Estradiol’s interesting life at the cell’s plasma membrane.
Caldwell, J. D. 1, Gebhart V. M.2 and Jirikowski G. F.2
1- Edward Via College of Osteopathic Medicine Dept. of Pharmacology, Spartanburg, SC, USA2- Jena University Hospital
Inst. Anatomie II, Jena, Germany
Highlights: Rapid effects of estrogens cannot exclusively be explained be nuclear receptor actions.
Membrane receptors may be distinct from classical steroid receptors Steroid binding globulins are expressed in the central nervous system Expression of steroids binding globulins is in part steroid dependent Cellular accumulation of steroid hormones depends on binding globulins
Keywords: Gonadal steroids, Adrenal hormones, non genomic effects, membrane receptor, steroid binding globulins
CBG- Corticosteroid binding globulin, CHO cell- Chinese hamster ovary cell, CNS- central nervous system,
ERα – Estrogen receptor alpha , ERβ estrogen receptor- beta,
Erk-1 / 2 Extracellular-signal Regulated Kinase, GC- glucocorticoid,
GPR 30 G-protein coupled receptor 30, RBP- Retinol binding protein,
SHBG- Sex hormone binding globulin,
Stra6- Stimulated by retinoic acid 6 transmembrane protein,
The key ovarian steroid in humans, estradiol, is perhaps the most potent single molecule in the human body. It demonstrates a plethora of physiological effects on almost every organ in the body and
it seems to have more pathways of physiological action than any other molecule. We are still discovering whole avenues of its effects. Many physicians and endocrinologists devote any attention that they pay
to estradiol entirely to its manifold effects in the cell’s nucleus. There is a pharmacology review for preparing students for their medical exams that has this line, “Mechanism of action utilizes intracellular receptors” as the only thing medical students need to know about steroids. In contrast, this review will
focus on some little noted, but still extremely fascinating, effects of estradiol on the cell’s plasma membrane envelope and will suggest there are effects of the membrane back on estradiol. In short, it will review the very interesting life of estradiol at the cell’s plasma membrane.
Well, it’s in all of the textbooks
As indicated above, the currently taught version of how all steroids work is dictated by what endocrinologists call the “Free Steroid Hypothesis”. Briefly, this hypothesis states that steroids are carried in the blood by proteins known as steroid-binding globulins. Around their target organs, these steroids exist in equilibrium with their binding proteins and as in any system in equilibrium, if the free moiety is being drawn down, by for example diffusion into another compartment, more of the bound element is released into the free reservoir. This free moiety of steroids, since everyone knows that steroids are lipophilic, passes freely through the plasma lipid layer of the cell membrane and into the cytosol where it is grabbed by important cytosolic receptors. A number of events may occur at this point, but basically the steroid with its receptor passes into the cell nucleus where it has many, many effects on transcription of genes. In other reviews [1-3] we have argued against the viability of the Free Steroid Hypothesis, and we will not reiterate in this paper either the cogent arguments of numerous researchers or the chronology of our own path to believing that there are fallacies in the Free Steroid Hypothesis.
There may be many places where this model does not work very well, but the most jarringly illogical point is on the inside of the plasma membrane. What could possibly induce a steroid, sitting snuggly in its lipid bilayer to come out of that layer and jump into the aqueous cytosolic environment? The answer to that question is—nothing. No self-respecting steroid would leave the lipid bilayer, without some help, to go into the water inside the cell. Allera and Wildt  clearly demonstrated that without some protein element, steroids did not even pass through the lipid bilayer. So, if steroids do not just pass through the cell membrane, what happens to them there? This question is the basis of this entire paper. What happens to the steroid at the level of the cell’s plasma membrane envelope?
Estradiol is being grabbed
One possibility as to what is happening to the ovarian steroid estradiol (E2) at the membrane is that, unlike what is suggested in the Free Steroid Hypothesis, it is grabbed by a membrane-associated receptor. That is the reason that there need to be proteins in the plasma membrane for E2 to move through the membrane. This could be a membrane-associated receptor for E2. Many excellent laboratories have been engaged in trying to determine what this receptor or these receptors are.
One candidate for a grabber of E2 in the cell’s membrane is GPR30. In the 1990s it was found that E2 stimulated cellular functions via second messengers Erk-1 and Erk-2 . This study demonstrated that this effect could be seen in a cell line that did not express either estradiol receptor alpha (ERα) or ERβ suggesting that the membrane-associated GPR30 receptor not only mediates rapid actions of E2, but that it does so without the assistance of either of the intracellular ERs α or β. The possibility that many of the proteins involved in binding and carrying steroids such as E2 interact to mediate even a single action of a steroid is a relevant consideration and some models of such combined
and synergistic action will be presented later in this review. Actions of E2 via GPR30 have been demonstrated in cell lines linked to breast cancer  and thyroid cancer . Oddly, however, even though there are antibodies that apparently have specificity for GPR30 at least in blot material , there has been no attempt to map the localization of GPR30 in a range of organs. Interestingly, with regard to the issue of interactions among various grabbers of steroids is evidence that GPR30 and ERβ may be found in the same cells  where they may interact to mediate E2’s effects.
Martin Kelly has proposed a separate membrane-associated estradiol receptor [10-17]. Their ER has rapid effects that are mediated both by interaction with GABA receptors [13, 18] and associated with opiate neurons . They have found a synthetic agonist that is specific to their ER called ST-X [15, 19]. Unlike GPR30, Kelly has not yet presented evidence that their ER is associated with other known response factors for E2.
What about the inside of the plasma membrane?
It should be mentioned that it is controversial whether GPR30 even has a role in E2 actions. Kang et al.  claim that many of the actions attributed to GPR30 are actually mediated by a splice variant of ERα called ERalpha-36. Ellis Levin is a prominent researcher in the field and he has claimed that ERα is selectively moved to the inside of the plasma membrane [21-23]. They have even defined the elements that are required to direct ERα to the inner side of the plasma membrane. In spite of this, Levin does not claim that ERα binds extracellular E2 from this position.
