- Email: [email protected]
To be or not to be (a receptor)
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
To be or not to be (a receptor) ¨ Martin Wehling ∗ , Armin Schultz, Ralf Losel Department of Clinical Pharmacology, Medical School Mannheim, University of Heidelberg, D-68167 Mannheim, Germany
a r t i c l e
i n f o
a b s t r a c t
Article history:
In addition to cellular responses that are elicited by steroids involving the modulation
Published on line 17 January 2007
of transcription in the nucleus, it is now generally accepted that additional phenomena occur that do not depend on the genome. However, there is a puzzling variety of candidate
Keywords:
receptors described in the literature. © 2007 Published by Elsevier Inc.
Receptor Steroid Nongenomic effect
1.
Introduction
In addition to cellular responses that are elicited by steroids involving the modulation of transcription in the nucleus, it is now generally accepted that additional phenomena occur that do not depend on the genome. Hence, those actions are called nongenomic or extranuclear. In the absence of a mechanistic model, such responses have already been observed – and carefully described – as early as the 1940s. However, the properties observed for many nongenomic actions of steroids significantly differ from the pattern seen with the receptors that mediate the genomic (classic) responses. Several lines of evidence have prompted the hypothesis that many or most nongenomic responses start at membrane, presumably the plasma membrane in most cases. However, the identities of receptors that mediate nongenomic phenomena largely are a matter of considerable controversy. While some rapid responses have been shown to involve classic steroid receptors, no receptors have yet been identified for perhaps the majority of such actions, although the number of nonclassic receptor candidates keeps growing. In the following we want to briefly review the sometimes puzzling data on putative nonclassic receptors that have been obtained from very different experimental approaches, and discuss some technical issues and conflicting observations reported in the literature over the past twenty years.
∗
2.
Steroid binding sites
There are numerous reports on “membrane” binding sites for various steroids in a wide variety of tissues, cells or subcellular fractions, assumed to represent nonclassical steroid receptors transmitting nongenomic responses. In many cases, only dissociation constants (KD od pKD ) are reported. However, sometimes pK values for cellular effects are given, which may even differ significantly from pKD for steroid binding, posing the claimed link between binding and effects as a major prerequisite for the receptor definition in the limelight. Sak et al. evaluated 40 studies on membrane sites for estrogen and found that pK values almost continuously span seven orders of magnitude from pK = 4 to 11 [1]. Even if we consider experimental error and species or cell type dependent differences, this is a very wide range. It is, though, unlikely that any particular steroid has a large number of different nonclassic receptors. A similar situation is found with regard to progesterone binding sites. Using different derivatives of progesterone and progesterone analogs, a zoo of binding proteins with molecular weights between 30 and 315 kDa (in the nondenatured state) was found in different studies [2]. Again, these differences are far beyond experimental error. Therefore, one might ask: of what nature are those binding sites? One answer to the conundrum may lie in the nature of steroids themselves. In general, steroids are very sticky
Corresponding author. Tel.: +49 621 383 4058; fax: +49 621 383 2024. E-mail address: [email protected] (M. Wehling). 0039-128X/$ – see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.steroids.2006.11.007
108
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
molecules, with progesterone and estradiol possibly being more sticky than others. This may be the physicochemical base of low affinity binding to numerous proteins, which renders ligand blotting experiments difficult to evaluate, as well as binding to non-protein cellular structures, most importantly to membranes. Manipulation of the sterol content of membranes by various methods leads to a major increase in progesterone binding in some cells, and this binding even exhibits (limited) specificity. This is complemented by the observation of progesterone binding enhancement by digitonin treatment (an agent that is known to efficiently sequester cholesterol), but not other surface active compounds, which was seen in bovine luteal membranes [3] and, repeatedly, in spermatozoa (e.g. [4]). Steroids exhibit specific diverse effects on membrane fluidity in artificial and reconstituted membranes as well, although all steroids increased the activity of an embedded model membrane enzyme similarly [5]. The membrane localization of steroid binding sites or receptors is often supported by experiments using steroid conjugated to a (labeled) macromolecular carrier supposed not to enter the cell. Although this approach is straightforward, it is sometimes troublesome that the preparation may contain residual free steroid as it is left over from the synthetic process or result from break down to release free steroid. Furthermore, even molecules that are presumably membrane nonpermeant such as steroid-albumin conjugates, have been found inside