Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2018/04/19/mol.117.110510.DC1 1521-0111/94/1/665–673$35.00 https://doi.org/10.1124/mol.117.110510 MOLECULAR PHARMACOLOGY Mol Pharmacol 94:665–673, July 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Insulin Receptor Plasma Membrane Levels Increased by the Progesterone Receptor Membrane Component 1 s Kaia K. Hampton, Katie Anderson, Hilaree Frazier, Olivier Thibault, and Rolf J. Craven Department of Pharmacology and Nutritional Sciences, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky Received September 7, 2017; accepted April 13, 2018 Downloaded from ABSTRACT The insulin receptor (IR) is a ligand-activated receptor tyrosine therapeutic applications because a small-molecule PGRMC1 kinase that has a key role in metabolism, cellular survival, and ligand, AG205, also decreases plasma membrane IR levels. proliferation. Progesterone receptor membrane component However, PGRMC1 knockdown via short hairpin RNA expres- 1 (PGRMC1) promotes cellular signaling via receptor trafficking sion and AG205 treatment potentiated insulin-mediated phos- and is essential for some elements of tumor growth and phorylation of the IR signaling mediator AKT. Finally, PGRMC1 metastasis. In the present study, we demonstrate that PGRMC1 also increased plasma membrane levels of two key glucose molpharm.aspetjournals.org coprecipitates with IR. Furthermore, we show that PGRMC1 transporters, GLUT-4 and GLUT-1. Our data support a role for increases plasma membrane IR levels in multiple cell lines and PGRMC1 maintaining plasma membrane pools of the receptor, decreases insulin binding at the cell surface. The findings have modulating IR signaling and function. Introduction 2014). PGRMC1 localizes to endosomes, the endoplasmic reticulum (Nölte et al., 2000; Ahmed et al., 2010a), and the Changes in insulin signaling have been linked to multiple plasma membrane (Krebs et al., 2000), consistent with its diseases, most typically diabetes (Alghamdi et al., 2014), but highly conserved role in trafficking. In cancer, the best at ASPET Journals on September 24, 2021 also loss of cognitive function (de la Monte, 2012) and cancer characterized trafficking target for PGRMC1 is the epidermal progression (Malaguarnera and Belfiore, 2011; Sciacca et al., growth factor receptor (EGFR) tyrosine kinase (Ahmed et al., 2014; Forest et al., 2015). Insulin receptor (IR) is a tetramer 2010a; Mir et al., 2012; Aizen and Thomas, 2015; Kabe et al., a b comprised of 2 (ligand binding) and 2 (kinase domain) chains 2016), although PGRMC1 also increases plasma membrane that is expressed in numerous tissues. The human IR encodes pools of glucagon-like peptide-1 receptor and membrane pro- two isoforms, IR-A (lacking exon 11) and IR-B, which have a gestin receptor a1, a plasma membrane progesterone receptor predominant role in metabolism (Belfiore et al., 2009). IR is (Thomas et al., 2014; Zhang et al., 2014) that may be the target transported in a cycle of plasma membrane export, activation, for PGRMC1-dependent progesterone signaling (Bashour and and internalization (McClain, 1992; Goh and Sorkin, 2013; Wray, 2012; Peluso, 2013; Guo et al., 2016; Sun et al., 2016). Boothe et al., 2016; Posner, 2017). Signaling from the IR PGRMC1 has numerous other binding partners, including through the IR substrate-1/phosphatidylinositol 3-kinase/AKT cytochrome P450 proteins (Hughes et al., 2007; Szczesna- pathway causes the glucose transporter (GLUT)-4 to trans- Skorupa and Kemper, 2011; Oda et al., 2011), plasminogen locate from intracellular vesicles to the plasma membrane activator inhibitor RNA-binding protein 1 (Peluso et al., 2005, (Huang and Czech, 2007), increasing glucose uptake (Stöckli 2013), and a-tubulin (Lodde and Peluso, 2011). PGRMC1 is et al., 2011). attractive as a therapeutic target because it has a small- Progesterone receptor membrane component 1 (PGRMC1) molecule ligand, called AG-205 (Ahmed et al., 2010b; Xu et al., (Cahill, 2007) contributes to signaling by stabilizing trans- 2011; Zhang et al., 2014; Aizen and Thomas, 2015; Piel et al., membrane receptors at the plasma membrane (Hampton and 2016). Craven, 2014; Thomas et al., 2014), and these receptors In the present study, we have investigated the role of include tyrosine kinases (Thomas et al., 2014; Zhang et al., PGRMC1 in maintaining IR at the plasma membrane. PGRMC1 has been associated with insulin signaling in a This work was supported by Washington University Diabetes Research clinical study of insulin-resistant, high body mass index Center Grant P30 DK020579-40; University of Kentucky CTSA Grant UL1 subjects, which demonstrated decreased PGRMC1 RNA levels TR001998; and National Institutes of Health National Institute on Aging [Grant 5R01AG033649-08] and National Institutes of Health [Grant T32 compared with insulin-sensitive subjects (Elbein et al., 2011). DK007778 (to K.K.H. and H.F.)]