This raises the obvious question, if cells go through all of the energy expenditure to shunt ERs to the membrane, what are they doing there? Although Levin’s laboratory does not claim that ERs bind extracellular E2, other laboratories do. Mermelstein et al. [24, 25] have demonstrated that ERα is shunted to the cell’s membrane where it interacts with glutamate receptors to influence cell excitability. A process known as palmitoylation is essential for this to occur . The Micevych laboratory has also studied the role of ERα at the membrane as well, which they suggest is sometimes mediated by a splice variant of ERα . Their laboratory has done extensive work to examine the role of membrane ERs interacting with the brain’s opiate system [28-30], which they link to central control of female sexual receptivity [30, 31].
Another possibility suggested by the presence of ERs on the inside of the plasma membrane, but not having a function to bind extracellular E2, is that they are there as part of a larger complex of binding proteins and response proteins that either help to internalize E2 or help to mediate some function of E2 at the plasma membrane. This idea will be further explored below.
A collection of proteins in caveolae
Several of the studies cited above suggest that ERs are shunted to the level of the cell membrane, from which site they may function as part of a complex involved in either E2 uptake or intracellular responses to E2. Could the cell membrane really have so many proteins just involved in
uptake of E2? The answer is “yes and it is more crowded in specific areas than even that suggests”. Caveolae are structurally-defined, protein-rich areas of the plasma membrane . There are proteins that are associated with the caveolae and these are called caveolins [32, 33]. Caveolin-1 seems to be most closely associated with E2 receptors ERα and β [33, 34]. There are several tissues in which caveolin-1 and ERβ are particularly closely associated . Levin has described the presence of ERα associated with caveolae . There has been considerable research on the importance of the non- genomic interaction of ERs in the caveolae in the control of nitric oxide production in vascular endothelial cells [35, 36]. Chambliss et al.  have made the point that ERβ is very important in caveolae in controlling this response. Therefore, it is clear that caveolae contain a concentration and perhaps complexes of proteins that seem to interact in important ways to control cellular function; for example, maintenance of vascular patency. It also seems clear that something or some things in the caveolae is/are binding E2.
What is estradiol doing in the plasma membrane?
Therefore, it is clear that there are elements associated with the cell’s plasma membrane that bind E2 and it seems likely that these binding elements are concentrated, perhaps with other important proteins, in specific areas of the plasma membrane, such as the caveolae. But, does E2 have any action at the membrane level or are these complexes only involved in delivering E2 to the nucleus? Most early studies of E2 examined its long-term developmental effects. However, Clara Szego had conducted many experiments in the 1970s showing that E2 had receptors in  and had actions on the growth of uterine endometrium [39, 40]. Then in the early 1980s her laboratory conducted a series of experiments that turned ideas about the actions of E2 upside down. They found that E2 treatment of uterus in vitro increased the presence of microvilli in endometrial cells within seconds, not hours . They clearly showed that E2 treatment resulted in a very rapid appearance of microvilli that stuck out of the endometrial cells into the uterine lumen. This finding sat fallow for more than a decade, perhaps because there was no model at the time for such a rapid action of E2 and partly because no one could imagine a mechanism by which such a rapid, non-genomic action of steroids could occur.
Kipp and Ramirez  suggest a possible mechanism of the very rapid effect of E2 to produce evaginations from the cell’s surface. They found that E2 was bound to one end of the cellular structural elements tubulin (whereas the testicular steroid testosterone was bound at the other end). They demonstrated that from this position, E2 regulated microtubule extension which drives distortion of the cell surface to produce microvilli or any similar cellular evagination. It seems that one non-genomic action of E2 that has tremendous potential to affect basic cellular functions: It is to alter the cell’s shape by making such evaginations. Below we see that such evaginations from neurons have dramatic significance for memory and other brain functions.
Things that stick out of neurons are called processes and can be either dendrites or axons. Perhaps more equivalent to Szego’s endometrial microvilli are tiny projections particularly from dendrites called spines. Woolley’s laboratory has studied E2-induced production of dendritic spines and their relationship to neuronal function [43-45]. However, they have never examined these effects of E2
in the timeframe of seconds. It would be fascinating to examine whether, in fact, E2 induces a very rapid proliferation of numerous spines and that what is most commonly examined after 24 hours are those few spines that remain after many have long since receded. If this is correct, is E2 a trophic factor that encourages outgrowth and exploration out from the neuron? The implications of such neuronal outgrowth are critical for the laying down of new memories [46, 47].
Our laboratory has used the recently available E2 pre-linked to a fluorophor called E2Glow™ . E2Glow™ is designed to maintain the basic chemical characteristics of E2 and thus to be available for binding by SHBG and cytosolic ERs. We have examined the uptake of E2Glow™ into the classically estradiol-sensitive cell line from Chinese hamster ovaries- CHO [48-50]. Figure 1 shows time-lapse confocal micrographs of binding and uptake of E2Glow™ over a period of 30 seconds. Already, after only
10seconds, in Figure 1A, the CHO cell is almost completely outlined by fluorescence. This probably indicates the initial accumulation of E2 on the plasma membrane of the CHO cell. Such accumulation could be due to binding of E2 to membrane-associated receptors such as GPR30, or membrane- associated ERα or β, or perhaps this represents the binding of E2 associated with SHBG to a receptor for SHBG. Because the CHO cells were raised and kept in serum, which contains steroid-binding globulins [48, 49], it is likely that SHBG in the medium quickly binds up the added E2Glow™ and thus the SHBG may help it bind the exogenous E2Glow™ to the membrane of the cell. Even at the earliest latency shown (see arrow, Figure 1A) there is an indication of the presence of membrane-associated outgrowths that are fluorescing, suggesting that these outgrowths very quickly accumulate and/or bind E2. This outgrowth, which we will call a sustentacle for reasons that will be made clear below, is even brighter and clearer in Figure 1B taken 20 seconds after addition of E2Glow™, perhaps indicating that by 20 seconds, the sustentacle has accumulated even more E2. It should be noted that even at this point, there is very little indication of fluorescence in the middle of the cell perhaps indicating that E2 is just starting to be taken up into the cytoplasm. At this point there is still a large dark area in the middle of the cell, which presumably represents the nucleus. Even in Figure 1C, where the entire cytoplasm seems to be filled with fluor, there is no fluorescence in this area, suggesting that there still has been no translocation of E2 into the nucleus. Therefore, it appears that E2 first accumulates at the cell membrane and most particularly in outgrowths that we are calling sustentacles. Only after considerable accumulation has occurred at the plasma membrane level does the cytoplasm seem to fill with E2 and, even then, no E2 is detectable in the cell nucleus.
