madman
Super Moderator
Abstract
Over the last decades, a great body of evidence has defined a novel view of the cellular mechanism of action of the steroid hormone 17β-estradiol (E2) through its estrogen receptors (i.e., ERα and ERβ). It is now clear that the E2-activated ERs work both as transcription factors and extranuclear plasma membrane-localized receptors. The activation of a plethora of signal transduction cascades follows the E2-dependent engagement of plasma membrane-localized ERs and is required for the coordination of gene expression, which ultimately controls the occurrence of the pleiotropic effects of E2. The definition of the molecular mechanisms by which the ERs locate at the cell surface (i.e., palmitoylation and protein association) determined the quest for understanding the specificity of the extra-nuclear E2 signaling. The use of mice models lacking the plasma membrane ERα localization unveiled that the extra-nuclear E2 signaling is operational in vivo but tissue-specific. However, the underlying molecular details for such ERs signaling diversity in the perspective of the E2 physiological functions in the different cellular contexts are still not understood. Therefore, to gain insights into the tissue specificity of the extra-nuclear E2 signaling to physiological functions, here we reviewed the known ERs extra-nuclear interactors and tried to extrapolate from available databases the ERα and ERβ extra-nuclear interactomes. Based on literature data, it is possible to conclude that by specifically binding to extra-nuclear localized proteins in different sub-cellular compartments, the ERs fine-tune their molecular activities. Moreover, we report that the context-dependent diversity of the ERs-mediated extra-nuclear E2 actions can be ascribed to the great flexibility of the physical structures of ERs and the spatial-temporal organization of the logistics of the cells (i.e., the endocytic compartments). Finally, we provide lists of proteins belonging to the potential ERα and ERβ extra-nuclear interactomes and propose that the systematic experimental definition of the ERs extra-nuclear interactomes in different tissues represents the next step for the research in the ERs field. Such characterization will be fundamental for the identification of novel druggable targets for the innovative treatment of ERs-related diseases.
In ‘The Structure of Scientific Revolutions’, Dr. Thomas Khun has clarified that the definition of scientific truth does not solely derive by applying objective criteria, but it is established by a shared consensus of a scientific community. Thus, scientific knowledge does not progress linearly and continuously but rather it undergoes periodic paradigm shifts that open up new approaches to understanding what scientists would never have considered valid before (Khun, 1962). In other words, the understanding of a natural phenomenon is an integrated mixture between the ‘objective’ results arising from experimentations and their ‘social’ interpretation in the context of a specific scientific historical background
The estrogen and estrogen receptor field represents a great example of how novel discoveries contribute to paradigm definitions and paradigm shifts. However, this same field also nicely shows how difficult is to introduce in the scientific community unconventional findings, which may not reconcile with the ‘social aspect of science but are fitting in the ‘objective’ understanding of the natural phenomenon.
In this respect, the Wikipedia definition of estrogens reads that ‘they are a group of compounds [i.e., estrone (E1), estriol (E3) and 17β-estradiol (E2), the most effective one] named for their importance in both menstrual and estrous reproductive cycles. They are the primary female sex steroids. […] Their name comes from the Greek οἶστρος (oistros), literally meaning verve or inspiration but figuratively sexual passion or desire, and the suffix –gen, meaning producer of’. The basic textbook definition of estrogens, instead, reads that ‘they are steroid hormones produced in ovary and adrenal cortex and are the dominant steroid in females’ (Silverthon, 2007).
As steroid hormones (i.e., small lipophilic molecules that freely diffuse across cell membranes), estrogens act by engaging the estrogen receptors (ERs) that, as again defined by Wikipedia, ‘are a group of proteins found inside cells that are members of the nuclear hormone family of intracellular receptors. Once activated by estrogen, the ERs can translocate into the nucleus and bind to DNA to regulate the activity of different genes. A very similar definition can be also found in textbooks where the ERs are ‘found within cells, either in the cytoplasm or in the nucleus. The ultimate destination of ER-hormone complexes is the nucleus, where the complex acts as a transcription factor, binding to DNA and regulating gene transcription (Silverthon, 2007)
Consequently, the ‘social’ perception of estrogens is clearly that they are female sex steroid hormones acting exclusively in female reproductive tissues and organs by triggering the activity of their receptors, which are transcription factors. The effects of estrogens then derive from a change in the gene expression profile of the target cells that results in a phenotypic alteration of their specific cellular behavior.
