title: Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1.
abstract: A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued.
authors.affiliation: Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy.
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Pati
authors.affiliation: Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center of the Medical University, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.
authors.forename: Diana
authors.initials: D
authors.lastname: Groza
authors.affiliation: Dipartimento di Oncologia, Università degli Studi di Torino, Via Santena 5/bis, 10126 Torino, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Riganti
authors.affiliation: Dipartimento di Oncologia, Università degli Studi di Torino, Via Santena 5/bis, 10126 Torino, Italy.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kopecka
authors.affiliation: Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Niso
authors.affiliation: Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Berardi
authors.affiliation: Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center of the Medical University, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hager
authors.affiliation: Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center of the Medical University, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Heffeter
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan.
authors.forename: Miwa
authors.initials: M
authors.lastname: Hirai
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tsugawa
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan; Japan Agency for Medical Research and Development (AMED), Core Research for Evolutional Science and Technology (CREST), Tokyo 160-8582, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kabe
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Suematsu
authors.affiliation: Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy. Electronic address: carmen.abate@uniba.it.
authors.forename: Carmen
authors.initials: C
authors.lastname: Abate
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D008024:Ligands; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000378:metabolism*; D017480:Receptors, sigma / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D008565:Membrane Proteins; C115296:PGRMC1 protein, human; D011980:Receptors, Progesterone; D017480:Receptors, sigma; C106107:sigma-2 receptor
keywords: Confocal microscopy; Flow-cytometry; Heme-PGRMC1 dimer; PGRMC1; Sigma-2 receptor; apo-PGRMC1 monomer