SHBG is involved in uptake of E2
Figure 2A gives some idea of the process by which E2Glow™ is taken up into the cell. First, it appears that the addition of 10-5 M unlabeled E2 competes with E2Glow™ for whatever mechanism is involved in internalization of E2 into the cell. This alone suggests that the internalization of E2 involves a saturable receptor-type mechanism rather than passive diffusion across the plasma membrane. In figure 2B it is clear that the addition of antibody to SHBG to the medium blocks or at least delays the uptake of E2Glow™, clearly indicating that SHBG is part of the uptake process for E2 into CHO cells. As indicated above, many laboratories have suggested SHBG is important for E2 uptake, but this is the first
demonstration that blocking SHBG activity in CHO cells affects E2 uptake. Since CHO cells are an important model for cytoplasmic estradiol receptor action, our findings suggest that SHBG is part of a multi-protein system that is responsible for E2 uptake; a system that likely includes membrane- associated ERs. Such a multi-protein system is delineated for another steroid below.
A further indication that SHBG is important for the internalization of E2 is shown in figure 3: Co- localization of SHBG immunostaining and E2Glow™ accumulation occurs within cytoplasmic granules in CHO cells. Combined with the indication that SHBG is necessary for E2 uptake shown in figure 2, this may suggest that SHBG is internalized with E2 bound to it and then the combination can be found in some secretory vesicles . Since we have already demonstrated the presence of SHBG in neuronal axons, it is possible that these organelles are responsible for shunting E2 throughout the cell.
Role of Sustentacles
It might be suggested that the sustentacles that are seen to accumulate E2 in figure 1, may actually have been created by E2. That is, Clara Szego and coworkers suggested that very soon after exposure to E2, endothelial cells of the uterus showed an increased level of microvilli [38, 39, 51]. We suggested above that the Kipp and Ramirez data  showing E2 binding to tubulin could be a rapid, non-genomic mechanism whereby microtubules are activated, exactly the sort of structural microtubules that would effect the evagination of cell membranes to make microvilli or sustentacles. Is it possible that the first effect of exposure to E2 is that outgrowths are produced on the surface of the estradiol-sensitive cell that aid in the internalization of E2?
But, how does E2 get into the nucleus after it is accumulated at the outside of the cell? A clue to the answer for this question is in Figure2. Figure 2B was taken 30 sec. after another CHO cell was exposed to E2Glow™, so at a time when an untreated cell would have been filled with cytoplasmic E2. However, these cells had been pretreated with a 1:200 concentration of SHBG antibody. Clearly there is almost no accumulation of E2 in this cell suggesting that blocking of SHBG with antibody dramatically inhibited the entrance of E2Glow™ into the cell. This would indicate that SHBG is a critical factor in the internalization of all E2 into the cell. We have previously demonstrated that fluor-labeled SHBG was internalized into specific cells in the brain  within 10 minutes, suggesting that SHBG itself is internalized rather rapidly in some brain cells. So, it is very possible that without SHBG, either in the media surrounding the cell or SHBG on the cell membrane extracellular surface is critical for internalization of E2 into the cell. Further, it is possible that there are specialized outgrowths that we are calling sustentacles, from cells that may concentrate these internalization mechanisms.
What are SHBG and CBG doing in the nose?
We have evidence that steroid-binding globulins such as SHBG and corticosteroid binding globulin (CBG) are found in the brain [53-60]. We have also found evidence that both CBG and SHBG are
found in both the main olfactory epithelium (look this up) and in the epithelium of the vomeronasal organ of rats [59, 61]. We have found these steroid-binding globulins in the nasal mucus of rats ) suggesting that they are excreted into the mucus by epithelial olfactory cells in order to bind aerosolized steroids [63, 64]. Ploss et al.  showed that this SHBG is made in the olfactory epithelium as shown by RT-PCR and found the presence of SHBG in the axons of sensory olfactory cells suggesting that SHBG, and possibly bound steroids, are moved out of the epithelial cells toward the olfactory bulb. Figure 2 shows a model that we are proposing for the action of CBG in the main olfactory area. In this model CBG is found in the nasal mucus of rats, where it could bind aerosolized steroids such as a fear pheromone [64, 66-68] or a glucocorticoid (GC) or even possibly progestins. CBG with a bound steroid then binds to a putative CBG receptor on the surface of the nasal sensory cell. CBG with its bound steroid is then internalized into the olfactory cell (it is also possible that these cells produce CBG). Our evidence that CBG is found in the axons efferent from these olfactory epithelial cells suggests that CBG (and probably olfactory SHBG) is important in moving the steroid within the cell. It likely moves CBG along the axon and into the olfactory glomerulum where the CBG and its associated steroid may be released to either affect mitral cells in that area or perhaps to enter blood vessels at this point to affect the CNS via the blood system. In this way, the nose can inform the brain, via nervous conduction or via the blood, of the presence of aerosolized steroids in the environment. Although, we are not sure what these steroids are, there is evidence that rodents can detect ovarian-steroid-like pheromones [69-73], which might be bound by SHBG in the mucus. There is also evidence for “fear pheromones” in rodents and humans [63, 64, 66, 74] that are perhaps bound by CBG and passed along to the CNS. Berglund et al.  have suggested that lesbian women detect a progesterone-like steroid, which might be bound by CBG.