Although these definitions are undoubtedly true, they represent only a part of the complex mode of estrogen action through the ERs (Table 1). Indeed, the actual inclusive definition of this particular scientific field that takes into account all the ‘objective’ knowledge accumulated during years should read as follows. In target cells, estrogens specifically act through at least two ERs subtypes, which work both as transcription factors and as plasma membrane receptors and mediate the physiological effects of these sex steroid hormones in reproductive and non-reproductive tissues both in females and in males. This latter definition underscores a shift from a limited perspective (i.e., traditional view) to a wider one in which estrogens in general and 17β-estradiol (E2), in particular, are global regulators of integrated physiology and thus of body homeostasis.
This definition derives from the concepts demonstrated by the contribution of different investigators in the last 60 years or so, which can be organized in historical periods. Clearly, such a timeline does not have to be considered as composed of isolated periods in which only one specific line of research was pursued. Rather, we name them after the main discoveries that occurred in that particular period of time, but we additionally stress the concept that the accumulation of the knowledge of the different aspects of E2 signaling occurred (and still occurs) in parallel (Figure 1).
The Biochemical Age (1960s-1970s) represents the infancy of the estrogen and estrogen receptor field and has been instrumental in the definition that E2 specifically binds to a ‘receptor protein’ inside the target cell and also to understand that E2-induced administration causes changes in gene transcription because this hormone elicits transcriptional effects (Buller & O'Malley, 1976; Jensen & Jacobson, 1962; Noteboom & Gorski, 1965). This period set the pace for the Nuclear Age (the 1980s-2000) where most of the knowledge in the field accumulated. Indeed, by the end of the last century, it became clear that the ‘receptor protein’ in estrogen target cells is indeed one of the two ERs subtypes (i.e., ERα and ERβ) and that the effects of estrogens in vivo are dependent on both ERα and ERβ that work as transcription factors in the nucleus (Brzozowski et al, 1997; Dupont et al, 2000; Green et al, 1986a; Green et al, 1986b; Kuiper et al, 1996; Lubahn et al, 1993; Ogawa et al, 1998; Schwabe et al, 1993a; Schwabe et al, 1993b).
The subsequent Extra-Nuclear Age (2001-2015) has demonstrated that the same ERs are located at the cell plasma membrane from which they signal for the activation of rapid effects (AKA extra-nuclear/non-genomic/membrane-initiated starting signals) that regulate diverse cellular processes, integrate with the ERs transcriptional actions, and are operational in vivo (Acconcia et al, 2005a; Acconcia et al, 2004; Adlanmerini et al, 2014; Jakacka et al, 2002; La Rosa et al, 2012; Li et al, 2003; Pedram et al, 2014; Pietras & Szego, 1977; Razandi et al, 1999).
And yet, this might still represent only a part of the entire story as the research performed during the last five years (2016-2021) has shown the harmonization of the nuclear and extra-nuclear ERs field (i.e., the age of cross-talk) thanks to the deep analysis of the phenotypes of the mice models in which the extra-nuclear E2 signaling has been selectively knocked out (Adlanmerini et al., 2014; Farman et al, 2018; Fontaine et al, 2020; Gustafsson et al, 2016; Nanjappa et al, 2016; Pedram et al., 2014; Vinel et al, 2016; Yu et al, 2020). Therefore, this period lays the foundations for the next future paradigm shifts. Indeed, reports indicate that E2 and the membrane-bound ERα could be internalized through an active mechanism for hormone uptake by complex endocytic trafficking routes (Adams, 2005; Hammes et al, 2005; Sampayo et al, 2018; Scheidt et al, 2010; Totta et al, 2016; Totta et al, 2015a; Totta et al, 2015b; Totta et al, 2014). Moreover, it is now clear that the extra-nuclear rapid effects mediated by the ERs can be specifically modulated by different ERs exogenous ligands (e.g., xenoestrogen like nutritional flavonoids) determining a modification of ERs-based signaling to physiological functions (Acconcia et al, 2016; Acconcia et al, 2015; La Rosa et al, 2014; Marino et al, 2012).
