Benzodiazepines and steroidogenesis B. J. Whitehouse
Over the last 3 years, two rather disparate areas of biological research have come together unexpectedly, and the information that is emerging may not only fill in some of the gaps in our knowledge of the mechanism of stimulation of steroidogenesis, but also gives new insights into potential interactions between the nervous and endocrine systems. Benzodiazepine drugs are widely used therapeutically because of their sedative and anxiolytic effects, which are known to be mediated through modulation of GABAA receptors in the central nervous system. However, there is another recognition site for these drugs, termed the peripheral (or mitochondrial) benzodiazepine receptor, which is found in a wide variety of non-neural sites in the body, including the pituitary, testis, ovary and adrenal cortex. It has recently been proposed that this benzodiazepine receptor plays a central role in the regulation of steroidogenesis by controlling the rate-limiting step in the steroid biosynthetic pathway the transport of cholesterol to the inner mitochondrial membranes. A further possibility is that the endogenous polypeptide ligand for the peripheral benzodiazepine receptor (known as diazepam-binding inhibitor or endozepine) may play a part in mediating the effects of trophic hormones on steroidogenesis. The discus¬ sion that follows attempts to explain the background to these findings, and to assess the significance of these \p=n-\
new
hypotheses.
Peripheral benzodiazepine receptors The peripheral benzodiazepine receptors (PBR) were
first recognized as distinct from the central receptor because they did not appear to be functionally linked to the GABA-regulated chloride channel and by the different selectivity of ligands binding to them (Braestrup & Squires, 1977; Costa & Guidotti, 1979). Anxiolytic or anticonvulsant drugs, such as clonazapam, which bind with high affinity to the central benzodiazepine receptor interact only weakly with the peripheral receptor. In contrast, certain benzodiaze¬ pines, which have no anxiolytic activity and which interact only weakly with the central receptor, have
nanomolar binding affinities for the peripheral binding site. The compound known as Ro5-4864(7-
chloro-l,3-dihydro-l-methyl-5-/7-chlorophenyI)-2H1,4-benzodiazepin-2-one) falls into this category, and has been used to localize and characterize peripheral receptor sites (e.g. De Souza, Anholt, Murphy et al. 1985). The best known benzodiazepine drug, diazepam, binds rather more strongly to the central than the peripheral receptor (Braestrup & Squires,
1977). It should be noted that PBR are also found in glial cells in the central nervous system, and that some authorities, therefore, consider the
term mitochon¬
benzodiazepine receptor to be more appropriate (Costa & Guidotti, 1991). Until recently no clearly
drial
defined function for the PBR could be established, and they tended to be viewed as an incidental drugbinding site of no particular physiological significance. However, since they were found in high concentrations in the mitochondria of endocrine tissues such as testis, adrenal cortex and anterior pituitary, the possibility of a role in the control of hormone secretion had been suggested (DeSouza et al. 1985; Anholt, Pedersen, DeSouza & Snyder, 1986).
