Pgrmc1 increases plasma membrane-associated EGFR levels. In panels A–E, the cell surface proteins of A549/con and A549/RNAi cells were biotin-labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified using avidin-agarose columns. Lanes 1 and 2 of panels A and B are Western blots of the proteins that failed to bind to the avidin-agarose columns (unbtn), whereas lanes 3 and 4 are Western blots of the avidin-bound proteins (biotin). The Western blots were probed for EGFR (A), PCNA (B), E-cadherin (C), and CXCR4/fusin (D). In panel E, one-tenth of the total pool of avidin-bound proteins was separated by SDS-PAGE and stained with Coomassie Blue. The 7.4-fold decrease in EGFR at the plasma membrane was repeated in triplicate biotinylation reactions and was statistically significant (p = 0.01). In panels F and G, Pgrmc1 expression was inhibited by siRNA transfection, and the cells were labeled and analyzed similarly to panels A–D for EGFR (F) and E-cadherin (G). In H and I, EGFR was stained by immunofluorescence (IF) in A549/con and A549/RNAi cells, respectively, indicating an increased intracellular pool of the receptor.

EGFR and Pgrmc1 co-fractionate in a high density vesicle fraction. A549 cells were lysed and fractionated on an Opti-prep density gradient then analyzed by Western blot for EGFR (A), Pgrmc1 (B), calnexin (C, a marker for the endoplasmic reticulum), Rab5 (D, a marker for secretory vesicles), CD9 (E, a plasma membrane protein), and caveolin (lower panel). The results suggest that Pgrmc1 and EGFR co-fractionate primarily in intracellular microsomes.

Pgrmc1 increases EGFR levels in MDA-MB-468 breast cancer cells. Panel A is a diagram indicating the positions of the siRNA molecules targeting Pgrmc1. Exons 1, 2, and 3 are indicated by ex. MDA-MB-468 cells were transfected with a control siRNA (siCON, lanes 1 and 3) or two separate siRNAs targeting Pgrmc1 (siPGR and siPGR2, lanes 2 and 4, respectively). In panels B–E, protein levels were analyzed by Western blot for EGFR (B), phosphotyrosine (C), Pgrmc1 (D), and ku70 (E) as a control for protein loading. Panels F–H show Western blot analyses for insulin-like growth factor 1 receptor (IGF-1R; F), c-Kit (G), and transferrin receptor (tfR) (H). I, MDA-MB-468 cells transfected with siCON (solid line) or siPGR (dashed line) were incubated with increasing doses of the EGFR inhibitor AG1478, and viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. % viability refers to absorbance after treatment relative to untreated control cells. In panels J-K, MDA-MB-468 cells were treated with increasing doses of the Pgrmc1 ligand AG-205. Protein expression was analyzed by Western blot for EGFR (J) and ku70 (K). Cells were treated with 0 (lanes 1), 2 (lanes 2), 10 (lanes 3), or 50 (lanes 4) μm AG-205 for 24 h in serum-free medium. In panels L–M, A549 cells were treated with the same doses and analyzed for EGFR (L) and ku70 (M). The results show that the Pgrmc1 ligand AG-205 decreases EGFR protein levels. Expression analyses were repeated at least in duplicate throughout.

Disrupting Pgrmc1 function suppresses sensitivity to EGFR inhibitors. A, A549/con (solid line) or A549/RNAi (dashed line) cells were maintained in media lacking serum and treated with 2.5–10 μm EGFR inhibitor AG1478 for 96 h. Percent viability was determined by cell counting, and for all of the panels % viability refers to the cell density relative to untreated cells. B, AG1478 susceptibility in Ad-LacZ and Ad-Pgr-hbd-infected A549 cells, were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay 4 days after infection in serum-free media containing increasing doses of AG1478. Solid lines represent cells infected with the control Ad-LacZ, whereas Ad-Pgr-hbd-infected cells are indicated by a dashed line. C, A549 cells were treated with vehicle (solid line) or 10 μm AG-205 (dashed line) plus increasing doses of erlotinib and counted. D, MDA-MB-231 breast cancer cells were treated with AG-205 and erlotinib as described in panel C. Each of the experiments is representative of experiments performed at least in triplicate. The results indicate that increases in proliferation in Pgrmc1-expressing cells are reversed by EGFR inhibitors.

EGFR and Pgrmc1 co-precipitate and co-localize. A, EGFR was precipitated with the antibody (Ab) IMC-C225 from serum-starved A549 cells and probed for EGFR (top panel) or Pgrmc1 (second panel). Lane 1 is a control precipitation with an irrelevant antibody. For the inverse experiment, Pgrmc1 was precipitated from A549 cells with pre-immune serum (PIS, lane 3) or an anti-Pgrmc1 antibody (α-Pgr, lane 4). EGFR was detected in the latter reaction (upper panel, lane 4). WB, Western blot. B, Pgrmc1 was precipitated from A549 cells before (lane 1) and after (lane 2) stimulation with 50 ng/ml EGF for 15 min, and precipitates were probed for Pgrmc1 (upper panel) or EGFR (lower panel). C, EGFR was immunoprecipitated (IP) from serum-starved A549/con (lane 1) or A549/RNAi (lane 2) cells. Immunoprecipitation reactions were probed for EGFR (top) or Pgrmc1 (bottom). D, EGFR was immunoprecipitated with IMC-C225 from MDA-MB-231 breast cancer cells, and precipitation reactions were analyzed by Western blot for EGFR (upper panel) or Pgrmc1 (lower panel). E, the upper panels show fluorescence of Pgrmc1- green fluorescent protein expressed in A549 cells and immunofluorescence (IF) for EGFR, which was detected with a rhodamine-labeled secondary antibody. The lower panel shows a merged image, indicating that Pgrmc1 and EGFR co-localize to an intracellular region adjacent to the nuclear membrane. The bar indicates 25 μm.

Pgrmc1 localizes to the microsomal lumen. A, shown is a diagram of the protease digestion experiments used in panels B–E. An isolated microsomal fraction was incubated with proteinase K (dark gray circles) in the absence (left) and presence (right) of detergents to distinguish proteins that were protected by the microsomal membranes from cytoplasmic proteins. B–E, Western blots of microsomal fractions incubated without protease (lanes 1 and 6) or with 0.01 (lanes 2 and 7), 0.1 (lanes 3 and 8), 1 (lanes 4 and 9), or 5 μg/ml (lanes 5 and 10) proteinase K in the absence (lanes 1–5) or presence (lanes 6–10) of 1% Triton X-100 are shown. Blots were probed for Pgrmc1 (B), calnexin (cytoplasmic epitope (C) and lumen epitope (D)) and protein disulfide isomerase (PDI) (E, which localizes to the endoplasmic reticulum lumen). The results indicate that Pgrmc1 is protected from digestion by the microsomal membrane (compare lanes 3 and 8) but localizes to microsomes that are sensitive to high levels of proteinase K.