cells, even in the nuclei [6]. Nongenomic phenomena often follow a very rapid time course, and so the binding (and dissociation) kinetics of putative binding sites is assumed to be fast as well. T1/2 values in the range of few minutes (or even seconds) have been reported for most membrane binding sites. However, measuring binding in this time frame (which is still slow compared to most enzyme reactions) may be difficult, or error-prone, using non-equilibrium methods that employ separation steps taking much more time than the half life of dissociation. The dextran charcoal assay, which has been extremely valuable for classic steroid receptors with much longer dissociation times, is thus questionable in this context. In general, this may also add uncertainty to KD determinations. Independent from the demonstration of ligand binding, some researchers have tried to identify nonclassic receptors using antibodies raised against the ligand binding domains of classic nuclear receptors. In the case of progesterone receptors, the most popular approach has been to use an antibody called c262 that has been suggested to mimic progesterone at the ligand binding site of the classic progesterone receptor (PR). However, this approach also yielded many different candidates [2], even residing in the same cells. Some of those proteins have been subjected to identification attempts, but only hitherto unknown proteins unrelated to PR could be detected [7], which suggests that candidates identified by this method may merely reflect antibody cross-reactivity.
3.
Steroid effects
Even in the absence of receptor proteins, steroids may exert biological effects. Progesterone, when applied at micromo-
lar concentration, induces fusion of vesicles prepared from purified lipids. Other steroids were far less effective [8]. This finding may have significance for the acrosome reaction in spermatozoa that also occurs in the upper nanomolar/lower micromolar range, although there are several traditional protein receptor candidates that may be involved (see below). Complicating the plot, spermatozoa may contain more than one progesterone receptor [9,10]. Despite considerable efforts in this regard, only few proteins have been identified and subsequently profiled to convincingly qualify as nonclassic steroid receptors. In the wider sense, such receptors include proteins, which are modulated by steroids but have other “main tasks”. These include GABAA receptors, Na+ /K+ -ATPase and Sigma-1 receptors. The brassinosteroid receptor kinases, which actually represent the first specialised membrane steroid receptor ever cloned, do not have orthologs in animals; their occurence is restricted to plants. A novel, hitherto unknown membrane protein that was reported to bind progesterone and trigger intracellular signalling has been originally isolated from seatrout and termed membrane progestin receptor (mPR) [11]. Subsequently, homologs in several other species including man have been identified and represent a new family of steroid membrane receptors [12]. The three known family members exhibit tissue-specific expression, including the ovaries and spermatozoa, which are known places of progesterone action. These proteins appear to possess seven transmembrane domains, and thus resemble G-protein coupled receptors. Their subcellular localization is controversial; while mPRs were first described to be in the plasma membrane, other studies found predominant localization in the endoplasmic reticulum [13,14] (see also Fig. 1). Nevertheless, an intracellular location does not preclude any physiological response to steroids, as those molecules cross the cell membrane easily and almost freely diffuse throughout the cell. However, there are conflicting results on mPRs’ effect on cellular signaling as well. None of the members of this family of receptors signaled in response to progesterone as tested in different expression systems, a yet unexplained discrepancy [14]. For estradiol, a G-protein coupled receptor has been identified and termed GPR30 [15]. This receptor is thought to activate EGF receptors by triggering EGF release. Similar to those mPRs mentioned above, the subcellular localization of GPR30 has been attributed to the plasma membrane [16] or endoplasmic reticulum [17]. Again, the physiological significance is a matter of controversy [18], though a recent study showed the de novo occurence of estrogen-induced responses of intracellular calcium by a GPR30-construct transfected in HeLa cells [19]. Last in this incomplete list of receptor candidates is a protein now termed PGRMC1 that had been isolated in our laboratory. It is abundantly expressed in liver, but recombinant expression only moderately increases progesterone binding. On the other hand, it occurs in spermatozoa, and antibodies against it mitigate the progesterone-induced acrosome reaction. Unfortunately, spermatozoa as terminally differentiated cells do not lend themselves to techniques such as overexpression or knockdown. In ovary cells, overexpression of PGRMC1 enhanced progesterone responsiveness, while PGRMC1 antibody blocked the antiapoptotic action [20]. However, similar
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
109
Fig. 1 – Left: immunofluorescent image of HA-tagged mPR␣ (Fugu) transfected into MDA231 cells, stained with anti-HA antibody. Right: counterstained with DAPI. The construct was kindly provided by Dr. T. Krietsch, Endokrinologikum Hamburg.