Benzodiazepines and steroidogenesis Evidence has accumulated in the last 3 years sug¬ gesting that the benzodiazepine receptors which are located in the mitochondria of the testis and adrenal cortex (Anholt et al. 1986) play a part in the regulation of steroidogenesis. Initially, it was reported that two benzodiazepines, diazepam and Ro5-4864, stimulated side-chain cleavage of cholesterol in bovine adrenal mitochondria (Yanagibashi, Ohno, Nakamichi et al. 1989). Following this, Mukhin, Papadopoulos, Costa & Krueger (1989) showed that seven different benzo¬ diazepines stimulated steroidogenesis in the Y-l mouse adrenal tumour cell line, and that the potency of the stimulation of steroid production correlated well with their relative affinity for the peripheral receptor. It has also been reported that benzo¬
diazepines stimulate pregnenolone biosynthesis from exogenous cholesterol by testicular mitochondria
(Papadopoulos, Mukhin, Costa & Krueger, 1990) and progesterone production in ovarian cell cultures (Amsterdam & Suh, 1991). Studies with adrenocort¬ ical cells suggest that benzodiazepine receptors mediate translocation of cholesterol from the outer to the inner mitochondrial membrane (Krueger & Papadopulos, 1990). Thus, there is now a substantial body of evidence which suggests that PBR are involved in the acute stimulation of steroidogenesis by increasing the availability of cholesterol to cyto¬ chrome P-450SCC. There are, however, some inconsistencies which are worth noting, in that benzodiazepines have also been found to inhibit steroidogenesis under some condi¬ tions. In fact, the first reported action of diazepam on the adrenal cortex was a decreased aldosterone response to potassium ion stimulation in bovine zona glomerulosa cells, which was attributed to action as a calcium channel antagonist (Shibata, Kojima & Ogata, 1986). Subsequently, it was found that high doses (> 10 pmol/1) of diazepam also inhibited adrenocorticotrophin (ACTH)- and precursorstimulated steroidogenesis. This effect did not seem to be receptor-mediated ; instead of direct inhibition of microsomal hydroxylations by interaction with cyto¬ chrome P-4502i and cytochrome P-45017o was postula¬ ted (Holloway, Kenyon, Dowie et al. 1989). In addition, even the pharmacology of the PBRmediated effect on steroidogenesis is not straight¬ forward, since one benzodiazepine, flunitrazepam, has been found to inhibit tropic hormone stimulation of steroidogenesis in the Y-l adrenocortical and MA-10 Leydig cell lines (Papadopoulos, Nowzari & Krueger, 1991ft).
Diazepam-binding inhibitor/endozepine The second aspect of this story emerges from the fact that it is now recognized that there are endogenous ligands for the benzodiazepine receptor. In 1983, a 9 kDa polypeptide was isolated from rat brain and referred to as diazepam-binding inhibitor (DBI) to reflect its activity (Guidotti, Forchetti, Corda et al. 1983); later similar active substances were isolated from bovine and human brains and named endozepine (Shoyab, Gentry, Marquardt & Todaro, 1986). DBI also inhibits binding to the peripheral benzodia¬ zepine receptor, and is found in neurones of the cen¬ tral nervous system, in some specialized glial cells and in peripheral tissues, with particularly high concentra¬ tions in the adrenal cortex and testicular Leydig cells (Bovolin, Schlichting, Miyata et al. 1990; Rheaume, Tonon, Smih et al. 1990). Previously, a steroidogenic protein (termed '8-2 K') had been isolated from bovine adrenal cortex, this stimulated the delivery of cholesterol to the inner mitochondrial membrane and
synthesis was controlled by ACTH (Yanagibashi, Ohno, Kawamura & Hall, 1988). The sequence of this protein was found to be identical to that of bovine its
brain DBI, except that it lacked the last two residues at the C terminus (Besman, Yanagibashi, Lee et al. 1989). Recently, DBI itself and its two major process¬ ing products DBI 33-550 and DBI 17-50 have recently been found to stimulate pregnenolone formation in adrenocortical and testicular mitochondria (Papadopoulos, Berkovich, Krueger et al. 1991 ). It is interesting that DBI in the adrenal appears to be under the control of ACTH, the tissue content declines after hypophysectomy and increases with ACTH treatment (Massotti, Slobodyanski, Konkel et al. 1991).