Above we used the newly created term sustentacles to describe outgrowths from CHO cells that concentrated fluorescence very quickly when E2Glow™ was added to the cell medium. This term is derived from the term sustentacular cells, which are olfactory epithelial cells that are not part of the neurogenic progression [76-78]. Sustentacular cells are characterized by being in the olfactory epithelium, not being of neuronal developmental origin, and in that they have considerable tiny, narrow outgrowths of uncertain purpose. These outgrowths roughly resemble the outgrowths seen in figure 1 that concentrate E2. Therefore, we are naming these outgrowths sustentacles. It is perhaps interesting that we have also identified similar outgrowths, or sustentacles, in neurons near the third ventricle that have SHBG in them but do not produce SHBG [52, 54]. We found that these periventricular cells internalized SHBG, perhaps via their sustentacles . It is perhaps a function of sustentacles that they have SHBG receptors on them and that they, therefore, aid in the internalization of steroids.
Is there another player?
For the uninitiated, all of these various proteins that seem to live at the plasma membrane and interact to affect various steroid-related physiological functions may seem rather dizzying. Well, hold on to your hats. There is at least one more (and perhaps many more) player on this stage. In the Free Steroid Model of steroid action, the steroid binding globulins such as SHBG and CBG carry steroids in the blood and mostly just release them in the vicinity of target cells (purists would say that there is an equilibrium at all times near a target cell wherein removal of steroids from the milieu by diffusion across
the membrane results in continued release from stores , they diffuse passively across the plasma membrane and into the cytosol where they are bound by cytoplasmic receptors. One glaringly obvious flaw in this model has always been, once lipophilic steroids are cozily ensconced in the lipid bilayer of the plasma membrane what would possibly coax them to jump out of that layer and into the aqueous environment of the cytosol? As indicated above, many researchers, such as Allera and Wildt  claim that this does not happen without the presence of specific proteins for steroids. Above, this review has already delineated many potential proteins that might live at the inner edge of the plasma membrane. Razandi et al.  found that ERα was specifically targeted to go there. Mermelstein and Micevych [25, 25, 80, 80, 81] have evidence that ERβ is there. Recent work in this laboratory has suggested that the kind of increase in spines induced by E2 and discussed above is mediated via glutamate receptors . Several researchers have identified either ERα or β in caveolae in plasma membrane [21, 36, 37, 83].
However, we have claimed for some time  that steroid-binding globulins are involved in active responses to steroids. We have evidence that they are internalized into neurons and other brain cells in vivo  and that they are internalized by hippocampal and fibroblast cells in vitro [52, 84]. Others have evidence of SHBG internalization in other tissues [85-87]. Rosner’s laboratory has presented extensive evidence that SHBG is bound by a putative receptor at the plasma membrane level in the prostate [88- 93]. Above we presented evidence (Figure 2) that E2 is not internalized without functional SHBG present suggesting that not only is SHBG and likely an SHBG receptor found in the membrane, but that these are critical elements in internalization of E2 and perhaps of other steroids. Below is a model for another steroid-binding globulin that may serve, at least for now, as a model for how many steroids are internalized.
Do steroids live in the membrane for a while?
We  and others [90, 94-99] have searched for the putative SHBG receptor for some time. However, undoubtedly the best characterized system involving internalization of a steroid by a steroid- binding globulin is that of the Sun laboratory [100-102]. This laboratory first discovered that a membrane-associated protein originally associated with control of cancer , called Stra6 was involved in binding of retinol binding protein (RBP) [100-102]. In their model  RBP is bound by the Stra6 in the membrane. Then the steroid (retinol) is released into the lipid bilayer, which should be quite commodious for a steroid. Retinol then can remain in the plasma membrane until it is picked up by a protein. One such protein is enumerated by them as “cytosolic RBP”. Therefore, in their model, the steroid-binding globulin is essential to deliver the steroid to the lipid bilayer where the binding globulin itself is bound by a receptor; and a steroid-binding globulin is also essential in getting the steroid actually into the cytoplasm of the cell. This agrees with our evidence that CBG can be found in the axons of olfactory neurons  and figure 2. We suggested that steroid-binding globulins are a part of the system that internalizes steroids as well as responsible for moving them around within the cell. Therefore, the Sun model may serve as a model for steroids other than retinol in that it shows the critical nature of steroid-binding globulins in steroid internalization into cells.
Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their actions.
1.J.D. Caldwell, A sexual arousability model involving steroid effects at the plasma membrane, Neurosci. Biobehav. Rev. 26 (2002) 13-30.
2.J.D. Caldwell, F. Suleman, SH.H. Chou, R.A. Shapiro, Z. Herbert, G.F. Jirikowski, Emerging Roles of Steroid Binding Globulins, Horm. Metab. Res. 38 (2006) 206-218.
3.J.D. Caldwell, G.F. Jirikowski, An active role for steroid-binding globulins: an update, Horm. Metab. Res. 45 (2013) 477-484.
4.A. Allera, L. Wildt, Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles–II. Comparative influx and efflux, J. Steroid Biochem. Mol. Biol. 42 (1992) 757-771.
5.E.J. Filardo, J.A. Quinn, K.I. Bland, A.R. Frackelton, Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans- activation of the epidermal growth factor receptor through release of HB-EGF, Mol. Endocrinol. 14 (2000) 1649-1660.
6.T.M. Ahola, T. Manninen, N Alkio, T. Ylikomi, G protein-coupled receptor 30 is critical for a progestin-induced growth inhibition in MCF-7 breast cancer cells, Endocrinology 143 (2002) 3376-3384.
7.A. Vivacqua, D. Bonofiglio, L. Albanito, A. Madeo, V. Rago, A. Carpino, A.M. Musti D. Picard, S. Ando, M. Maggiolini, 17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30, Mol. Pharmacol. 70 (2006) 1414-1423.
8.Y.R. Li, C.E. Ren, Q. Zhang, J.C. Li, R.C. Chian, Expression of G protein estrogen receptor (GPER) on membrane of mouse oocytes during maturation, J. Assist. Reprod. Genet. 30 (2013) 227-232.
9.G.S. Huang, M.J. Gunter, R.C. Arend, M. Li, H. Arias-Pulido, E.R. Prossnitz, G.L. Goldberg, H.O. Smith, Co-expression of GPR30 and ERbeta and their association with disease progression in uterine carcinosarcoma, Am. J. Obstet. Gynecol. 203 (2010) 242 e1-242 e5.