to other receptor candidates, this protein remains elusive; it is predominantly localized to the endoplasmic reticulum, contains heme and is transcriptionally regulated in a manner that is similar to a cytochrome. Therefore, its physiological role still remains to be elucidated.
4.
Conclusions
In equilibrium thermodynamics, a receptor is often defined as a larger molecule binding another (smaller) molecule called ligand. According to this definition, most of the binding sites discussed above probably are receptors. In the context of physiology and pharmacology, a protein also needs to induce a physiological process or signal that is modulated depending on ligand binding, to be termed receptor. This link is sometimes missing, or ambiguous. However, even receptor candidates that are characterized at the molecular level (the list in the previous paragraph is not complete) yield conflicting results in different laboratories as shown not only for the mPR example. Thus, the current answer to the title question still has to be “maybe” for almost all candidates. Exceptions apply to receptors with other main functions (e.g., GABAA , Na+ , K+ ATPase) and the brassinosteroid receptor in plants. Many more experiments will be needed to primarily detect and, most important, understand the physiological/pathophysiological implications of receptor activation. As elsewhere, knockout mice that have the respective classic recpetors deleted have been used to further corroborate a putative receptor’s candidacy. Yet, even that seemingly clear condition can be blurred when unexpected variants of the original receptor under study are still expressed [21], which complicate the physiological characterization. As those novel membrane receptors could provide new therapeutic options yet to be discovered, the search for and further characterization of promising candidates may still be rewarding though having been extremely difficult, lengthy and frustrating in many instances so far. However, first pharmaceutical applications seem to loom around the corner, with a purely nongenomically acting estrogenic compound, STX, having just appeared on the scene as a promising candidate [22].
references
[1] Sak K, Everaus H. Nongenomic effects of 17beta-estradiol—diversity of membrane binding sites. J Steroid Biochem Mol Biol 2004;88:323–35. [2] Bramley T. Non-genomic progesterone receptors in the mammalian ovary: some unresolved issues. Reproduction 2003;125:3–15. [3] Menzies GS, Howland K, Rae MT, Bramley TA. Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin. Mol Cell Endocrinol 1999;153:57–69. [4] Ambhaikar M, Puri C. Cell surface binding sites for progesterone on human spermatozoa. Mol Hum Reprod 1998;4:413–21. [5] Whiting KP, Restall CJ, Brain PF. Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms. Life Sci 2000;67:743–57. [6] Moats RK, 2nd, Ramirez VD. Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells. J Endocrinol 2000;166:631–47. [7] Luconi M, Bonaccorsi L, Bini L, Liberatori S, Pallini V, Forti G, et al. Characterization of membrane nongenomic receptors for progesterone in human spermatozoa. Steroids 2002;67:505–9. [8] Shivaji S, Jagannadham MV. Steroid-induced perturbations of membranes and its relevance to sperm acrosome reaction. Biochim Biophys Acta 1992;1108:99–109. [9] Tesarik J. Progesterone-induced acrosome reaction: one receptor is not enough. Fertil Steril 1996;65:1265–7. ¨ [10] Losel R, Dorn-Beineke A, Falkenstein E, Wehling M, Feuring M. Porcine spermatozoa contain more than one membrane progesterone receptor. Int J Biochem Cell Biol 2004;36:1532–41. [11] Zhu Y, Rice CD, Pang Y, Pace M, Thomas P. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc Natl Acad Sci USA 2003;100:2231–6. [12] Zhu Y, Bond J, Thomas P. Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Proc Natl Acad Sci USA 2003;100:2237–42. [13] Ashley RL, Clay CM, Farmerie TA, Niswender GD, Nett TM. Cloning and characterization of an ovine intracellular seven
110
[14]
[15]
[16]
[17]
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
transmembrane receptor for progesterone that mediates calcium mobilization. Endocrinology 2006;147:4151–9. ¨ Krietsch T, Fernandes MS, Kero J, Losel R, Heyens M, Lam EW, et al. Human homologs of the putative G protein-coupled membrane progestin receptors (mPR␣, , ␥) localize to the endoplasmic reticulum and are not activated by progesterone. Mol Endocrinol 2006;20:3146–64. Filardo EJ, Quinn JA, Bland KI, Frackelton Jr AR. 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 2000;14:1649–60. Thomas P, Pang Y, Filardo EJ, Dong J. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 2005;146:624–32. Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 2005;307:1625–30.