Significance of DBI and the benzodiazepine receptors The final question which must be considered is whether DBI is the long sought 'rapidly turning-over protein' which mediates the response to ACTH in the adrenal cortex and luteinizing hormone (LH) in the testis and ovary. Although initially this seemed a strong possibility, there are a number of facts which argue against DBI having a unique role, of which probably the most telling is that the time-course of its
appearance after ACTH treatment does not correlate with steroidogenic response either in vitro or in vivo (Massotti et al. 1991; Brown, Hall, Shoyab & Papadopoulos, 1992). It may be simply that process¬ ing of the molecule is required, since there is some evidence that the smaller DBI-derived peptides are more active (Papadopoulos et al. 1991 ). However, in view of the known role of cyclic AMP-dependent protein kinases in ACTH and LH action, the involve¬ ment of a phosphorylated product in the stimulation of steroidogenesis seems likely (see Vinson, 1987). Recently, phosphoproteins with short half-lives have been isolated from adrenal and testicular mitochon¬ drial preparations following stimulation with trophic hormones (Epstein & Orme-Johnson, 1991 ; Stocco & Sodeman, 1991). At present, these would seem to be better candidates than DBI to be the specific intracel¬ lular mediators of ACTH or LH action. However, an alternative role for DBI in the nervous system appears possible with the recent demonstration that glial cells can metabolize cholesterol to yield steroids such as pregnenolone (and its sulphate), progesterone, 3ahydroxy-5a-pregnan-20-one, and 3a,21-dihydroxy-
5a-pregnan-20-one (Hu, Bourreau, Jung-Testas et al. 1987; Baulieu & Robel, 1990). The presence of DBI and benzodiazepine receptors within the same cells begs the question of whether DBI plays a role in the control of the synthesis of these steroids (Costa & Guidotti, 1991). This taken together with the know¬ ledge that these so-called 'neurosteroids', like the
benzodiazepines,
can act as modulators of GABAA receptor function (Majewska, Harrison, Schwartz et al. 1986) suggests that a series of totally unsuspected interactions between the nervous and endocrine sys¬ tems remains to be explored.
Krueger, . E. & Papadopoulos, V. (1990). Journal of Biological Chemistry 265, 15015-15022. Majewska, M. D., Harrison, N. L., Schwartz, R. D., Barker, J. L. & Paul, S. (1986). Science 232, 1004-1007. Massotti, M., Slobodyanski, E., Konkel, D., Costa, E. & Guidotti, A. (1991). Endocrinology 129, 591-596. Mukhin, ., Papadopoulos, V., Costa, E. & Krueger, . E. (1989). Proceedings of the National Academy of Sciences of the
REFERENCES
Papadopoulos, V., Berkovich, ., Krueger, K. E., Costa, E. & Guidotti, . (1991a). Endocrinology 129, 1481-1488. Papadopoulos, V., Mukhin, A. G, Costa, E. & Krueger, . E. (1990). Journal of Biological Chemistry 265, 3772-3779. Papadopoulos, V., Nowzari, F. B. & Krueger, . E. (19916). Journal of Biological Chemistry 266, 3682-3687. Rheaume, E., Tonon, M. C, Smih, F., Simard, J., Desy, L., Vaudry, H. & Pelletier, G. (1990). Endocrinology 111,
Amsterdam, A. & Suh, B. S. (1991). Endocrinology 129, 503-510. Anholt, R. R. H., Pedersen, P. L., De Souza, . B. & Snyder, S. H. (1986). Journal of Biological Chemistry 261, 576-583. Baulieu, E. E. & Robel, P. (1990). Journal of Steroid Biochemistry and Molecular Biology 37, 395-403. Besman, M. J., Yanagibashi, K., Lee, T. D., Kawamura, M., Hall, P. F. & Shively, J. E. (1989). Proceedings of the National Academy of Sciences of the U.S.A. 86, 4897-4901. Bovolin, P., Schlichting, J. L., Miyata, M., Ferrarese, C, Guidotti, A. & Alho, H. (1990). Regulatory Peptides 29, 267-281.
Braestrup, C. & Squires, R. F. (1977). Proceedings of the National Academy of Sciences of the U.S.A. 74, 3805-3809. Brown, A. S., Hall, P. F., Shoyab, M. & Papadopoulos, V. (1992). Molecular and Cellular Endocrinology 83, 1-9. Costa E. & Guidotti, A. (1979). Annual Review of Pharmacology and Toxicology 19, 531-545. Costa, E. & Guidotti, A. (1991). Life Sciences 49, 325-344. De Souza, E. B., Anholt, R. R. H., Murphy, K. M. M., Snyder, S. H. & Kuhar, M. J. (1985). Endocrinology 116, 567-573. Epstein, L. F. & Orme-Johnson, N. R. (1991). Journal of Biological Chemistry 266, 19739-19745. Guidotti, ., Forchetti, C. M., Corda, M. G, Konkel, D., Bennet, C. D. & Costa, E. (1983). Proceedings of the National Academy of Sciences of the U.S.A. 80, 3531-3533. Holloway, C. D., Kenyon, C. J., Dowie, L. J., Corrie, J. E. T., Gray, C. E. & Fraser, R. (1989). Journal of Steroid Biochemistry 33, 219-225. Hu, Z.-Y., Bourreau, E., Jung-Testas, I., Robel, P. & Baulieu, E.-E. (1987). Proceedings of the National Academy of Sciences of the U.S.A. 84, 8215-8219.
U.S.A.
86, 9813-9816.
1986-1994.
Shibata, H., Kojima,
I. & Ogata, E. (1986). Biochemical and Biophysical Research Communications 135, 994-999. Shoyab, M., Gentry, L. E., Marquardt, H. & Todaro, G. (1986). Journal of Biological Chemistry 261, 11968-11973. Stocco, D. M. & Sodeman, T. C. (1991). Journal of Biological Chemistry 166, 17931-17938. Vinson, G P. (1987). Journal of Endocrinology 114, 163-165. Yanagibashi, K„ Ohno, Y., Kawamura, M. & Hall, P. F. (1988). Endocrinology 123, 2075-2082. Yanagibashi, K., Olino, Y., Nakamichi, N., Matsui, T., Hayashida, K., Takmura, M., Yamada, K., Tou, S. & Kawamura, M. (1989). Journal of Biochemistry 106, 1026-1029.
Physiology Group,
Biomedicai Sciences Division,
King's College London, Kensington Campus, London W8 U.K.
7AH,
Over the last 3 years, two rather disparate areas of biological research have come together unexpectedly, and the information that is emerging may not only fill in some of the gaps in our knowledge of the mechanism of stimulation of steroidogenesis, but also gives new insights into potential interactions between the nervous and endocrine systems. Benzodiazepine drugs are widely used therapeutically because of their sedative and anxiolytic effects, which are known to be mediated through modulation of GABAA receptors in the central nervous system. However, there is another recognition site for these drugs, termed the peripheral (or mitochondrial) benzodiazepine receptor, which is found in a wide variety of non-neural sites in the body, including the pituitary, testis, ovary and adrenal cortex. It has recently been proposed that this benzodiazepine receptor plays a central role in the regulation of steroidogenesis by controlling the rate-limiting step in the steroid biosynthetic pathway the transport of cholesterol to the inner mitochondrial membranes. A further possibility is that the endogenous polypeptide ligand for the peripheral benzodiazepine receptor (known as diazepam-binding inhibitor or endozepine) may play a part in mediating the effects of trophic hormones on steroidogenesis. The discus¬ sion that follows attempts to explain the background to these findings, and to assess the significance of these \p=n-\
new
hypotheses.