Notwithstanding all this evidence, still, the concept that E2 elicits rapid actions modulating a different aspect of cell physiology by acting through the same nuclear receptor, which is located outside of the nucleus is a debated issue as witnessed by the fact that in the last year's research about the novel additional membrane receptor for estrogens (i.e., GPR30 also known as GPER1) and its connections with the E2-regulated effects in diverse cellular contexts (Pepermans et al, 2021) has exponentially exploded (Fig. 1B). The accumulation of novel information regarding the role of GPER1 in E2 signaling has driven the ‘social’ perception of E2 signaling towards the picture that, while the E2-dependent regulation of gene expression is due to the functions of the classic ERs, which can only work as nuclear transcription factors, the E2-elicited extra-nuclear effects are mostly ascribed to the activity of the non-classic seven-spanning membrane receptor for E2 (i.e., GPER1).
Perhaps, one of the reasons for which the above-mentioned ‘objective’ knowledge (i.e., the same nuclear ERs are also located at the plasma membrane and trigger E2 extra-nuclear signaling per se) is being underestimated resides in the fact that on the contrary to the knowledge regarding the molecular interactions the ERs exploit to regulate gene expression in the nucleus (i.e., the ERs nuclear interactome) (Cirillo et al, 2013; Gigantino et al, 2020; Giurato et al, 2018; Metivier et al, 2003; Nassa et al, 2011a; Nassa et al, 2011b; Reid et al, 2003; Tarallo et al, 2011), there is a lack of understanding of the molecular mechanisms activated by the extra-nuclear-localized ERs in association with other proteins (i.e., the ERs extra-nuclear interactome) for generating the diversity of rapid signal transduction pathways and physiological effects in the different cellular contexts.
*Therefore, we reviewed here the reported physical interactions of the ERs with other proteins in the extra-nuclear compartment to provide a picture of the extra-nuclear ERs interactomes and their possible physiological implications in the appearance of the E2-dependent effects.
2. The relationship among the pleiotropic effects of E2 and the extra-nuclear signaling.
2.1 The pleiotropic effects of E2
2.2 The E2 signaling diversity
2.3 The dynamics of E2 signaling
2.4 The topology of the origin of the extra-nuclear E2 signaling.
3. The problem of the cell-type specificity of the extra-nuclear E2 signaling.
3.1 The ERs biochemical structure.
3.2 The ERs structural plasticity in the subcellular compartments.
3.3 The nature of the E2 stimulation.
4. The ERα and ERβ interactomes.
4.1 The nuclear binding partners of the ERs.
4.2 The extra-nuclear binding partners of the ERs.
4.2.1 Signaling molecules and adaptors
4.2.1.1 Src and PI3K
4.2.1.2 Receptor tyrosine kinase (RTKs) and Shc
4.2.1.3 p38/MAPK
4.2.1.4 Other receptors
4.2.2 Plasma membrane scaffolding proteins
4.2.2.1 Caveolins
4.2.2.2 Striatin
4.2.3 Cytoplasmic scaffolding proteins
4.2.3.1 Calmodulin
4.2.3.2 p130Cas/BCAR
4.2.3.3 HPIP
4.2.3.4 PELP1/MNAR
4.2.3.5 MTA1s
5. High-throughput analyses of the extra-nuclear ERs interactome.
5.1 Bioinformatic identification of the extra-nuclear ERs interactomes.
5.2 The relationships among endocytic proteins and the ERs
5.3 Endocytic proteins and E2 extra-nuclear signaling
5.4 E2 internalization via membrane-located ERs
6. Discussion.
7. Future perspectives.
This outlined concept can be immediately challenged because there is not a systematic experimental characterization of the extra-nuclear ERs interactomes. This caveat has to be considered as the recognition of a novel starting point from which to derive ‘objective’ knowledge (Khun, 1962).