[18] Pietras RJ, Levin ER, Szego CM. Estrogen receptors and cell signaling. Science 2005;310:50–1. [19] Funakoshi T, Yanai A, Shinoda K, Kawano MM, Mizukami Y. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane. Biochem Biophys Res Commun 2006;346:904–10. [20] Peluso JJ, Pappalardo A, Losel R, Wehling M. Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone’s antiapoptotic action. Endocrinology 2006;147:3133–40. [21] Kos M, Denger S, Reid G, Korach KS, Gannon F. Down but not out? A novel protein isoform of the estrogen receptor alpha is expressed in the estrogen receptor alpha knockout mouse. J Mol Endocrinol 2002;29:281–6. [22] Qiu J, Bosch MA, Tobias SC, Krust A, Graham SM, Murphy SJ, et al. A G-protein-coupled estrogen receptor is involved in hypothalamic control of energy homeostasis. J Neurosci 2006;26:5649–55.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
To be or not to be (a receptor) ¨ Martin Wehling ∗ , Armin Schultz, Ralf Losel Department of Clinical Pharmacology, Medical School Mannheim, University of Heidelberg, D-68167 Mannheim, Germany
a r t i c l e
i n f o
a b s t r a c t
Article history:
In addition to cellular responses that are elicited by steroids involving the modulation
Published on line 17 January 2007
of transcription in the nucleus, it is now generally accepted that additional phenomena occur that do not depend on the genome. However, there is a puzzling variety of candidate
Keywords:
receptors described in the literature. © 2007 Published by Elsevier Inc.
Receptor Steroid Nongenomic effect
1.
Introduction
In addition to cellular responses that are elicited by steroids involving the modulation of transcription in the nucleus, it is now generally accepted that additional phenomena occur that do not depend on the genome. Hence, those actions are called nongenomic or extranuclear. In the absence of a mechanistic model, such responses have already been observed – and carefully described – as early as the 1940s. However, the properties observed for many nongenomic actions of steroids significantly differ from the pattern seen with the receptors that mediate the genomic (classic) responses. Several lines of evidence have prompted the hypothesis that many or most nongenomic responses start at membrane, presumably the plasma membrane in most cases. However, the identities of receptors that mediate nongenomic phenomena largely are a matter of considerable controversy. While some rapid responses have been shown to involve classic steroid receptors, no receptors have yet been identified for perhaps the majority of such actions, although the number of nonclassic receptor candidates keeps growing. In the following we want to briefly review the sometimes puzzling data on putative nonclassic receptors that have been obtained from very different experimental approaches, and discuss some technical issues and conflicting observations reported in the literature over the past twenty years.
∗
2.