10.M.J. Kelly, A.H. Lagrange, E.J. Wagner, O.K. Ronnekleiv, Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways, Steroids 64 (1999) 64-75.
11.M.J. Kelly, O.K. Ronnekleiv, N. Ibrahim, A.H. Lagrange, E.J. Wagner, Estrogen modulation of K channel activity in hypothalamic neurons involved in the control of the reproductive axis, Steroids 429 (2004) 447-456.
12.A.H. Lagrange, O.K. Ronnekliev, M.J. Kelly, Modulation of G protein-coupled receptors by an estrogen receptor that activates protein kinase A, Mol. Pharmacol. 51 (1997) 605-612.
13.A.H. Lagrange, E.J. Wagner, O.K. Ronnekleiv, M.J. Kelly, Estrogen rapidly attenuates a GABAB response in hypothalamic neurons, Neuroendocrinology 64 (1996) 114-123.
14.A.H. Lagrange, O.K. Ronnekleiv, M.J. Kelly, Estradiol-17 beta and mu-opioid peptides rapidly hyperpolarize GnRH neurons: a cellular mechanism of negative feedback? Endocrinology 136 (1995) 2341-2344.
15.J. Qiu, M.A. Bosch, S.C. Tobias, A. Krust, S.M. Graham, S.J. Murphy, K.S. Korach, T.S. Chambon, O.K. Ronnekleiv, M.J. Kelly, A G-protein-coupled estrogen receptor is involved in hypothalamic control of energy homeostasis, J. Neurosci. 26 (2006) 5649-5655.
16.J. Qiu, O.K. Ronnekleiv, M.J. Kelly, Modulation of hypothalamic neuronal activity through a novel G-protein-coupled estrogen membrane receptor, Steroids 73 (2008) 985-991.
17.J. Qiu, M.A. Bosch, S.C. Tobias, D.K. Grandy, T.S. Scanlan, O.K. Ronnekleiv, M.J. Kelly, Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C, J. Neurosci. 23 (2003) 9529-9540.
18.M.J. Kelly, M.D. Loose, O.K. Ronnekleiv, Estrogen suppresses mu-opioid- and GABAB-mediated hyperpolarization of hypothalamic arcuate neurons, J.Neurosci. 12 (1992) 2745-2750.
19.S.C. Tobias, J. Qiu, M.J. Kelly, Synthesis and Biological Evaluation of SERMs with Potent Nongenomic Estrogenic Activity, Chem. Med. Chem. 1 (2006) 565-571.
20.L. Kang, X. Zhang, Y. Xie, Y. Tu, D. Wang, Z. Liu, Z.Y. Wang, Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling, Mol. Endocrinol. 24 (2010) 709-721.
21.E.R. Levin, Cellular functions of plasma membrane estrogen receptors, Steroids 429 (2002) 471- 475.
22.A. Pedram , M. Razandi, E.R. Levin, Nature of Functional Estrogen Receptors at the Plasma Membrane, Mol. Endocrinol. 20 (2006) 1996-2009.
23.M. Razandi, G. Alton, A. Pedram, S. Ghonshani, P. Webb, E.R. Levin, Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane, Mol. Cell. Biol. 23 (2003) 1633-1646.
24.M.I. Boulware, J.P. Weick, B.R. Beckland, S.P. Kuo, R.D. Groth, P.G. Mermelstein, Estradiol Activates Group I and II Metabotropic Glutamate Receptor Signaling, Leading to Opposing Influences on cAMP Response Element-Binding Protein, J. Neurosci. 25 (2005 5066-5078.
25.P. Dewing , M.I. BoulwareI, K. Sinchak, A. Christensen, P.G. Mermelstein, P. Micevych, Membrane estrogen receptor-alpha interactions with metabotropic glutamate receptor 1a modulate female sexual receptivity in rats, J. Neurosci. 27 (2007) 9294-9300.
26.Meitzen J, Luoma JI, Boulware MI, Hedges VL, Peterson BM, Tuomela K, Britson KA, Mermelstein PG. Palmitoylation of estrogen receptors is essential for neuronal membrane signaling. Endocrinology 2013; 154 (11):4293-4304
27.Dominguez R, Dewing P, Kuo J, Micevych P. Membrane-initiated estradiol signaling in immortalized hypothalamic N-38 neurons. Steroids 2013; 78 (6):607-613
28.Eckersell CB, Popper P, Micevych PE. Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala. J.Neurosci. 1998; 18:3967-3976
29.Mills RH, Sohn RK, Micevych PE. Estrogen-induced mu-opioid receptor internalization in the medial preoptic nucleus is mediated via neuropeptide Y-Y1 receptor activation in the arcuate nucleus of female rats. J.of Neuroscience 2004; 24 (4):947-955
30.Sinchak K, Eckersell C, Quezada V, Norell A, Micevych P. Preproenkephalin mRNA levels are regulated by acute stress and estrogen stimulation. Physiol.Behav.2000.Jun.1-15;69(4- 5):425-32. 2001; 69:425-432
31.Sinchak K, Hendricks DG, Baroudi R, Micevych PE. Orphanin FQ/nociceptin in the ventromedial nucleus facilitates lordosis in female rats. Neuroreport. 1997; 8:3857-3860
32.Stan RV. Structure of caveolae. Biochimica et Biophysica Acta 2005; 1746 (334):348
33.Mendez-Bolaina E, Sanchez-Gonzalez A, Ramirez-Sanchez I, Nunez-Sanchez M. Effect of
caveolin-1 scaffolding peptide and 17beta-estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells. Am J Physiol Cell Physiol. 2007; 293 (6):C1953-C1961
34.Gilad LA, Schwartz B. Association of estrogen receptor beta with plasma-membrane caveola components: implication in control of vitamin D receptor. J Mol Endocrinol. 2007; 38 (6):603-618
35.Kim KH, Bender JR. Rapid, estrogen receptor-mediated signaling: why is the endothelium so special? Sci STKE 2005; 288:pe28
36.Zhu W, Smart EJ. Caveolae, estrogen and nitric oxide. Trends Endocrinol Metab. 2003; 14 (3):114-117
37.Chambliss KL, Yuhanna IS, Anderson RG, Mendelsohn ME, Shaul PW. ERbeta has nongenomic action in caveolae. Mol Endocrinol.. 2002; 16 (5):938-946
38.Pietras RJ, Szego CM. Estrogen receptors in uterine plasma membrane. J.Steroid Biochem. 1979;
39.Pietras RJ, Szego CM. Surface modifications evoked by estradiol and diethylstilbestrol in isolated endometrial cells: evidence from lectin probes and extracellular release of lysosomal protease. Endocrinology 1975; 97 (6):1445-1454
40.Pietras RJ, Szego CM. Steroid hormone-responsive, isolated endometrial cells. Endocrinology 1975; 96 (4):946-954
41.Rambo CO, Szego CM. Estrogen action at endometrial membranes: alterations in luminal surface detectable within seconds. J.Cell Biol. 1983; 97 (3):679-685
42.Kipp JL, Ramirez VD. Estradiol and testosterone have opposite effects of microtubule polymerization. Neuroendocrinology 2003; 77:258-272
43.Woolley CS, Weiland NG, McEwen BS, Schwartzkroin PA. Estradiol increases the sensitivity of hippocampal CA1 pyramidal cells to NMDA receptor-mediated synaptic input: correlation with dendritic spine density. J.Neurosci. 1997; 17 (5):1848-59.