We are aware that great technical limitations can underly the substantial lack of evidence about the specific interactions of the ERs in the cytoplasm. Indeed, the amount of the ERs localized in the extra-nuclear compartment is much less (approximately 10-15%) than the amount of the receptors in the nuclei of ERs expressing cells (Acconcia & Marino, 2011).
However, we propose different strategies to discover and characterize the extra-nuclear ERs interactome.
i) The most direct approach would be the overexpression of the ERs. However, this cellular manipulation would artificially increase the amount of the ERs in all subcellular compartments, increasing the possibility of artifacts.
ii) It would be possible to evaluate the extra-nuclear ERs interactome by directly targeting the ERs in the extra-nuclear compartments. This solution would require either a modification of the structure of the ERs or their labeling with specific extra-nuclear targeting protein tags. However, such modifications could themselves affect the physiological associations of the ERs with the specific interactors. In this respect, an ERα variant in which all the nuclear localization signals have been mutated has been characterized for an increased-to-exclusive extra-nuclear localization (Burns et al, 2011; Totta et al., 2014) but these reagents have not been used to study the extra-nuclear ERα interactome.
iii) Perhaps the most physiological approach (although expensive and time-consuming) to study the context-dependent extra-nuclear ERs interactome could be the use of wild type and palmitoylation defective mice models (Adlanmerini et al., 2014; Pedram et al., 2014) where the extra-nuclear interactome could be derived in each tissue as the difference between the identified ERα interacting partners in intact and mutated mice.
Other strategies tackling this problem (e.g., the development of specific inhibitors of membrane ERs signaling; the evaluation of rapid E2 actions underpinning disease and sexual dimorphism) could be developed as a function of specific technical advancements (e.g., SILAC mass spectrometry), but, whatever the case, the solution to this challenging issue will eventually clarify the picture of the ERs (and possibly also of other steroid receptors) functioning, thus allowing, on one hand, the definitive proofs for a physiological role of extra-nuclear ERs actions in health and disease and on the other hand the identification of novel druggable targets for the innovative treatment of diseases where the ERs play fundamental roles (e.g., breast cancer).
Over the last decades, a great body of evidence has defined a novel view of the cellular mechanism of action of the steroid hormone 17β-estradiol (E2) through its estrogen receptors (i.e., ERα and ERβ). It is now clear that the E2-activated ERs work both as transcription factors and extranuclear plasma membrane-localized receptors. The activation of a plethora of signal transduction cascades follows the E2-dependent engagement of plasma membrane-localized ERs and is required for the coordination of gene expression, which ultimately controls the occurrence of the pleiotropic effects of E2. The definition of the molecular mechanisms by which the ERs locate at the cell surface (i.e., palmitoylation and protein association) determined the quest for understanding the specificity of the extra-nuclear E2 signaling. The use of mice models lacking the plasma membrane ERα localization unveiled that the extra-nuclear E2 signaling is operational in vivo but tissue-specific. However, the underlying molecular details for such ERs signaling diversity in the perspective of the E2 physiological functions in the different cellular contexts are still not understood. Therefore, to gain insights into the tissue specificity of the extra-nuclear E2 signaling to physiological functions, here we reviewed the known ERs extra-nuclear interactors and tried to extrapolate from available databases the ERα and ERβ extra-nuclear interactomes. Based on literature data, it is possible to conclude that by specifically binding to extra-nuclear localized proteins in different sub-cellular compartments, the ERs fine-tune their molecular activities. Moreover, we report that the context-dependent diversity of the ERs-mediated extra-nuclear E2 actions can be ascribed to the great flexibility of the physical structures of ERs and the spatial-temporal organization of the logistics of the cells (i.e., the endocytic compartments). Finally, we provide lists of proteins belonging to the potential ERα and ERβ extra-nuclear interactomes and propose that the systematic experimental definition of the ERs extra-nuclear interactomes in different tissues represents the next step for the research in the ERs field. Such characterization will be fundamental for the identification of novel druggable targets for the innovative treatment of ERs-related diseases.