Steroid binding sites
There are numerous reports on “membrane” binding sites for various steroids in a wide variety of tissues, cells or subcellular fractions, assumed to represent nonclassical steroid receptors transmitting nongenomic responses. In many cases, only dissociation constants (KD od pKD ) are reported. However, sometimes pK values for cellular effects are given, which may even differ significantly from pKD for steroid binding, posing the claimed link between binding and effects as a major prerequisite for the receptor definition in the limelight. Sak et al. evaluated 40 studies on membrane sites for estrogen and found that pK values almost continuously span seven orders of magnitude from pK = 4 to 11 [1]. Even if we consider experimental error and species or cell type dependent differences, this is a very wide range. It is, though, unlikely that any particular steroid has a large number of different nonclassic receptors. A similar situation is found with regard to progesterone binding sites. Using different derivatives of progesterone and progesterone analogs, a zoo of binding proteins with molecular weights between 30 and 315 kDa (in the nondenatured state) was found in different studies [2]. Again, these differences are far beyond experimental error. Therefore, one might ask: of what nature are those binding sites? One answer to the conundrum may lie in the nature of steroids themselves. In general, steroids are very sticky
Corresponding author. Tel.: +49 621 383 4058; fax: +49 621 383 2024. E-mail address: [email protected] (M. Wehling). 0039-128X/$ – see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.steroids.2006.11.007
108
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
molecules, with progesterone and estradiol possibly being more sticky than others. This may be the physicochemical base of low affinity binding to numerous proteins, which renders ligand blotting experiments difficult to evaluate, as well as binding to non-protein cellular structures, most importantly to membranes. Manipulation of the sterol content of membranes by various methods leads to a major increase in progesterone binding in some cells, and this binding even exhibits (limited) specificity. This is complemented by the observation of progesterone binding enhancement by digitonin treatment (an agent that is known to efficiently sequester cholesterol), but not other surface active compounds, which was seen in bovine luteal membranes [3] and, repeatedly, in spermatozoa (e.g. [4]). Steroids exhibit specific diverse effects on membrane fluidity in artificial and reconstituted membranes as well, although all steroids increased the activity of an embedded model membrane enzyme similarly [5]. The membrane localization of steroid binding sites or receptors is often supported by experiments using steroid conjugated to a (labeled) macromolecular carrier supposed not to enter the cell. Although this approach is straightforward, it is sometimes troublesome that the preparation may contain residual free steroid as it is left over from the synthetic process or result from break down to release free steroid. Furthermore, even molecules that are presumably membrane nonpermeant such as steroid-albumin conjugates, have been found inside cells, even in the nuclei [6]. Nongenomic phenomena often follow a very rapid time course, and so the binding (and dissociation) kinetics of putative binding sites is assumed to be fast as well. T1/2 values in the range of few minutes (or even seconds) have been reported for most membrane binding sites. However, measuring binding in this time frame (which is still slow compared to most enzyme reactions) may be difficult, or error-prone, using non-equilibrium methods that employ separation steps taking much more time than the half life of dissociation. The dextran charcoal assay, which has been extremely valuable for classic steroid receptors with much longer dissociation times, is thus questionable in this context. In general, this may also add uncertainty to KD determinations. Independent from the demonstration of ligand binding, some researchers have tried to identify nonclassic receptors using antibodies raised against the ligand binding domains of classic nuclear receptors. In the case of progesterone receptors, the most popular approach has been to use an antibody called c262 that has been suggested to mimic progesterone at the ligand binding site of the classic progesterone receptor (PR). However, this approach also yielded many different candidates [2], even residing in the same cells. Some of those proteins have been subjected to identification attempts, but only hitherto unknown proteins unrelated to PR could be detected [7], which suggests that candidates identified by this method may merely reflect antibody cross-reactivity.
3.