44.Woolley CS, Cohen RS. Sex steroids and neuronal growth in adulthood. Hormones, Brain and Behavior 2002; 4:717-777
45.Woolley SC, O’Malley BW, Lydon JP, Crews D. Genotype differences in behavior and tyrosine hydroxylase expression between wild-type and progesterone receptor knockout mice. Behavioural Brain Research 2006; 167 (2):197-204
46.Frankfurt M, Luine V. The evolving role of dendritic spines and memory: Interaction(s) with estradiol. Hormones & Behavior 2015; doi: 10.1016/j.yhbeh.2015.05.004 (epub)
47.Giese KP, Aziz W, Kraev I, Stewart MG. Generation of multi-innervated dendritic spines as a novel mechanism of long-term memory formation. Neurobiol Learn Mem. 2015; S1074- 7427 (15):72-76
48.FORTUNATI N, Fissore F, FAZZARI A, Piovano F, Catalano M, Becchis M, Berta L, FRAIRIA R. Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG). J Steroid Biochem Mol Biol. 1999; 70 (1- 3):73-80
49.Kushner PJ, Hort E, Shine J, Baxter JD, Greene GL. Construction of cell lines that express high levels of the human estrogen receptor and are killed by estrogens. Mol Endocrinol. 1990; 4 (10):1465-1473
50.Greene G, Gilna P, Waterfield M, Baker A, Hort Y, Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science.1986 Mar 7;231(4742):1150-4. 1986; 231 (4742):1150-1154
51.Pietras RJ, Szego CM. Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane binding-sites specific for estradiol- 17beta. J.Cell Physiol. 1979; 98 (1):145-159
52.Caldwell JD, Shapiro RA, Jirikowski GF, Suleman F. Internalization of Sex Hormone-Binding Globulin into Neurons and Brain cells In vitro and In vivo. Neuroendocrinology 2007; 86 (2):84-93
53.Wang YM, Bayliss DA, Millhorn DE, Petrusz P, Joseph DR. The androgen-binding protein gene is expressed in male and female rat brain. Endocrinology 1990; 127 (6):3124-3130
54.Herbert Z, Jirikowski GF, Petrusz P, Englof I, Caldwell JD. Distribution of androgen binding globulin in the rat hypothalamo-hypophyseal system, co-localization with oxytocin. Brain Research 2003; 992 (2):151-158
55.Herbert Z, Pollak EI, Caldwell JD, Jirikowski GF. Subcellular localization of SHBG in rat hypothalamus and pituitary. Society for Neurosci.Abs 2003; 610.3
56.Herbert Z, Gothe S, Caldwell JD, Bernstein HG, Jirikowski GF. Sex hormone-binding globulin/SHBG is produced in human hypothalamic neurons. FENS Abstracts 2004;
57.Herbert Z, Gothe S, Caldwell JD, Bernstein HG, Melle C, Lewis J, Jirikowski GF. Characterization of sex hormone binding globulin (SHBG) in the human hypothalamus. Neuroendocrinology 2005; 81:286-293
58.Jirikowski GF, Pusch L, Mopert B, Herbert Z, Caldwell JD. Expression of corticosteroid binding globulin in the rat central nervous system. J.Chem.Neuroanat. 2007; 34 (1-2):22-28
59.Mopert B, Herbert Z, Caldwell JD, Jirikowski GF. Distribution of corticosteroid-binding globulin in the rat hypothalamus, colocalization with oxytocin and vasopressin. FENS Abstracts 2004; vol.2:A089.11
60.Mopert B, Herbert Z, Caldwell JD, Jirikowski GF. Expression of Corticosterone Binding Globulin CBG in the rat hypothalamus. Hormone and Metabolic Research 2006; 38:246-252
61.Sivukhina EV, Schaefer HH, Jirikowski GF. Differences in colocalization of corticosteroid-binding globulin and glucocorticoid receptor immunoreactivity in the rat brain. Ann Anat.2012 Dec 8.pii: S0940-9602(12)00162-8 2012; S0940-9602 (12):162-168
62.Dolz W, Eitner A, Caldwell JD, Jirikowski GF. Expression of corticosteroid binding globulin in the rat olfactory system. Acta Histochem. 2012; in press
63.Ackerl K, Aztmueller M, Grammer K. The scent of fear. Neuroendocrinol.Letters 2002; 23:79-84
64.Chen D, Katdare A, Lucas N. Chemosignals of Fear Enhance Cognitive Performance in Humans. Chem.Senses 2006; 31:415-423
65.Ploss V, Gebhart VM, Dolz W, Jirikowski GF. Sex hormone binding globulin in the rat olfactory system. J Chem Neuroanat. 2014; 57-58:10-14
66.Zhou W, Chen D. Fear-Related Chemosignals Modulate Recognition of Fear in Ambiguous Facial Expressions. Psychological Science 2009; 20 (2):177-183
67.Schulkin J, Morgan MA, Rosen JB. A neuroendocrine mechanism for sustaining fear. Trends in Neurosciences 2005; 28 (12):629-635
68.Rasia-Filho AA, Londero RG, Achaval M. Functional activities of the amygdala: an overview. J.Psychiatry Neurosci.2000.Jan;25(1):14-23. 2000; 25:14-23
69.Beckman M. Pheromone reception. When in doubt, mice mate rather than hate. Science 2002; 295 (5556):782.