In ‘The Structure of Scientific Revolutions’, Dr. Thomas Khun has clarified that the definition of scientific truth does not solely derive by applying objective criteria, but it is established by a shared consensus of a scientific community. Thus, scientific knowledge does not progress linearly and continuously but rather it undergoes periodic paradigm shifts that open up new approaches to understanding what scientists would never have considered valid before (Khun, 1962). In other words, the understanding of a natural phenomenon is an integrated mixture between the ‘objective’ results arising from experimentations and their ‘social’ interpretation in the context of a specific scientific historical background
The estrogen and estrogen receptor field represents a great example of how novel discoveries contribute to paradigm definitions and paradigm shifts. However, this same field also nicely shows how difficult is to introduce in the scientific community unconventional findings, which may not reconcile with the ‘social aspect of science but are fitting in the ‘objective’ understanding of the natural phenomenon.
In this respect, the Wikipedia definition of estrogens reads that ‘they are a group of compounds [i.e., estrone (E1), estriol (E3) and 17β-estradiol (E2), the most effective one] named for their importance in both menstrual and estrous reproductive cycles. They are the primary female sex steroids. […] Their name comes from the Greek οἶστρος (oistros), literally meaning verve or inspiration but figuratively sexual passion or desire, and the suffix –gen, meaning producer of’. The basic textbook definition of estrogens, instead, reads that ‘they are steroid hormones produced in ovary and adrenal cortex and are the dominant steroid in females’ (Silverthon, 2007).
As steroid hormones (i.e., small lipophilic molecules that freely diffuse across cell membranes), estrogens act by engaging the estrogen receptors (ERs) that, as again defined by Wikipedia, ‘are a group of proteins found inside cells that are members of the nuclear hormone family of intracellular receptors. Once activated by estrogen, the ERs can translocate into the nucleus and bind to DNA to regulate the activity of different genes. A very similar definition can be also found in textbooks where the ERs are ‘found within cells, either in the cytoplasm or in the nucleus. The ultimate destination of ER-hormone complexes is the nucleus, where the complex acts as a transcription factor, binding to DNA and regulating gene transcription (Silverthon, 2007)
Consequently, the ‘social’ perception of estrogens is clearly that they are female sex steroid hormones acting exclusively in female reproductive tissues and organs by triggering the activity of their receptors, which are transcription factors. The effects of estrogens then derive from a change in the gene expression profile of the target cells that results in a phenotypic alteration of their specific cellular behavior.
Although these definitions are undoubtedly true, they represent only a part of the complex mode of estrogen action through the ERs (Table 1). Indeed, the actual inclusive definition of this particular scientific field that takes into account all the ‘objective’ knowledge accumulated during years should read as follows. In target cells, estrogens specifically act through at least two ERs subtypes, which work both as transcription factors and as plasma membrane receptors and mediate the physiological effects of these sex steroid hormones in reproductive and non-reproductive tissues both in females and in males. This latter definition underscores a shift from a limited perspective (i.e., traditional view) to a wider one in which estrogens in general and 17β-estradiol (E2), in particular, are global regulators of integrated physiology and thus of body homeostasis.
This definition derives from the concepts demonstrated by the contribution of different investigators in the last 60 years or so, which can be organized in historical periods. Clearly, such a timeline does not have to be considered as composed of isolated periods in which only one specific line of research was pursued. Rather, we name them after the main discoveries that occurred in that particular period of time, but we additionally stress the concept that the accumulation of the knowledge of the different aspects of E2 signaling occurred (and still occurs) in parallel (Figure 1).
The Biochemical Age (1960s-1970s) represents the infancy of the estrogen and estrogen receptor field and has been instrumental in the definition that E2 specifically binds to a ‘receptor protein’ inside the target cell and also to understand that E2-induced administration causes changes in gene transcription because this hormone elicits transcriptional effects (Buller & O'Malley, 1976; Jensen & Jacobson, 1962; Noteboom & Gorski, 1965). This period set the pace for the Nuclear Age (the 1980s-2000) where most of the knowledge in the field accumulated. Indeed, by the end of the last century, it became clear that the ‘receptor protein’ in estrogen target cells is indeed one of the two ERs subtypes (i.e., ERα and ERβ) and that the effects of estrogens in vivo are dependent on both ERα and ERβ that work as transcription factors in the nucleus (Brzozowski et al, 1997; Dupont et al, 2000; Green et al, 1986a; Green et al, 1986b; Kuiper et al, 1996; Lubahn et al, 1993; Ogawa et al, 1998; Schwabe et al, 1993a; Schwabe et al, 1993b).
