed when the control and oestrogen treated cells are compared. More information on the oestrogen receptor monomer Steroid receptors belong to the same family of proteins, have a very similar structure and share many properties. The have a similar domain structure as follows: N C A B C D E F A and B are highly variable between different steroid receptors but contain sites which can modulate transcriptional activation e.g. protein kinas phosphorylation motifs C is a 66 aminoacid highly conserved sequence involved in binding to the Hormone Response Element (HRE –see below) and also in the dimerization of rhe receptor. D is a hinge region very susceptible to proteolysis contributing to the instability of the receptor. This region also contains a nuclear localization site E is the steroid binding domain and is also involved in the dimerization F is the C –terminal domain and has no known function. Many steroid receptors have now been cloned and sequenced. In order to ascribe functions to the various domains, deletion mutants have been produced lacking particular sequences, the corresponding cDNA transfected into HeLa cells and oestrogen-inducible gene transcription measured after gene transcription. Hormone Response Elements (HRE) or ERE (Oestrogen Response Elements) GRE (Glucocorticoid Response Elements) etc. There is a hormone response element located upstream (up to several hundred base pairs upstream) of the transcription start site of all hormone responsive genes where the promoter region has been sequenced. This is the site at which the hormone receptor dimeric complex binds specifically to DNA.This HRE is identified by a DNAase footprinting technique where the receptor is allowed to bind to the DNA and thus protects a particular sequence from the action of DNAase. When these sequences have been characterized they are found to consist of a palindromic sequence of bases which read the same from the 5’ to 3’ as they do on the opposing strand of DNA. These notes are intended to be used in conjunction with Lecture Handouts and appropriate chapters in textbooks (e.g Molecular Cell Biology, Lodish et al. 4th edition, Chapter 20, Cell-to Cell Signalling: Hormones and Receptors; Biochemistry with Clinical Correlations, Devlin TM . 4th edition, Chapt 21, Biochemistry of Steroid Hormones which show chemical structures and have excellent diagrams illustrating most of the points made in these lecture notes.