Peripheral benzodiazepine receptors The peripheral benzodiazepine receptors (PBR) were
first recognized as distinct from the central receptor because they did not appear to be functionally linked to the GABA-regulated chloride channel and by the different selectivity of ligands binding to them (Braestrup & Squires, 1977; Costa & Guidotti, 1979). Anxiolytic or anticonvulsant drugs, such as clonazapam, which bind with high affinity to the central benzodiazepine receptor interact only weakly with the peripheral receptor. In contrast, certain benzodiaze¬ pines, which have no anxiolytic activity and which interact only weakly with the central receptor, have
nanomolar binding affinities for the peripheral binding site. The compound known as Ro5-4864(7-
chloro-l,3-dihydro-l-methyl-5-/7-chlorophenyI)-2H1,4-benzodiazepin-2-one) falls into this category, and has been used to localize and characterize peripheral receptor sites (e.g. De Souza, Anholt, Murphy et al. 1985). The best known benzodiazepine drug, diazepam, binds rather more strongly to the central than the peripheral receptor (Braestrup & Squires,
1977). It should be noted that PBR are also found in glial cells in the central nervous system, and that some authorities, therefore, consider the
term mitochon¬
benzodiazepine receptor to be more appropriate (Costa & Guidotti, 1991). Until recently no clearly
drial
defined function for the PBR could be established, and they tended to be viewed as an incidental drugbinding site of no particular physiological significance. However, since they were found in high concentrations in the mitochondria of endocrine tissues such as testis, adrenal cortex and anterior pituitary, the possibility of a role in the control of hormone secretion had been suggested (DeSouza et al. 1985; Anholt, Pedersen, DeSouza & Snyder, 1986).
Benzodiazepines and steroidogenesis Evidence has accumulated in the last 3 years sug¬ gesting that the benzodiazepine receptors which are located in the mitochondria of the testis and adrenal cortex (Anholt et al. 1986) play a part in the regulation of steroidogenesis. Initially, it was reported that two benzodiazepines, diazepam and Ro5-4864, stimulated side-chain cleavage of cholesterol in bovine adrenal mitochondria (Yanagibashi, Ohno, Nakamichi et al. 1989). Following this, Mukhin, Papadopoulos, Costa & Krueger (1989) showed that seven different benzo¬ diazepines stimulated steroidogenesis in the Y-l mouse adrenal tumour cell line, and that the potency of the stimulation of steroid production correlated well with their relative affinity for the peripheral receptor. It has also been reported that benzo¬
diazepines stimulate pregnenolone biosynthesis from exogenous cholesterol by testicular mitochondria
(Papadopoulos, Mukhin, Costa & Krueger, 1990) and progesterone production in ovarian cell cultures (Amsterdam & Suh, 1991). Studies with adrenocort¬ ical cells suggest that benzodiazepine receptors mediate translocation of cholesterol from the outer to the inner mitochondrial membrane (Krueger & Papadopulos, 1990). Thus, there is now a substantial body of evidence which suggests that PBR are involved in the acute stimulation of steroidogenesis by increasing the availability of cholesterol to cyto¬ chrome P-450SCC. There are, however, some inconsistencies which are worth noting, in that benzodiazepines have also been found to inhibit steroidogenesis under some condi¬ tions. In fact, the first reported action of diazepam on the adrenal cortex was a decreased aldosterone response to potassium ion stimulation in bovine zona glomerulosa cells, which was attributed to action as a calcium channel antagonist (Shibata, Kojima & Ogata, 1986). Subsequently, it was found that high doses (> 10 pmol/1) of diazepam also inhibited adrenocorticotrophin (ACTH)- and precursorstimulated steroidogenesis. This effect did not seem to be receptor-mediated ; instead of direct inhibition of microsomal hydroxylations by interaction with cyto¬ chrome P-4502i and cytochrome P-45017o was postula¬ ted (Holloway, Kenyon, Dowie et al. 1989). In addition, even the pharmacology of the PBRmediated effect on steroidogenesis is not straight¬ forward, since one benzodiazepine, flunitrazepam, has been found to inhibit tropic hormone stimulation of steroidogenesis in the Y-l adrenocortical and MA-10 Leydig cell lines (Papadopoulos, Nowzari & Krueger, 1991ft).