Steroid effects
Even in the absence of receptor proteins, steroids may exert biological effects. Progesterone, when applied at micromo-
lar concentration, induces fusion of vesicles prepared from purified lipids. Other steroids were far less effective [8]. This finding may have significance for the acrosome reaction in spermatozoa that also occurs in the upper nanomolar/lower micromolar range, although there are several traditional protein receptor candidates that may be involved (see below). Complicating the plot, spermatozoa may contain more than one progesterone receptor [9,10]. Despite considerable efforts in this regard, only few proteins have been identified and subsequently profiled to convincingly qualify as nonclassic steroid receptors. In the wider sense, such receptors include proteins, which are modulated by steroids but have other “main tasks”. These include GABAA receptors, Na+ /K+ -ATPase and Sigma-1 receptors. The brassinosteroid receptor kinases, which actually represent the first specialised membrane steroid receptor ever cloned, do not have orthologs in animals; their occurence is restricted to plants. A novel, hitherto unknown membrane protein that was reported to bind progesterone and trigger intracellular signalling has been originally isolated from seatrout and termed membrane progestin receptor (mPR) [11]. Subsequently, homologs in several other species including man have been identified and represent a new family of steroid membrane receptors [12]. The three known family members exhibit tissue-specific expression, including the ovaries and spermatozoa, which are known places of progesterone action. These proteins appear to possess seven transmembrane domains, and thus resemble G-protein coupled receptors. Their subcellular localization is controversial; while mPRs were first described to be in the plasma membrane, other studies found predominant localization in the endoplasmic reticulum [13,14] (see also Fig. 1). Nevertheless, an intracellular location does not preclude any physiological response to steroids, as those molecules cross the cell membrane easily and almost freely diffuse throughout the cell. However, there are conflicting results on mPRs’ effect on cellular signaling as well. None of the members of this family of receptors signaled in response to progesterone as tested in different expression systems, a yet unexplained discrepancy [14]. For estradiol, a G-protein coupled receptor has been identified and termed GPR30 [15]. This receptor is thought to activate EGF receptors by triggering EGF release. Similar to those mPRs mentioned above, the subcellular localization of GPR30 has been attributed to the plasma membrane [16] or endoplasmic reticulum [17]. Again, the physiological significance is a matter of controversy [18], though a recent study showed the de novo occurence of estrogen-induced responses of intracellular calcium by a GPR30-construct transfected in HeLa cells [19]. Last in this incomplete list of receptor candidates is a protein now termed PGRMC1 that had been isolated in our laboratory. It is abundantly expressed in liver, but recombinant expression only moderately increases progesterone binding. On the other hand, it occurs in spermatozoa, and antibodies against it mitigate the progesterone-induced acrosome reaction. Unfortunately, spermatozoa as terminally differentiated cells do not lend themselves to techniques such as overexpression or knockdown. In ovary cells, overexpression of PGRMC1 enhanced progesterone responsiveness, while PGRMC1 antibody blocked the antiapoptotic action [20]. However, similar
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
109
Fig. 1 – Left: immunofluorescent image of HA-tagged mPR␣ (Fugu) transfected into MDA231 cells, stained with anti-HA antibody. Right: counterstained with DAPI. The construct was kindly provided by Dr. T. Krietsch, Endokrinologikum Hamburg.
to other receptor candidates, this protein remains elusive; it is predominantly localized to the endoplasmic reticulum, contains heme and is transcriptionally regulated in a manner that is similar to a cytochrome. Therefore, its physiological role still remains to be elucidated.
4.
Conclusions
In equilibrium thermodynamics, a receptor is often defined as a larger molecule binding another (smaller) molecule called ligand. According to this definition, most of the binding sites discussed above probably are receptors. In the context of physiology and pharmacology, a protein also needs to induce a physiological process or signal that is modulated depending on ligand binding, to be termed receptor. This link is sometimes missing, or ambiguous. However, even receptor candidates that are characterized at the molecular level (the list in the previous paragraph is not complete) yield conflicting results in different laboratories as shown not only for the mPR example. Thus, the current answer to the title question still has to be “maybe” for almost all candidates. Exceptions apply to receptors with other main functions (e.g., GABAA , Na+ , K+ ATPase) and the brassinosteroid receptor in plants. Many more experiments will be needed to primarily detect and, most important, understand the physiological/pathophysiological implications of receptor activation. As elsewhere, knockout mice that have the respective classic recpetors deleted have been used to further corroborate a putative receptor’s candidacy. Yet, even that seemingly clear condition can be blurred when unexpected variants of the original receptor under study are still expressed [21], which complicate the physiological characterization. As those novel membrane receptors could provide new therapeutic options yet to be discovered, the search for and further characterization of promising candidates may still be rewarding though having been extremely difficult, lengthy and frustrating in many instances so far. However, first pharmaceutical applications seem to loom around the corner, with a purely nongenomically acting estrogenic compound, STX, having just appeared on the scene as a promising candidate [22].
