The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells

ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

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Tetrapeptides Modelled to the Androgen Binding Site of ZIP9 Stimulate Expression of Tight Junction Proteins and Tight Junction Formation in Sertoli Cells

Biology ◽

10.3390/biology11010055 ◽

2021 ◽

Vol 11 (1) ◽

pp. 55

Author(s):

Marie-Louise Möller ◽

Ahmed Bulldan ◽

Georgios Scheiner-Bobis

Keyword(s):

Androgen Receptor ◽

Tight Junction ◽

Binding Site ◽

Sertoli Cells ◽

Alternative Pathway ◽

Tight Junction Formation ◽

Associated Proteins ◽

A Cell ◽

Androgen Binding

Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation.

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Differences in the steroid-binding site specificities of rat prostate androgen receptor and epididymal androgen-binding protein (ABP)

Journal of Steroid Biochemistry ◽

10.1016/0022-4731(79)90209-7 ◽

1979 ◽

Vol 10 (5) ◽

pp. 487-497 ◽

Cited By ~ 32

Author(s):

Josef Kirchhoff ◽

Monique Soffie ◽

Guy G. Rousseau

Keyword(s):

Androgen Receptor ◽

Binding Site ◽

Binding Protein ◽

Androgen Binding Protein ◽

Steroid Binding ◽

Rat Prostate ◽

Androgen Binding

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Differences in the steroid-binding site specificities of rat prostate androgen receptor and epididymal androgen binding protein (ABP)

Journal of Steroid Biochemistry ◽

10.1016/0022-4731(80)90125-9 ◽

1980 ◽

Vol 13 (1) ◽

pp. vi

Keyword(s):

Androgen Receptor ◽

Binding Site ◽

Binding Protein ◽

Androgen Binding Protein ◽

Steroid Binding ◽

Rat Prostate ◽

Androgen Binding

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Steroid hormone receptors and the sexual phenotype of the Harderian gland in hamsters

Journal of Endocrinology ◽

10.1677/joe.0.1210149 ◽

1989 ◽

Vol 121 (1) ◽

pp. 149-156 ◽

Cited By ~ 39

Author(s):

F. Vilchis ◽

G. Pérez-Palacios

Keyword(s):

Androgen Receptor ◽

Binding Site ◽

Hormone Receptors ◽

Steroid Hormone ◽

Sedimentation Coefficient ◽

Harderian Gland ◽

Steroid Hormone Receptors ◽

Progestin Receptors ◽

Female Gland ◽

Androgen Binding

ABSTRACT To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The in-vitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [3H]7α,17α-dimethyl-17β-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (Kd) of 0·7 nmol/l and maximal saturation binding capacity of 84·0 ± 3·0 (s.d.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5α-dihydrotestosterone, testosterone and 3α-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17β, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8–9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species. Journal of Endocrinology (1989) 121, 149–156

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Acute effect of PMSG on ovarian androgen-binding sites in the intact immature female rat

Reproduction Fertility and Development ◽

10.1071/rd9920055 ◽

1992 ◽

Vol 4 (1) ◽

pp. 55 ◽

Cited By ~ 4

Author(s):

S Campo ◽

RS Carson ◽

JK Findlay

Keyword(s):

Androgen Receptor ◽

Binding Site ◽

Binding Sites ◽

Nuclear Dna ◽

Acute Effect ◽

Binding Activity ◽

Female Rat ◽

Preovulatory Follicles ◽

Ovulatory Follicles ◽

Androgen Binding

Androgen-binding activity in ovaries containing only immature follicles is compared with that in ovaries stimulated with pregnant mare serum gonadotrophin to induce development of large preovulatory follicles. The androgen-binding sites present in cytosols prepared from unstimulated ovaries exhibited kinetics, affinity and a range of specificities for natural and synthetic androgens consistent with those exhibited by the androgen receptor isolated from androgen-sensitive tissues. Studies of sedimentation and DNA binding suggest that the receptor-like androgen-binding site present in unstimulated ovaries exists as the 'non-activated' form which is able to undergo transformation and bind to nuclear DNA. Properties of the androgen-binding sites isolated from ovaries stimulated with gonadotrophin were very different from those of the androgen receptor present in unstimulated ovaries. Specificity for androgens was reduced, capacity for dihydrotestosterone (DHT) and methyltrienolone (R1881) increased and androgen-binding activity was associated exclusively with the 4.5S form which was not able to bind to nuclear DNA. These data confirm that a shift from a receptor-like to a nonfunctional androgen-binding site is associated with the development of ovulatory follicles and suggest that shifts in androgen-binding populations will determine the response of developing ovarian follicles to androgens.

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Identification of the Deoxyribonucleic Acid-Binding Site of a Regulatory Protein Involved in Prostate-Specific and Androgen Receptor-Dependent Gene Expression1

Endocrinology ◽

10.1210/endo.140.5.6696 ◽

1999 ◽

Vol 140 (5) ◽

pp. 2063-2070 ◽

Cited By ~ 12

Author(s):

Lila Patrikainen ◽

Jingdong Shan ◽

Katja Porvari ◽

Pirkko Vihko

Keyword(s):

Androgen Receptor ◽

Binding Site ◽

Deoxyribonucleic Acid ◽

Regulatory Protein

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Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer

Cancers ◽

10.3390/cancers13143488 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3488

Author(s):

Fuqiang Ban ◽

Eric Leblanc ◽

Ayse Derya Cavga ◽

Chia-Chi Flora Huang ◽

Mark R. Flory ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Splice Variants ◽

Full Length ◽

Cancer Resistance ◽

Castration Resistant Prostate Cancer ◽

Terminal Domain ◽

Castration Resistant ◽

Coregulatory Proteins ◽

Androgen Binding

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

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Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

Journal of Endocrinology ◽

10.1677/joe.0.1080267 ◽

1986 ◽

Vol 108 (2) ◽

pp. 267-273 ◽

Cited By ~ 11

Author(s):

S. Kyakumoto ◽

R. Kurokawa ◽

Y. Ohara-Nemoto ◽

M. Ota

Keyword(s):

Androgen Receptor ◽

Nuclear Receptors ◽

Binding Sites ◽

Androgen Receptors ◽

Dissociation Constants ◽

Scatchard Analysis ◽

Male And Female ◽

Short Period ◽

Almost All ◽

Androgen Binding

ABSTRACT Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 °C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5′-phosphate (5 mmol/l) from nuclear fractions with 93–95% efficiency. The exchange of the bound steroids occurred by 24–48 h at 0 °C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0·8 and 0·9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females. J. Endocr. (1986) 108, 267–273

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