Expresión and Subcellular Localization of 17BETA-estradiol Binding proteins in C2C12 Cell Line?.

MILANESI, LORENA; RUSSO DE BOLAND, ANA; BOLAND, RICARDO

Congreso; AAOMM; 2004

AAOMM

Expression and Subcellular Localization of 17B-Estradiol Binding Proteins in C2C12 Cell Line Milanesi Lorena M, Russo de Boland Ana, Boland Ricardo L. Laboratorio de Quý´mica Biolo´gica, Universidad Nacional del Sur, Bahý´a Blanca. The classical cellular mechanism of 17h-estradiol action is assumed to involve the steroid diffusion through the plasma membrane, its binding to a cytoplasmic/nuclear receptor (ER), and subsequent transcription and protein synthesis regulation. In addition to this mechanism, increasing evidence for non-genomic estrogen short-term effects in different cells types has been accumulated and led to hypothesize the existence of cellsurface resident receptor forms triggering membrane events. These cellsurface protein receptors have been detected in different cell lines (MCF-7, SHM, HT-29) and recently, the expression of an estrogen binding protein and its effects on the development and function of human and bovine skeletal muscle was reported. The present study was focused to analyze the presence and subcellular localization of ER binding proteins in C2C12 cells (a murine skeletal muscle cell line). By competition assays we have detected specific binding sites for [3H]17hestradiol in total homogenates. The subcellular localization of these binding sites were predominantly mithocondrial-microsomal. Immunochemical studies employing specific monoclonal antibodies against different domains of the classical ER (ERa) detected the 67 kDa immunoreactive band expected for the ERa in nuclear and cytosolic fractions and, additionally, low molecular weight bands present in the mitochondrial and microsomal fractions. These reactive bands were able to bind the steroid hormone in Ligand blot assays. Also these studies showed a clear surface labeling of the macromolecular complexes E2- BSA-FITC in non-permeabilized living cells. The characterization of these estrogen binding proteins and the signaling cascades activated thereby by the steroid hormone in skeletal muscle cells may provide information about molecular events implicated in alterations in muscle contractility and development which occur in menopausia.