Characteristics of membrane progestin receptor alpha (mPRalpha) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions - PubMed (original) (raw)
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Characteristics of membrane progestin receptor alpha (mPRalpha) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions
Peter Thomas. Front Neuroendocrinol. 2008 May.
Abstract
Rapid, progestin actions initiated at the cell surface that are often nongenomic have been described in a variety of reproductive tissues, but until recently the identities of the membrane receptors mediating these nonclassical progestins actions remained unclear. Evidence has been obtained in the last 4-5 years for the involvement of two types of novel membrane proteins unrelated to nuclear steroid receptors, progesterone membrane receptors (mPRs) and progesterone receptor membrane component 1 (PGMRC1), in progestin signaling in several vertebrate reproductive tissues and in the brain. The mPRs, (M(W) approximately 40 kDa) initially discovered in fish ovaries, comprise at least three subtypes, alpha, beta and gamma and belong to the seven-transmembrane progesterone adiponectin Q receptor (PAQR) family. Both recombinant and wildtype mPRs display high affinity (K(d) approximately 5 nM), limited capacity, displaceable and specific progesterone binding. The mPRs are directly coupled to G proteins and typically activate pertussis-sensitive inhibitory G proteins (G(i)), to down-regulate adenylyl cyclase activity. Recent studies suggest the alpha subtype (mPRalpha) has important physiological functions in variety of reproductive tissues. The mPRalpha is an intermediary in progestin induction of oocyte maturation and stimulation of sperm hypermotility in fish. In mammals, the mPRalphas have been implicated in progesterone regulation of uterine function in humans and GnRH secretion in rodents. The single-transmembrane protein PGMRC1 (M(W) 26-28 kDa) was first purified from porcine livers and its cDNA was subsequently cloned from porcine smooth muscle cells and a variety of other tissues by different investigators. PGMRC1 and the closely-related PGMRC2 belong to the membrane-associated progesterone receptor (MAPR) family. The PGMRC1 protein displays moderately high binding affinity for progesterone which is 2- to 10-fold greater than that for testosterone and glucocorticoids, and also can bind to other molecules such as heme, cholesterol metabolites and proteins. The signal transduction pathways induced by binding of progesterone to PGMRC1 have not been described to date, although motifs for tyrosine kinase, kinase binding, SH2 and SH3 have been predicted from the amino acid sequence. Evidence has been obtained that PGMRC1 mediates the antiapoptotic affects of progesterone in rat granulosa cells. The PGMRC1 protein may also be an intermediary in the progesterone induction of the acrosome reaction in mammalian sperm. Despite these recent advances, many aspects of progestin signaling through these two families of novel membrane proteins remain unresolved. Biochemical characterization of the receptors has been hampered by rapid degradation of the partially purified proteins. A major technical challenge has been to express sufficient amounts of the recombinant receptors on the plasma membranes in eukaryotic systems to permit investigations of their progestin binding and signal transduction characteristics. Additional basic information on the molecular and cellular mechanisms by which mPRs and PGMRC1 interact with progestins, signal transductions pathways and other proteins will be required to establish a comprehensive model of nontraditional progestin actions mediated through these novel proteins.
Figures
Figure 1
DEAE chromatography of solubilized seatrout ovarian membranes with step wise elution of protein with NaCl. Three fractions (I,II, III) were collected and PAGE was performed on aliquots from each fraction and silver stained. Reproduced from Zhu et al. [150], with permission.
Figure 2
Competition curves of steroid binding to plasma membranes of MDA-MB-231 (PR-) cells stably transfected with (A) human mPRα, (B) seatrout mPRα, (C) zebrafish mPRα, expressed as a percentage of maximum [3 H]progestin binding (A: [3 H]P4; B:[3 H]20β-S; C: [3 H]17α,20β-P). P4, progesterone; 20β-S, 17,20β,21-trihydroxy-4-pregnen-3-one; 17α,20β-P, 17,20β-dihydroxy-4-pregnen-3-one; RU486, mifepristone; R5020, promegestone; cort, cortisol; E2, estradiol-17β; T, testosterone. (A and B reproduced from Thomas et al., 2007 [122], C reproduced from Hanna et al., 2005 [37] with permission).
Figure 3
Co-immunoprecipitation of mPRα coupled to G protein α subunits in spotted seatrout oocyte membranes with specific inhibitory G protein (Gi 1,2, Go) antibodies followed by immunodetection of seatrout mPRα by Western blot analysis. Membranes (10 μg) were incubated for 45 min. with vehicle or 290 nM 20 β-S, solubilized and then incubated overnight with 5 μl of Gαi1,2 or Gαo antibody or nonimmune rabbit serum (niR) and 50 μl of Protein A/G Plus-agarose beads. Immunoprecipitated seatrout mPRα protein was detected using the monoclonal PR10-1 antibody described in Zhu et al. (2003). Representative immunoblot is shown. There was a strong and specific association of Gαi1,2 with seatrout mPRα, whereas the mPRα protein was not detected in the Gαo or nonimmune rabbit serum immunoprecipitated samples. The amount of seatrout mPRα associated with Gαi1,2 was decreased after treatment with 20β-S (reproduced from Pace, 2005 [81] with permission).
Figure 4
Down regulation of coactivator SRC2 mRNA expression in human pregnant myometrial cells treated with 100 nM progesterone-bovine serum albumen conjugate (P4-BSA) for 3hrs. * P<0.05 compared to no supplement (NS) controls, N=4. SRC2 protein expression (insert) was also decreased after 16hr treatment (reproduced from Karteris et al., 2006, with permission). CBP: cAMP response element binding protein coactivator. SRC: steroid receptor (PR) coactivator (reproduced from Karteris et al., 2006 [41] with permission).
Figure 5
Summary of the signal transduction pathways activated by progestins through mPRα (1-8) and PGMRC1 (8). mPRα pathways 1-4, 6,7 are mediated through activation of inhibitory G proteins (Gi), mPRα pathway 5 is mediated though an olfactory G protein (G olf). Pathway 1. Activation of Erk (Erk1, Erk2): Atlantic croaker oocytes during final maturation, probably through mPRα [82] and in MDA-MB-231 cells transfected with seatrout mPRα [150] and zebrafish mPRα and mPRβ [37]. Pathway 2: Activation of mitogen activated kinase p38: PTX-sensitive, via the mPRs in human myocytes resulting in phosphorylation of myosin light chain [41]. Pathway 3: Akt activation: Atlantic croaker oocytes during final maturation, probably through mPRα, resulting in upregulation of phosphodiesterase activity [82]. Pathway 4: Decrease cAMP levels: PTX-sensitive, in spotted seatrout oocytes during final maturation through mPRα [83, Fig.2], in MDA-MB-231 cells transfected with seatrout mPRα or human mPRα [122, 150], and in human myocytes via mPRα and mPRβ [41], in Jurkat cells via mPRα [21]. Pathway 5: Increase cAMP levels: in Atlantic croaker sperm through mPRα [135]. Pathway 6: Potentiation of PR-B transactivation: through mPRα- and mPRβ-mediated pathways in human myocytes [41]. Pathway 7: Down-regulation SRC2 coactivator expression: through mPRα in human myocytes [41]. Pathway 8: Increase intracellular free calcium concentrations: from internal calcium stores in CHO cells transfected with ovine mPRα [1] and in CHO cells transfected with PGMRC1 [26]. PM: plasma membrane, NE: nuclear envelope, ER: endoplasmic reticulum.
Figure 6
Detection of mPRα and PGMRC1 mRNAs in various cell lines after 35 cycles of RT-PCR. MDA: MDA-MB-231, SKBR: SKBR3, MYO: human myometrial cells after several passages, HEK: HEK293, RT-: lacking reverse transcriptase for actin controls, showing no DNA contamination.
Figure 7
Selection of membrane progestin receptor-transfected cells for receptor characterization and signal transduction studies. Binding of [3 H]-P4 to plasma membranes of MDA-MB-231 cells stably transfected with human mPRα. A and B: Binding to ten subclones (a1-a10), obtained by limiting dilution, was assessed after three weeks selection. High [3 H]-P4 binding was associated with greater mPRα protein expression in the membrane fractions (insert) as assessed by Western blot analysis. C: [3 H]-P4 binding increased upon further selection for two weeks of the clones which had the highest binding in A&B (a1,a3,a5,a10). The receptor binding assay was conducted as described in Thomas et al., 2007. * P<0.05 compared to untransfected control cells (231-ve), Student's paired t test, N=4.
Figure 8
Loss of [3 H]-P4 binding to plasma membranes prepared from MDA-MB-231 cells stably transfected with human mPRα during incubation at room temperature for 1hr (RT-1hr) or in the absence of protease inhibitors (-prot.inhib). The receptor binding assay was conducted as described in Thomas et al., 2007 [122] with modifications from the normal 30 min incubation procedure at 4C in the presence of protease inhibitors as indicated. Significant differences identified with a multiple range test, Fisher PLSD, are indicated with different letters (P<0.05), N=4.
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