. We posited that downregulated PGRMC1 could disrupt https://doi.org/10.1124/mol.117.110510. s This article has supplemental material available at molpharm. normal IR function, and we show in this work that PGRMC1 aspetjournals.org. has a direct role in regulating IR trafficking and signaling. ABBREVIATIONS: Cy5, cyanine 5; EGFR, epidermal growth factor receptor; GLUT, glucose transporter; IR, insulin receptor; PBS, phosphate- buffered saline; PGRMC1, progesterone receptor membrane component 1. 665 666 Hampton et al. Materials and Methods cell scraper. Lysates were centrifuged at 13,000 rpm to clear cell debris and assayed for protein by bicinchoninic acid assay. Immunofluores- Tissue Culture. A549 cells were obtained from American Type cence was performed, as previously described (Crudden et al., 2005), Culture Collection (Manassas, VA) and verified by Genetica (Cincin- fixing cells in 3.7% formaldehyde and permeabilizing in 1% Triton nati, OH). HUH7 cells were provided by B. Spear (University of X-100. The anti-IR antibody was NBP2-12793 (Novus Biologicals, Kentucky College of Medicine). Cells were maintained in Dulbecco’s Littleton, CO), and cells were imaged on a Nikon TE200 microscope. modified Eagle’s medium (Corning, Manassas, VA) containing 10% Cell Surface Labeling Assays. For a single experiment, four fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and antibiotics and dishes of 90%–95% confluent cells were used. After the removal were maintained at 37°C in 5% CO and air. The A549 derivatives 2 of media, cells were washed twice with ice-cold PBS (VWR, Radnar, infected with lentiviruses expressing short hairpin RNAs were pre- PA) and labeled with sulfo-NHS-SS-biotin [sulfosuccinimidhyl-2- pared from the plasmids pGIPZ (control) and V2LHS_90636 (biotinamido)-ethyl-1,3-dithipropionate] for 30 minutes at 4°C on a (PGRMC1-knockdown) and have been previously described (Ahmed et al., 2010b). rocking platform. The labeled proteins were purified with avidin Reagents and Treatments. AG205 was purchased from Timtec agarose using the Cell Surface Protein Isolation Kit (Thermo Scien- ’ (Newark, NJ). Dose response and time courses were performed tific, Waltham, MA), according to the manufacturer s instructions. For previously (data not shown) to establish the most effective concentra- comparison, the intracellular protein pool that did not bind avidin- tions and times. A549 and HUH7 cells were treated with AG205 agarose was also collected and stored as the unbound or cytoplasmic (20 mM) for 90 minutes and underwent protein analysis or cell surface fraction. Cell surface labeling reactions were performed at least in Downloaded from labeling. A549 control and PGRMC1-knockdown were plated on glass- triplicate, and fraction levels were confirmed via SDS-PAGE gel bottom microwell dishes (MatTek, Ashland, MA) for imaging. A549 separation and staining with Coomassie Blue, as previously described control and PGRMC1-knockdown were treated with recombinant (Ahmed et al., 2010a). Western blots of biotin-labeled eluates and human insulin, cyanine 5 (Cy5), labeled (Nanocs, New York, NY) at unbound fractions were performed at least in triplicate. a concentration of 100 nM for 5 and 15 minutes. Cells were visualized Immunologic Techniques. For Western blot analysis, cell ly- using a Nikon A1R1 resonant scanning confocal system (Nikon, Inc., sates were prepared by incubating cells in radioimmunoprecipitation Melville, NY) at the University of Kentucky Imaging Facility and assay buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% molpharm.aspetjournals.org analyzed with NIS-Elements C imaging software. sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl- For cell-signaling experiments, cells were incubated in RPMI methylsulfonyl fluoride, 1 mg/ml aprotinin, and 1 mg/ml leupeptin] for 1640 medium without serum for 6 hours. In some cases, cells were 10 minutes at 4°C. Lysates were cleared by centrifugation at 14,000g treated with vehicle control (dimethylsulfoxide) or AG-205 for the final for 10 minutes at 4°C, and proteins were separated by gel electropho- 90 minutes of the serum starvation period. For some experiments, resis. The antibodies used in this study were anti-IRb (Novus cells were then treated with 100 nM recombinant human insulin Biologicals), anti-IRb (Cell Signaling, Danvers, MA), PGRMC1 (Sigma-Aldrich) for 5 minutes. In other experiments, cells were (Abcam, Cambridge, MA), anti-proliferating cell nuclear antigen treated with 10 mM erlotinib or 10 mM