The subsequent Extra-Nuclear Age (2001-2015) has demonstrated that the same ERs are located at the cell plasma membrane from which they signal for the activation of rapid effects (AKA extra-nuclear/non-genomic/membrane-initiated starting signals) that regulate diverse cellular processes, integrate with the ERs transcriptional actions, and are operational in vivo (Acconcia et al, 2005a; Acconcia et al, 2004; Adlanmerini et al, 2014; Jakacka et al, 2002; La Rosa et al, 2012; Li et al, 2003; Pedram et al, 2014; Pietras & Szego, 1977; Razandi et al, 1999).
And yet, this might still represent only a part of the entire story as the research performed during the last five years (2016-2021) has shown the harmonization of the nuclear and extra-nuclear ERs field (i.e., the age of cross-talk) thanks to the deep analysis of the phenotypes of the mice models in which the extra-nuclear E2 signaling has been selectively knocked out (Adlanmerini et al., 2014; Farman et al, 2018; Fontaine et al, 2020; Gustafsson et al, 2016; Nanjappa et al, 2016; Pedram et al., 2014; Vinel et al, 2016; Yu et al, 2020). Therefore, this period lays the foundations for the next future paradigm shifts. Indeed, reports indicate that E2 and the membrane-bound ERα could be internalized through an active mechanism for hormone uptake by complex endocytic trafficking routes (Adams, 2005; Hammes et al, 2005; Sampayo et al, 2018; Scheidt et al, 2010; Totta et al, 2016; Totta et al, 2015a; Totta et al, 2015b; Totta et al, 2014). Moreover, it is now clear that the extra-nuclear rapid effects mediated by the ERs can be specifically modulated by different ERs exogenous ligands (e.g., xenoestrogen like nutritional flavonoids) determining a modification of ERs-based signaling to physiological functions (Acconcia et al, 2016; Acconcia et al, 2015; La Rosa et al, 2014; Marino et al, 2012).
Notwithstanding all this evidence, still, the concept that E2 elicits rapid actions modulating a different aspect of cell physiology by acting through the same nuclear receptor, which is located outside of the nucleus is a debated issue as witnessed by the fact that in the last year's research about the novel additional membrane receptor for estrogens (i.e., GPR30 also known as GPER1) and its connections with the E2-regulated effects in diverse cellular contexts (Pepermans et al, 2021) has exponentially exploded (Fig. 1B). The accumulation of novel information regarding the role of GPER1 in E2 signaling has driven the ‘social’ perception of E2 signaling towards the picture that, while the E2-dependent regulation of gene expression is due to the functions of the classic ERs, which can only work as nuclear transcription factors, the E2-elicited extra-nuclear effects are mostly ascribed to the activity of the non-classic seven-spanning membrane receptor for E2 (i.e., GPER1).
Perhaps, one of the reasons for which the above-mentioned ‘objective’ knowledge (i.e., the same nuclear ERs are also located at the plasma membrane and trigger E2 extra-nuclear signaling per se) is being underestimated resides in the fact that on the contrary to the knowledge regarding the molecular interactions the ERs exploit to regulate gene expression in the nucleus (i.e., the ERs nuclear interactome) (Cirillo et al, 2013; Gigantino et al, 2020; Giurato et al, 2018; Metivier et al, 2003; Nassa et al, 2011a; Nassa et al, 2011b; Reid et al, 2003; Tarallo et al, 2011), there is a lack of understanding of the molecular mechanisms activated by the extra-nuclear-localized ERs in association with other proteins (i.e., the ERs extra-nuclear interactome) for generating the diversity of rapid signal transduction pathways and physiological effects in the different cellular contexts.
