title: Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses.
abstract: The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing of other membrane receptors, such as TβRII, ALK5 or ALK1.
authors: Institute for Molecular and Cell Biology, University of Applied Sciences, Mannheim, Germany.
authors: Tassilo
authors: T
authors: Förg
authors: Institute for Molecular and Cell Biology, University of Applied Sciences, Mannheim, Germany; Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany; Institute for Medical Technology of Heidelberg University and University of Applied Sciences, Mannheim, Germany.
authors: Mathias
authors: M
authors: Hafner
authors: Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany; Q-bios GmbH Biotechnology, Mannheim, Germany.
authors: Andreas
authors: A
authors: Lux
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D000071063:Endoglin; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D020125:Mutation, Missense; D011956:Receptors, Cell Surface / Q000235:genetics*
chemical_list: D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface