Progesterone receptor membrane component-1 (PGRMC1) is the mediator of progesterone's antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic acid treatment and functional analysis of PGRMC1 mutations

Abstract

Progesterone (P4) receptor membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1) are thought to form a complex that functions as membrane receptor for P4. The present investigations confirm PGRMC1's role in this membrane receptor complex by demonstrating that depleting PGMRC1 with PGRMC1 small interfering RNA results in a 60% decline in [(3)H]P4 binding and the loss of P4's antiapoptotic action. Studies conducted on partially purified GFP-PGRMC1 fusion protein indicate that [(3)H]P4 specifically binds to PGRMC1 at a single site with an apparent K(d) of about 35 nm. In addition, experiments using various deletion mutations reveal that the entire PGRMC1 molecule is required for maximal [(3)H]P4 binding and P4 responsiveness. Analysis of the binding data also suggests that the P4 binding site is within a segment of PGRMC1 that is composed of the transmembrane domain and the initial segment of the C terminus. Interestingly, PAIRBP1 appears to bind to the C terminus between amino acids 70-130, which is distal to the putative P4 binding site. Taken together, these data provide compelling evidence that PGRMC1 is the P4 binding protein that mediates P4's antiapoptotic action. Moreover, the deletion mutation studies indicate that each domain of PGRMC1 plays an essential role in modulating PGRMC1's capacity to both bind and respond to P4. Additional studies are required to more precisely delineate the role of each PGRMC1 domain in transducing P4's antiapoptotic action.

Figure 1

The effect of scramble (A) and GAPDH siRNA (B) on GAPDH expression and the effect of scramble (C) or one of three different PGRMC1 (D–F) siRNAs on the expression of PGRMC1. Both GAPDH and PGRMC1 were assessed by immunofluorescence staining.

Figure 2

A, The effect of either scramble or PGRMC1 siRNA treatment on the expression of PGRMC1 (green) and PAIRBP1 (red) in SIGCs as assessed by immunofluorescence staining; B, quantitative estimate of immunofluorescent-stained PGRMC1 and PAIRBP1. Scramble control values were set to 100%. Values are means ± 1 se. *, Values significantly different from scramble control value (P < 0.05).

Figure 3

The effect of either scramble or PGRMC1 siRNA treatment on SIGC function. A, Effect of these siRNA treatments on P4’s ability to prevent apoptosis; B, effect of increasing [³H]P4 on specific [³H]P4 binding to intact SIGCs. The dashed line in B represents the best fit as described by a polynomial equation (r = 0.91). Note that maximal specific [³H]P4 binding is achieved with the addition of 1 × 10⁶ dpm [³H]P4 (i.e. 4.7 pmol). C, Effect of either scramble or PGRMC1 siRNA treatment on the capacity of intact SIGCs to specifically bind [³H]P4 when [³H]P4 is added at a saturating dose (1 × 10⁶ dpm). Values in A and C are means ± 1 se. *, Value significantly different from the appropriate control value (P < 0.05).

Figure 4

GFP-PGRMC1 expression in SIGCs. Overexpression of GFP-PGRMC1 increases the amount of a 56-kDa band that is detected by both the PGRMC1-NT antibody and the antibody to GFP (left). Note that this results in a dramatic increase in PGRMC1 levels compared with endogenous levels of PGRMC1 (faint 28-kDa band in upper left panel). The negative controls did not show any bands. Transfection of GFP-PGRMC1 results in 30–40% of the cells being transfected as judged by GFP fluorescence (right).

Figure 5

Isolation of GFP fusion proteins using the anti-GFP μMACS beads. SIGCs were transfected with either empty vector (pEGFP-N1) or GFP-PGRMC1. Twenty-four hours after transfection, cells were lysed and GFP-fusion proteins isolated as described in Materials and Methods. The GFP isolates were run on a gel and stained with either Coomassie Blue (A) or analyzed for the presence of GFP-fusion proteins by Western blot (B). Coomassie Blue-stained bands at 25, 48, and 62 kDa were detected among the eluted proteins isolated from cells transfected with empty vector and GFP-PGRMC1. However, a 28- and 56-kDa band was among the eluted proteins from empty vector and GFP-PGRMC1, respectively. These bands were also detected by Western blot using the GFP antibody (B). The negative control for the GFP Western blot did not show any bands. C, Total binding, nonspecific binding, and specific binding of [³H]P4 to partially purified GFP-proteins.

Figure 6

Ligand binding analysis of [³H]P4 to partially purified GFP-PGRMC1 fusion protein. Specific [³H]P4 binding decreased with the addition of nonradioactive P4. Hill plot analysis (inset) yielded a straight line with a slope of 1.08, indicting that [³H]P4 specifically bound to GFP-PGRMC1 in a competitive and reversible manner.

Figure 7

GFP-PGRMC1 mutants. A, Diagram that identifies each domain of PGRMC1, with numbers at the top representing amino acid numbers; B, Western blot probed with the GFP antibody that reveals that after transfection with the different GFP-PGRMC1 mutants, the appropriate-sized GFP-fusion protein is detected. The negative control for the GFP Western blot did not show any bands. Each GFP-PGRMC1 mutant was highly expressed and possessed the same cellular localization as revealed by GFP fluorescence (C).

Figure 8

A, Capacity of the various partially purified GFP-PGRMC1 fusion proteins to specifically binding [³H]P4. The numbers associated with each GFP-PGRMC1 fusion protein represent the amino acids that are encoded by the vector. B, Effect of transfecting the various GFP-PGRMC1 mutants on their ability to transduce P4’s antiapoptotic action in intact SIGCs. In both A and B, values are expressed as means ± 1 se. *, Value that is significantly different from either the wild-type (1–194) vector (A) or the empty vector (B).

Figure 9

A, PAIRBP1-PGRMC1 interaction as detected in the GFP pull-down assay. In this assay, SIGCs were transfected with GFP-PGRMC1 deletion mutants as well as the GFP-PGRMC1 D120G point mutation. GFP-PGRMC1 fusion proteins were isolated and Western blot performed using antibodies to GFP and PAIRBP1. Note that the GFP Western blot demonstrated that approximately the same amount of each GFP-PGRMC1 fusion protein was isolated for each vector. However, PAIRBP1 Western blot detected PAIRBP1 with all the mutants except the 1–70 deletion PGRMC1 mutant. B, Western blot analysis revealed that similar amounts of PAIRBP1 were present within all the lysates before the isolation of GFP-fusion proteins. The negative controls for the GFP and PAIRBP1 Western blots did not show any bands.

Figure 10

The expression, localization, and function of the GFP-PGRMC1 point mutant D120G. SIGCs were transfected with either GFP-PGRMC1 or GFP-PGRMC1-D120G. After 24 h, approximately the same amount of GFP-PGRMC1 fusion protein was detected in lysates from each treatment as revealed by Western blots using antibodies to either PGRMC1 or GFP. The negative controls for the PGRMC1 and GFP Western blots did not show any bands. The GFP-PGRMC1-D120G mutant has the same transfection efficiency and cellular localization as the other GFP-PGRMC1 fusion proteins (compare B with Fig. 4). The partially purified D120G point mutation has a reduced capacity to bind (C), and cells transfected with this mutant did not respond to a suboptimal concentration of P4 (1 nm) (D). Values are presented as means ± 1 se. *, Value that is significantly different from the appropriate control (P < 0.05).