Characterization of high affinity progesterone-binding membrane proteins by an anti-peptide antiserum

doi:10.1016/S0039-128X(97)00143-8

Steroids

Volume 63, Issue 2, February 1998, Pages 111-116

https://doi.org/10.1016/S0039-128X(97)00143-8 Get rights and content

Abstract

A chemically synthesized 15-mer oligopeptide derived from the N terminus of high affinity progesterone-binding membrane site(s) from porcine liver was used to generate site-specific antibodies. Western blotting experiments confirmed the specificity of the anti-peptide serum obtained. In further investigations this antiserum was used for the identification of the native progesterone-binding membrane protein complex that represents an oligomer with an apparent molecular mass of about 200 kDa. In temperature-induced Triton X-114 phase separation experiments combined with Western-blotting, the progesterone-binding site was identified as an hydrophobic (integral) membrane protein. In addition, in Western blotting analyses the antiserum reacted with the progesterone-binding or related proteins in membrane fractions from a wide array of different tissues in various species.

References (29)

E Baldi et al.
Nongenomic actions of progesterone on human spermatozoa
Trends Endocrinol Metab
(1995)
PF Blackmore et al.
Novel stimulators of calcium influx in human sperm
J Biol Chem
(1990)
PF Blackmore
Rapid non-genomic action of progesterone stimulate Ca²⁺ influx and the acrosome reaction in human sperm
Mol Signal
(1993)
KO Turner et al.
Progesterone-mediated efflux of cytosolic chloride during the human sperm acrosome reaction
Biochem Biophys Res Commun
(1995)
S Waldegger et al.
Electrophysiological effects of progesterone on hepatocytes
Biochim Biophys Acta
(1995)
M Wehling et al.
Aldosterone-specific membrane receptors and rapid non-genomic action of mineralocorticoids
Mol Cell Endocrinol
(1992)
M Christ et al.
Non-classical receptors for aldosterone in plasma membranes from pig kidneys
Mol Cell Endocrinol
(1994)
M Yamada et al.
Binding of sex hormones by male rat liver microsomes
J Steroid Biochem
(1982)
E Falkenstein et al.
Full-length cDNA sequence of a progesterone membrane-binding protein from porcine vascular smooth muscle cells
Biochem Biophys Res Commun
(1996)
MM Bradford
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)

C Bordier

Phase separation of integral membrane proteins in Triton X-114 solution

J Biol Chem

(1981)

A Mithöfer et al.

One-step purification of the β-glucan elicitor-binding protein from soybean (Glycine max L.) roots and characterization of an anti-peptide antiserum

FEBS Lett

(1996)

I Nemere et al.

Nontranscriptional effects of steroid hormones

Receptor

(1993)

M Wehling

Looking beyond the dogma of genomic steroid action: insights and facts of the 1990's

J Mol Med

(1995)

Cited by (48)

Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity
2023, Cell Reports
Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.
Loss of β-Arrestins or six Gα proteins in HEK293 cells caused Warburg effect and prevented progesterone-induced rapid proteasomal degradation of progesterone receptor membrane component 1
2021, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
In contrast to the mPRs and nuclear PGR, the PGRMC family consists of structurally similar PGRMC1 and PGRMC2 proteins [15], both characterized as single‐pass transmembrane receptors (SPTMRs) containing a short N terminus, a single-pass transmembrane domain, and a C terminus haem-binding domain [16–19]. Analysis of the radioactive P4 binding to PGRMC1 demonstrated that P4 specifically binds to PGRMC1 preferably at a site located within a segment that is composed of the transmembrane domain and the initial segment of the PGRMC1 C-terminus [20–22]. Nonetheless, there are conflicting reports suggesting P4 may or may not be the natural ligand for PGRMC1 [19,23].
Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.
New insights into progesterone actions on prolactin secretion and prolactinoma development
2019, Steroids
Citation Excerpt :
mPRδ and mPRε, on the other hand, show greatest expression in different regions of the human brain [58]. Progesterone receptor membrane component 1 (PGRMC1), which belongs to the b5-like heme/steroid-binding protein family, is another player in rapid P4 signaling [66–70]. PGRMC1 is a small, single transmembrane protein predominantly located in intracellular membranes, expressed in many different tissues [70].
Progesterone (P4) has controversial physiological effects on the regulation of the lactotroph population. While some studies have shown a negative role for P4 in prolactin secretion and lactotroph proliferation, antagonizing estradiol effects, others demonstrated a proliferative role of P4 at the pituitary level. Usually, progesterone actions in the pituitary gland were studied through their classical, genomic pathways triggered by nuclear progesterone receptors (nPRs). However, in 2003, the scene became more complex with the discovery of another group of progesterone receptors involved in rapid, non-genomic P4 effects: the membrane progesterone receptors (mPRs), which are members of the progesterone and adipoQ receptor (PAQR) family. This review examines the historical background and current data on the study of progesterone actions on PRL secretion providing new evidence of P4 effects at the hypothalamic and at the pituitary level through non-classic P4-receptors. In addition, we explore the role of progesterone in the development of experimental prolactinomas, a controversial topic in the literature.
Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary
2014, Theriogenology
The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.
Evidence for a genomic mechanism of action for progesterone receptor membrane component-1
2012, Steroids
Citation Excerpt :
Interestingly, PGRMC1 not only is detected within the plasma and endoplasmic reticular membranes but also within the nucleus [7,9]. Surprisingly, Western blot analysis not only detects PGRMC1 as the predicted ≈27 kDa band but also as higher molecular weight bands [10]. Although it is generally assumed that the higher molecular weight forms are oligomers of the ≈27 kDa form [11], this concept has not be rigorously tested.
Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the granulosa and luteal cells of rodent and primate ovaries. Interestingly, its molecular weight as assessed by Western blot is dependent on its cellular localization with a ≈27 kDa form being detected in the cytoplasm and higher molecular weight forms being detected in the nucleus. The higher molecular weight forms of PGRMC1 are sumoylated suggesting that they are involved in regulating gene transcription, since sumoylation of nuclear proteins often is associated with regulation of transcriptional activity of the sumoylated protein.
In order to identify a set of candidate genes that are regulated by PGRMC1, a human granulosa/luteal cell line (hGL5 cells) was treated with PGRMC1 siRNA and changes in gene expression monitored by microarray analysis. The microarray analysis revealed that PGRMC1 generally functioned as a repressor of transcription, since depletion of PGRMC1 resulted in a disproportionate increase in the number of transcripts. Moreover, a pathway analysis implicated PGRMC1 in the regulation of apoptosis, which is consistent with PGRMC1’s known biological action. More importantly these results support the concept that PGRMC1 influences gene transcription. Additional studies reveal that progesterone (P4) acting through a PGRMC1-dependent mechanism suppresses the activity of the transcription factor, Tcf/Lef, thereby identifying one molecular pathway through which P4–PGRMC1 can regulate gene transcription and ultimately apoptosis.
Progesterone receptor membrane component 1-Many tasks for a versatile protein
2008, Steroids
The protein now called Progesterone Receptor Membrane Component 1 (PGRMC1) has been described independently by many groups in different cellular contexts. As a result it has been given an impressive diversity of names. While this protein was initially described on the basis of a singular property, e.g. expression or steroid binding, its possible physiological roles have only recently been reported. Current evidence supports the perception that PGRMC1 may be involved in sterol metabolism or homeostasis and cell survival. Here, we summarize a few sometimes neglected pieces of evidence from the literature along with unpublished findings on the properties and functions of PGRMC1.

View all citing articles on Scopus

View full text

PaperCharacterization of high affinity progesterone-binding membrane proteins by an anti-peptide antiserum

Abstract

Trends Endocrinol Metab

J Biol Chem

Mol Signal

Biochem Biophys Res Commun

Biochim Biophys Acta

Mol Cell Endocrinol

Mol Cell Endocrinol

J Steroid Biochem

Biochem Biophys Res Commun

Anal Biochem

J Biol Chem

FEBS Lett

Nontranscriptional effects of steroid hormones

Receptor

Looking beyond the dogma of genomic steroid action: insights and facts of the 1990's

J Mol Med

Paper
Characterization of high affinity progesterone-binding membrane proteins by an anti-peptide antiserum