references
[1] Sak K, Everaus H. Nongenomic effects of 17beta-estradiol—diversity of membrane binding sites. J Steroid Biochem Mol Biol 2004;88:323–35. [2] Bramley T. Non-genomic progesterone receptors in the mammalian ovary: some unresolved issues. Reproduction 2003;125:3–15. [3] Menzies GS, Howland K, Rae MT, Bramley TA. Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin. Mol Cell Endocrinol 1999;153:57–69. [4] Ambhaikar M, Puri C. Cell surface binding sites for progesterone on human spermatozoa. Mol Hum Reprod 1998;4:413–21. [5] Whiting KP, Restall CJ, Brain PF. Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms. Life Sci 2000;67:743–57. [6] Moats RK, 2nd, Ramirez VD. Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells. J Endocrinol 2000;166:631–47. [7] Luconi M, Bonaccorsi L, Bini L, Liberatori S, Pallini V, Forti G, et al. Characterization of membrane nongenomic receptors for progesterone in human spermatozoa. Steroids 2002;67:505–9. [8] Shivaji S, Jagannadham MV. Steroid-induced perturbations of membranes and its relevance to sperm acrosome reaction. Biochim Biophys Acta 1992;1108:99–109. [9] Tesarik J. Progesterone-induced acrosome reaction: one receptor is not enough. Fertil Steril 1996;65:1265–7. ¨ [10] Losel R, Dorn-Beineke A, Falkenstein E, Wehling M, Feuring M. Porcine spermatozoa contain more than one membrane progesterone receptor. Int J Biochem Cell Biol 2004;36:1532–41. [11] Zhu Y, Rice CD, Pang Y, Pace M, Thomas P. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc Natl Acad Sci USA 2003;100:2231–6. [12] Zhu Y, Bond J, Thomas P. Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Proc Natl Acad Sci USA 2003;100:2237–42. [13] Ashley RL, Clay CM, Farmerie TA, Niswender GD, Nett TM. Cloning and characterization of an ovine intracellular seven
110
[14]
[15]
[16]
[17]
s t e r o i d s 7 2 ( 2 0 0 7 ) 107–110
transmembrane receptor for progesterone that mediates calcium mobilization. Endocrinology 2006;147:4151–9. ¨ Krietsch T, Fernandes MS, Kero J, Losel R, Heyens M, Lam EW, et al. Human homologs of the putative G protein-coupled membrane progestin receptors (mPR␣, , ␥) localize to the endoplasmic reticulum and are not activated by progesterone. Mol Endocrinol 2006;20:3146–64. Filardo EJ, Quinn JA, Bland KI, Frackelton Jr AR. 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 2000;14:1649–60. Thomas P, Pang Y, Filardo EJ, Dong J. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 2005;146:624–32. Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 2005;307:1625–30.
[18] Pietras RJ, Levin ER, Szego CM. Estrogen receptors and cell signaling. Science 2005;310:50–1. [19] Funakoshi T, Yanai A, Shinoda K, Kawano MM, Mizukami Y. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane. Biochem Biophys Res Commun 2006;346:904–10. [20] Peluso JJ, Pappalardo A, Losel R, Wehling M. Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone’s antiapoptotic action. Endocrinology 2006;147:3133–40. [21] Kos M, Denger S, Reid G, Korach KS, Gannon F. Down but not out? A novel protein isoform of the estrogen receptor alpha is expressed in the estrogen receptor alpha knockout mouse. J Mol Endocrinol 2002;29:281–6. [22] Qiu J, Bosch MA, Tobias SC, Krust A, Graham SM, Murphy SJ, et al. A G-protein-coupled estrogen receptor is involved in hypothalamic control of energy homeostasis. J Neurosci 2006;26:5649–55.