*Therefore, we reviewed here the reported physical interactions of the ERs with other proteins in the extra-nuclear compartment to provide a picture of the extra-nuclear ERs interactomes and their possible physiological implications in the appearance of the E2-dependent effects.
2. The relationship among the pleiotropic effects of E2 and the extra-nuclear signaling.
2.1 The pleiotropic effects of E2
2.2 The E2 signaling diversity
2.3 The dynamics of E2 signaling
2.4 The topology of the origin of the extra-nuclear E2 signaling.
3. The problem of the cell-type specificity of the extra-nuclear E2 signaling.
3.1 The ERs biochemical structure.
3.2 The ERs structural plasticity in the subcellular compartments.
3.3 The nature of the E2 stimulation.
4. The ERα and ERβ interactomes.
4.1 The nuclear binding partners of the ERs.
4.2 The extra-nuclear binding partners of the ERs.
4.2.1 Signaling molecules and adaptors
4.2.1.1 Src and PI3K
4.2.1.2 Receptor tyrosine kinase (RTKs) and Shc
4.2.1.3 p38/MAPK
4.2.1.4 Other receptors
4.2.2 Plasma membrane scaffolding proteins
4.2.2.1 Caveolins
4.2.2.2 Striatin
4.2.3 Cytoplasmic scaffolding proteins
4.2.3.1 Calmodulin
4.2.3.2 p130Cas/BCAR
4.2.3.3 HPIP
4.2.3.4 PELP1/MNAR
4.2.3.5 MTA1s
5. High-throughput analyses of the extra-nuclear ERs interactome.
5.1 Bioinformatic identification of the extra-nuclear ERs interactomes.
5.2 The relationships among endocytic proteins and the ERs
5.3 Endocytic proteins and E2 extra-nuclear signaling
5.4 E2 internalization via membrane-located ERs
6. Discussion.
7. Future perspectives.
This outlined concept can be immediately challenged because there is not a systematic experimental characterization of the extra-nuclear ERs interactomes. This caveat has to be considered as the recognition of a novel starting point from which to derive ‘objective’ knowledge (Khun, 1962).
We are aware that great technical limitations can underly the substantial lack of evidence about the specific interactions of the ERs in the cytoplasm. Indeed, the amount of the ERs localized in the extra-nuclear compartment is much less (approximately 10-15%) than the amount of the receptors in the nuclei of ERs expressing cells (Acconcia & Marino, 2011).
However, we propose different strategies to discover and characterize the extra-nuclear ERs interactome.
i) The most direct approach would be the overexpression of the ERs. However, this cellular manipulation would artificially increase the amount of the ERs in all subcellular compartments, increasing the possibility of artifacts.
ii) It would be possible to evaluate the extra-nuclear ERs interactome by directly targeting the ERs in the extra-nuclear compartments. This solution would require either a modification of the structure of the ERs or their labeling with specific extra-nuclear targeting protein tags. However, such modifications could themselves affect the physiological associations of the ERs with the specific interactors. In this respect, an ERα variant in which all the nuclear localization signals have been mutated has been characterized for an increased-to-exclusive extra-nuclear localization (Burns et al, 2011; Totta et al., 2014) but these reagents have not been used to study the extra-nuclear ERα interactome.
iii) Perhaps the most physiological approach (although expensive and time-consuming) to study the context-dependent extra-nuclear ERs interactome could be the use of wild type and palmitoylation defective mice models (Adlanmerini et al., 2014; Pedram et al., 2014) where the extra-nuclear interactome could be derived in each tissue as the difference between the identified ERα interacting partners in intact and mutated mice.
Other strategies tackling this problem (e.g., the development of specific inhibitors of membrane ERs signaling; the evaluation of rapid E2 actions underpinning disease and sexual dimorphism) could be developed as a function of specific technical advancements (e.g., SILAC mass spectrometry), but, whatever the case, the solution to this challenging issue will eventually clarify the picture of the ERs (and possibly also of other steroid receptors) functioning, thus allowing, on one hand, the definitive proofs for a physiological role of extra-nuclear ERs actions in health and disease and on the other hand the identification of novel druggable targets for the innovative treatment of diseases where the ERs play fundamental roles (e.g., breast cancer).