70.Collins SA, Gosling LM, Watkins RW, Cowan DP. Artificially increasing scent mark rate increases urogenital gland size in mice Mus musculus. Physiol.Behav. 2001; 74 (4-5):517-522
71.Kikusui T, Takigami S, Takeuchi Y, Mori Y. Alarm pheromone enhances stress-induced hyperthermia in rats [Abstract]. Physiology & Behavior 2001; 72:45-50
72.Nodari F, Hsu FF, Fu X, Holekamp TF, Kao LF, Turk J, Holy TE. Sulfated steroids as natural ligands of mouse pheromone-sensing neurons. J Neurosci. 2008; 28 (25):6407-6418
73.Sachs BD. Erection evoked in male rats by airborne scent from estrous females. Physiol.Behav. 1997; 62 (4):921-4.
74.Mujica-Parodi L, Strey HH, Frederick B, Savoy R, Cox D, Botanov Y, Tolkunov D, Rubin D, Weber J. Chemosensory Cues to Conspecific Emotional Stress Activate Amygdala in Humans. PLoS ONE 2009; 4 (7):1-14
75.Berglund H, Lindstrom P, Savic I. Brain response to putative pheromones in lesbian women. Proc Natl Acad Sci U S A. 2006; 103 (21):8269-8274
76.Krolewski RC, Packard A, Schwob JE. Global expression profiling of globose basal cells and neurogenic progression within the olfactory epithelium. J Comp Neurol. 2013; 521 (4):833-859
77.Moran DT, Rowley JC, Jafek BW. Electron microscopy of human olfactory epithelium reveals a new cell type: the microvillar cell. Brain Res. 1982; 253 (1-2):39-46
78.Pixley SK, Farbman A, Menco BP. Monoclonal antibody marker for olfactory sustentacular cell microvilli. Anat Rec. 1997; 248 (3):307-321
79.Hammond GL, Bocchinfuso WP. Sex hormone-binding globulin: gene organization and structure/function analyses. Horm.Res. 1996; 45 (3-5):197-201
80.Mermelstein PG, Micevych P. Nervous system physiology regulated by membrane estrogen receptors. Rev Neurosci. 2008; 19 (6):413-424
81.Srivastava DP, Waters EM, Mermelstein PG, Kramar EA, Shors TJ, Liu F. Rapid estrogen signaling in the brain: implications for the fine-tuning of neuronal circuitry. J Neurosci. 2011; 31 (45):16056-16063
82.Peterson BM, Mermelstein PG, Meisel RL. Estradiol mediates dendritic spine plasticity in the nucleus accumbens core through activation of mGluR5. Brain Struct Funct. 2014; May 31. [Epub ahead of print]
83.Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS, Mendelsohn ME, Anderson RG, Shaul PW. Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res. 2000; 87 (11):E44-E52
84.Caldwell JD, Suleman F, Jirikowski GF. Internalization of Sex Hormone Binding Globulin into Fibroblast 3T3 cells. Hormone and Metabolic Research 2007; 39:620-621
85.Avvakumov GV, Zhuk NI, Strel’chyonok OA. Subcellular distribution and selectivity of the
protein-binding component of the recognition system for sex-hormone-binding protein- estradiol complex in human decidual endometrium. Biochim.Biophys.Acta 1986; 881 (3):489-98.
86.Avvakumov GV, Krupenko SA, Strel’chyonok OA. Study of the transcortin binding to human endometrium plasma membrane. Biochim Biophys Acta. 1989; 984 (2):143-150
87.Strel’chyonok OA, Avvakumov GV, Survilo LJ. A recognition system for sex-hormone binding protein-estradiol complex in human decidual endometrium plasma membranes. Biochim.Biophys.Acta 1984; 802:459-466
88.Hryb DJ, Khan MS, ROMAS NA, Rosner W. Solubilization and partial characterization of the sex hormone- binding globulin receptor from human prostate. J.Biol.Chem. 1989; 264 (10):5378-5383
89.Hryb DJ, Khan MS, ROMAS NA, Rosner W. The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones. J.Biol.Chem. 1990; 265 (11):6048-6054
90.Hryb DJ, Khan MS, Rosner W. Testosterone-estradiol-binding globulin binds to human prostatic cell membranes. Biochem.Biophys.Res.Commun. 1985; 128 (1):432-440
91.NAKHLA AM, ROMAS NA, Rosner W. Estradiol Activates the Prostate Androgen Receptor and Prostate Specific Antigen Secretion through the Intermediacy of Sex Hormone Binding Globulin. J.of Biol.Chem 1997; 272 (11):6838-6841.
