However, a commercially available antibody (C3) raised against the N-terminal domain (amino acids 146) of PGRMC1 was not able to recognize csPGRMC1 at all on the surface of H9, NT-2/D1, HEK293T and HepG2 cells while 108-B6 and 4A68 were able to recognize csPGRMC1 around the cell surface of all cells (Fig

However, a commercially available antibody (C3) raised against the N-terminal domain (amino acids 146) of PGRMC1 was not able to recognize csPGRMC1 at all on the surface of H9, NT-2/D1, HEK293T and HepG2 cells while 108-B6 and 4A68 were able to recognize csPGRMC1 around the cell surface of all cells (Fig.1ad, upper panels). examine the epitope specificity of 108-B6 and 4A68, glutathione-S-transferase (GST)-fused PGRMC1 mutants were screened to identify the epitopes targeted by the antibodies. The result showed that 108-B6 and 4A68 acknowledged C-terminal residues 183195 and 171182, respectively, of PGRMC1, where trypsin-sensitive sites are located. A polyclonal anti-PGRMC1 antibody raised against the C-terminus of PGRMC1 could also acknowledged csPGRMC1 in a trypsin-sensitive manner, suggesting that this C-terminus of csPGRMC1 is usually exposed around the cell surface. This obtaining reveals that csPGRMC1 has a non-conventional plasma membrane topology, which is different from that of intracellular PGRMC1. == Introduction == Progesterone receptor membrane component 1 (PGRMC1) is usually a multifunctional protein with a C-terminal cytochromeb5domain name1. PGRMC1 is usually highly expressed in multiple types of malignancy, and represents a proliferation marker for numerous malignancy cells14. PGRMC1 also increases the neuronal toxicity of amyloid -peptides through binding to amyloid oligomer in Alzheimers disease5,6. PGRMC1 is also involved in diverse biological functions, such as regulation of cytochrome P450, progesterone signaling, vesicle trafficking, steroidogenesis, cell cycle regulation, anchorage-independent growth, invasive growth, angiogenesis, hypoxic biology, and autophagy promotion1,7. PGRMC1 consists of a short N-terminal luminal or extracellular domain name, a single membrane-spanning domain name, and a long cytoplasmic domain name811. Many studies have shown that PGRMC1 is usually localized at numerous subcellular compartments, such as endoplasmic reticulum, Golgi apparatus, plasma membrane, inner acrosomal membrane, nucleus, nucleolus, and mitochondria9,1215. PGRMC1 regulates cell proliferation and apoptosis in granulosa and luteal cells via conversation between its cytoplasmic cytochromeb5binding domain name (amino acids 70130) and plasminogen activator inhibitor RNA-binding protein-1 (PAIR-BP1)10,16. PGRMC1 can also interact or associate with other binding partners including epidermal growth factor receptor (EGFR)17, glucagon-like peptide-1 receptor (GLP-1R)18, insulin receptor19, glucose channels19, membrane progesterone receptor (mPR/PAQR7)20, and P450 proteins21,22. A recent study revealed that this heme-mediated dimerization of adjacent PGRMC1 monomers in the cytoplasmic side prospects PGRMC1 to interact with cytochromes P450 and EGFR, causing enhanced proliferation, anti-apoptosis, and chemoresistance of malignancy cells22. However, the conclusion is usually challenged with some observations and thoughts that tyrosine 113 phosphorylation of PGRMC1 is required for membrane trafficking to co-localize PGRMC1 and EGFR23, and Resatorvid the cytoplasmic domain name of PGRMC1 is located around the luminal side of microsomes in A549 cells17. Previously, we generated a panel of murine monoclonal antibodies (MAbs) against the surface molecules on undifferentiated human pluripotent stem cells (hPSCs) by using a altered decoy immunization strategy24. Subsequent studies showed that 108-B6 and 4A68, two of the MAbs, bind to cell surface expressed-PGRMC1 (csPGRMC1) on hPSCs and some malignancy cells25. PGRMC1 knockdown approach further revealed that PGRMC1 suppresses the p53 and Wnt/-catenin pathways to promote hPSC self-renewal25. Meanwhile, circulation cytometric analysis found that an anti-PGRMC1 antibody realizing the N-terminal domain name (residues 146) of Resatorvid PGRMC1 was not able to bind to csPGRMC1 on malignancy cells, although it was able to identify intracellular PGRMC1 in saponin-treated cells. Circulation cytometric analysis also showed that 108-B6 and 4A68 binding to csPGRMC1 was inhibited by trypsin treatment, suggesting that this epitopes of 108-B6 and 4A68 is usually outside the N-terminal Resatorvid domain name and have trypsin-sensitive sites within them. This observation led us to investigate the epitope of two MAbs on PGRMC1. The results revealed that 108-B6 acknowledged C-terminal residues 183195 of PGRMC1, and 4A68 acknowledged C-terminal residues 171182 of PGRMC1, where putative trypsin-sensitive sites are located. Thus, this obtaining reveals that this C-terminal domain name of PGRMC1 is usually exposed around the cell surface, instead of the N-terminal domain name of PGRMC1. A polyclonal anti-PGRMC1 raised against the C-terminal domain name of PGRMC1 also acknowledged csPGRMC1, supporting that this C-terminal domain name of PGRMC1 is usually exposed around the cell surface. Thus, epitope analysis of PGRMC1 antibodies reveals that csPGRMC1 has a different membrane topology from that of intracellular PGRMC1. == Results == == Monoclonal antibody against the N-terminus of PGRMC1 is not able MPL to identify csPGRMC1, whereas 108-B6 and 4A68 is able to identify csPGRMC1 == In the previous study, we generated two MAbs, 108-B6 and 4A68, against cell surface molecules on hPSCs, and found that both MAbs identify csPGRMC1 on hPSCs and some malignancy cells24,25. Many previous studies have shown that PGRMC1 has a short.