Diazepam-binding inhibitor/endozepine The second aspect of this story emerges from the fact that it is now recognized that there are endogenous ligands for the benzodiazepine receptor. In 1983, a 9 kDa polypeptide was isolated from rat brain and referred to as diazepam-binding inhibitor (DBI) to reflect its activity (Guidotti, Forchetti, Corda et al. 1983); later similar active substances were isolated from bovine and human brains and named endozepine (Shoyab, Gentry, Marquardt & Todaro, 1986). DBI also inhibits binding to the peripheral benzodia¬ zepine receptor, and is found in neurones of the cen¬ tral nervous system, in some specialized glial cells and in peripheral tissues, with particularly high concentra¬ tions in the adrenal cortex and testicular Leydig cells (Bovolin, Schlichting, Miyata et al. 1990; Rheaume, Tonon, Smih et al. 1990). Previously, a steroidogenic protein (termed '8-2 K') had been isolated from bovine adrenal cortex, this stimulated the delivery of cholesterol to the inner mitochondrial membrane and
synthesis was controlled by ACTH (Yanagibashi, Ohno, Kawamura & Hall, 1988). The sequence of this protein was found to be identical to that of bovine its
brain DBI, except that it lacked the last two residues at the C terminus (Besman, Yanagibashi, Lee et al. 1989). Recently, DBI itself and its two major process¬ ing products DBI 33-550 and DBI 17-50 have recently been found to stimulate pregnenolone formation in adrenocortical and testicular mitochondria (Papadopoulos, Berkovich, Krueger et al. 1991 ). It is interesting that DBI in the adrenal appears to be under the control of ACTH, the tissue content declines after hypophysectomy and increases with ACTH treatment (Massotti, Slobodyanski, Konkel et al. 1991).
Significance of DBI and the benzodiazepine receptors The final question which must be considered is whether DBI is the long sought 'rapidly turning-over protein' which mediates the response to ACTH in the adrenal cortex and luteinizing hormone (LH) in the testis and ovary. Although initially this seemed a strong possibility, there are a number of facts which argue against DBI having a unique role, of which probably the most telling is that the time-course of its
appearance after ACTH treatment does not correlate with steroidogenic response either in vitro or in vivo (Massotti et al. 1991; Brown, Hall, Shoyab & Papadopoulos, 1992). It may be simply that process¬ ing of the molecule is required, since there is some evidence that the smaller DBI-derived peptides are more active (Papadopoulos et al. 1991 ). However, in view of the known role of cyclic AMP-dependent protein kinases in ACTH and LH action, the involve¬ ment of a phosphorylated product in the stimulation of steroidogenesis seems likely (see Vinson, 1987). Recently, phosphoproteins with short half-lives have been isolated from adrenal and testicular mitochon¬ drial preparations following stimulation with trophic hormones (Epstein & Orme-Johnson, 1991 ; Stocco & Sodeman, 1991). At present, these would seem to be better candidates than DBI to be the specific intracel¬ lular mediators of ACTH or LH action. However, an alternative role for DBI in the nervous system appears possible with the recent demonstration that glial cells can metabolize cholesterol to yield steroids such as pregnenolone (and its sulphate), progesterone, 3ahydroxy-5a-pregnan-20-one, and 3a,21-dihydroxy-
5a-pregnan-20-one (Hu, Bourreau, Jung-Testas et al. 1987; Baulieu & Robel, 1990). The presence of DBI and benzodiazepine receptors within the same cells begs the question of whether DBI plays a role in the control of the synthesis of these steroids (Costa & Guidotti, 1991). This taken together with the know¬ ledge that these so-called 'neurosteroids', like the
benzodiazepines,
can act as modulators of GABAA receptor function (Majewska, Harrison, Schwartz et al. 1986) suggests that a series of totally unsuspected interactions between the nervous and endocrine sys¬ tems remains to be explored.