92.NAKHLA AM, Rosner W. Stimulation of prostate cancer growth by androgens and estrogens through the intermediacy of sex hormone-binding globulin. Endocrinology 1996; 137 (10):4126-4129
93.NAKHLA AM, Leonard J, Hryb DJ, Rosner W. Sex hormone-binding globulin receptor signal transduction proceeds via a G protein. Steroids 1999; 64:213-216
94.Krupenko NI, Avvakumov GV, Strel’chyonok OA. Binding of human sex hormone-binding globulin-androgen complexes to the placental syncytiotrophoblast membrane. Biochem.Biophys.Res.Commun. 1990; 171:1279-1283
95.Krupenko SA, Avvakumov GV, Strel’chyonok OA. A transcortin-binding protein in the plasma membrane of human syncytiotrophoblast. Biochem.Biophys.Res.Commun. 1991; 177:834-839
96.Khan MS, Hryb DJ, Hashim GA, ROMAS NA, Rosner W. Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin. J.Biol.Chem. 1990; 265 (30):18362-18365
97.Redondo C, Burke BJ, Findlay JB. The Retinol Binding Protein System: A potential paradigm for steroid binding globulins? Hormone and Metabolic Research 2006; 38:269-278
98.Redondo C, Vouropoulou M, Evans J, Findlay JB. Identification of the retinol-binding protein (RBP) interaction site and functional state of RBPs for the membrane receptor. FASEB J. 2008; 22 (4):1043-1054
99.Sundaram M, Sivaprasadarao A, Findlay JB. Expression and mutagenesis of retinol-binding protein. Methods Mol.Biol. 1998; 89:141-153
100.Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, Sun H. A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin A. Science 2007; 315:820-825
101.R. Kawaguchi, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, Sun H. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 2007; 315 (5813):820-825
102.R. Kawaguchi, J. Yu, M. Ter-Stepanian, M. Zhong, G. Cheng, Q. Yuan, M. Jin, G.H. Travis, D. Ong, H. Sun, Receptor-Mediated Cellular Uptake Mechanism that Couples to Intracellular Storage, ACS Chem. Biol. 6 (2011) 1041-1051.
103.W. Szeto W, Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid, Cancer Res. 61 (2001) 4197-4205.
104.R. Krieg R, G.F. Jirikowski, Steroid-Styrylfarbstoff-Konjugate zur Stimulation und direkten lichtoptischen Detektion des Verhaltens von Steroiden im lebenden biologischen Gewebe und in Gegenwart von steroidbindenden Proteinen, Patent J 090710 (2010.
105.Z. Herbert, E. Pollak, L. Molnar, J.D. Caldwell, G.F. Jirikowski, Co-transport of sex hormone- binding globulin SHBG with oxytocin in transport vesicles of the hypothalamo- hypophysial system, Horm. Metab. Res. 38 (2006) 291-293.
Figure 1. Sequential confocal laser micrographs of a live CHO cell over time after exposure to 10-9 M E2Glow™ demonstrating E2 entry into the cell. At 10 seconds (A) E2 seems to be concentrated around the edges of the cell, possibly indicating early concentration at the plasma membrane. Long moving membrane protrusions, “sustentacles” (white arrows), particularly have concentrated fluorescence and thus E2. At 20 seconds (B) more E2 is concentrated in the plasma membrane with very little in the cytoplasm yet. At this point even more sustentacles are apparent. Finally, after 30 seconds (C) much E2 is found in the cytoplasm as well as around the cell. As yet, however,
almost no E2 is found in the nucleus. Scalebar = 5µm
Figure 2. Pretreatment of CHO cells with 10-5 M E2 prevents accumulation and uptake of E2Glow™ (Fig. 2A). Preincubation of CHO cells with an antibody to SHBG (B) prevents movement and extension of sustentacles suggesting that the presence of SHBG is essential for uptake of E2. In both cases, fluorescent E2 is accumulated on the cell membrane, but entry into the cytoplasm is delayed.
Nuclear counterstain with Hoechst nuclear dye. Scalebars= 10 µm
Figure 3. After 10 min of treatment with E2Glow™, fluorescent steroid is accumulated in the nucleus and to a lesser extent in cytoplasm (A). Membrane protrusions are no longer visible. Fluorescence appears diminished due to histological fixation and subsequent immunostaining (B). Immunostaining for SHBG reveals that E2Glow™ is concentrated in SHBG-positive granules as visualized by immunoperoxidase
staining (Arrows) indicating a close association of internalized E2 with SHBG. Scalebar = 20µm
Figure 4: CBG in the olfactory system: CBG is expressed in goblet cells of the respiratory mucosa and in the Bowman Glands to enter the nasal mucus and to trap airborne glucocorticoids and possibly other aerosolic steroids. In this model sensory cells express CBG receptors (CBG-R). CBG expressed in mitral cells and in periglomerular cells, where it may aid comparison of systemic (endogenous) GC levels with exogenous GC concentrations. The interaction of olfactory and limbic circuits may be important for controlling social behaviors including dominance and stress response.
A B C
Fig. 1: Sequential confocal laser micrographs of life CHO cell over time after exposure to 10- 9M E2Glow™ demonstrating E2 entry into the cell. At 10 seconds (A) E2 seems to be concentrated around the edges of the cell, possibly indicating early concentration at the plasma membrane. Long moving membrane protrusions, “sustentacles” (white arrows) particularly have concentrated fluorescence and thus E2. At 20 seconds (B) more E2 is concentrated in the plasma membrane with very little in the cytoplasm yet. At this point even more sustentacles are apparent. Finally, after 30 seconds much E2 is found in the cytoplasm as well as around the cell. As yet, however, almost no E2 is found in the nucleus (N). Scale bar = 5µm
Fig. 2: Pretreatment of CHO cells with 10-5M Estradiol prevents accumulation and uptake of Estradiol Glow (Fig. 2A). Preincubarion of CHO cells with an antibody to SHBG prevents movement and extension of sustentacles. The fluorescent steroid is accumulated on the cell membrane, entry into the cytoplasm is delayed. Nuclear counterstain with Hoechst nuclear dye. Scalebars= 5µm
Fig. 3: After 10 min of treatment with E2Glow, the fluorescent steroid is accumulated in the nucleus and to a lesser extent in cytoplasm. Membrane protrusions are not visible anymore. Fluorescence appears dimished due to histological fixation and subsequent immunostaining (Fig.5). Imunostaining for SHBG reveals that E2Glow is concentrated in SHBG positive granules as visualized by immunoperoxidase staining (Arrows) indicating a close association of internalized E2 with SHBG.
Fig. 4: CBG in the olfactory system: CBG is expressed in goblet cells of the respiratory mucosa and in Bowman glands to enter the nasal mucus and to trap air borne GCs. Sensory cells express CBG receptor (CBG-R). CBG expressed in Mitral cells and in periglomerular cells may aid comparison of systemic (endogenous) GC levels with exogenous GC concentrations. The interaction of olfactory and limbic circuits may be
important for controlling social behaviors including dominance and stress response.
Abstract: Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their actions.