Krueger, . E. & Papadopoulos, V. (1990). Journal of Biological Chemistry 265, 15015-15022. Majewska, M. D., Harrison, N. L., Schwartz, R. D., Barker, J. L. & Paul, S. (1986). Science 232, 1004-1007. Massotti, M., Slobodyanski, E., Konkel, D., Costa, E. & Guidotti, A. (1991). Endocrinology 129, 591-596. Mukhin, ., Papadopoulos, V., Costa, E. & Krueger, . E. (1989). Proceedings of the National Academy of Sciences of the
REFERENCES
Papadopoulos, V., Berkovich, ., Krueger, K. E., Costa, E. & Guidotti, . (1991a). Endocrinology 129, 1481-1488. Papadopoulos, V., Mukhin, A. G, Costa, E. & Krueger, . E. (1990). Journal of Biological Chemistry 265, 3772-3779. Papadopoulos, V., Nowzari, F. B. & Krueger, . E. (19916). Journal of Biological Chemistry 266, 3682-3687. Rheaume, E., Tonon, M. C, Smih, F., Simard, J., Desy, L., Vaudry, H. & Pelletier, G. (1990). Endocrinology 111,
Amsterdam, A. & Suh, B. S. (1991). Endocrinology 129, 503-510. Anholt, R. R. H., Pedersen, P. L., De Souza, . B. & Snyder, S. H. (1986). Journal of Biological Chemistry 261, 576-583. Baulieu, E. E. & Robel, P. (1990). Journal of Steroid Biochemistry and Molecular Biology 37, 395-403. Besman, M. J., Yanagibashi, K., Lee, T. D., Kawamura, M., Hall, P. F. & Shively, J. E. (1989). Proceedings of the National Academy of Sciences of the U.S.A. 86, 4897-4901. Bovolin, P., Schlichting, J. L., Miyata, M., Ferrarese, C, Guidotti, A. & Alho, H. (1990). Regulatory Peptides 29, 267-281.
Braestrup, C. & Squires, R. F. (1977). Proceedings of the National Academy of Sciences of the U.S.A. 74, 3805-3809. Brown, A. S., Hall, P. F., Shoyab, M. & Papadopoulos, V. (1992). Molecular and Cellular Endocrinology 83, 1-9. Costa E. & Guidotti, A. (1979). Annual Review of Pharmacology and Toxicology 19, 531-545. Costa, E. & Guidotti, A. (1991). Life Sciences 49, 325-344. De Souza, E. B., Anholt, R. R. H., Murphy, K. M. M., Snyder, S. H. & Kuhar, M. J. (1985). Endocrinology 116, 567-573. Epstein, L. F. & Orme-Johnson, N. R. (1991). Journal of Biological Chemistry 266, 19739-19745. Guidotti, ., Forchetti, C. M., Corda, M. G, Konkel, D., Bennet, C. D. & Costa, E. (1983). Proceedings of the National Academy of Sciences of the U.S.A. 80, 3531-3533. Holloway, C. D., Kenyon, C. J., Dowie, L. J., Corrie, J. E. T., Gray, C. E. & Fraser, R. (1989). Journal of Steroid Biochemistry 33, 219-225. Hu, Z.-Y., Bourreau, E., Jung-Testas, I., Robel, P. & Baulieu, E.-E. (1987). Proceedings of the National Academy of Sciences of the U.S.A. 84, 8215-8219.
U.S.A.
86, 9813-9816.
1986-1994.
Shibata, H., Kojima,
I. & Ogata, E. (1986). Biochemical and Biophysical Research Communications 135, 994-999. Shoyab, M., Gentry, L. E., Marquardt, H. & Todaro, G. (1986). Journal of Biological Chemistry 261, 11968-11973. Stocco, D. M. & Sodeman, T. C. (1991). Journal of Biological Chemistry 166, 17931-17938. Vinson, G P. (1987). Journal of Endocrinology 114, 163-165. Yanagibashi, K„ Ohno, Y., Kawamura, M. & Hall, P. F. (1988). Endocrinology 123, 2075-2082. Yanagibashi, K., Olino, Y., Nakamichi, N., Matsui, T., Hayashida, K., Takmura, M., Yamada, K., Tou, S. & Kawamura, M. (1989). Journal of Biochemistry 106, 1026-1029.
Physiology Group,
Biomedicai Sciences Division,
King's College London, Kensington Campus, London W8 U.K.
7AH,
