Establishment of a Split-LUX Assay to Determine Protein Interaction of the BMP-Receptor mAlk2R206H (FOP Mutant) with the Rapamycin-Associated Protein FKBP12

Sandra Kronimus
2014 unpublished
Fibrodysplasia Ossificans Progressiva (FOP) is a devastating disease caused by various mutations in the BMP receptor mAlk2, which leads to heterotopic ossification. The mutation this thesis is focusing on is an arginine to histidine substitution on position 206 in the GS (glycine-serine-motif) domain of the mAlk2 receptor. Due to this mutation, the receptors inhibition is drastically altered, leading to constitutive activity and permanently activated BMP (bone morphogenic protein) signaling,
more » ... ch consequently leads to pathological bone cell differentiation. Finding a cure for this disease or a way to decelerate its progression, while gaining an understanding of its cause, is crucial. In this thesis, a protein complementation system (PCA) based on split-luciferase reassembling is established. To develop and further optimize a split-luciferase assay, a rapamycin inducible hetero-dimerization approach namely FRB (FKBP-rapamycin binding domain of FRAP) and FKBP12 (FK506 binding protein 12), fused to N- and C-terminal fragments of a luciferase, respectively, is used. Based on this method the interaction of the FOP mutant receptor (mAlk2R206H) with it´s wild type inhibitor FKBP12 and interactions of wild type mAlk2 as well as wild type mAlk3 with FKBP12 are examined. This work suggests, that mAlk2R206H inhibition by FKBP12 is weakened whereas the interaction of mAlk2wt as well as mAlk3wt can be observed by reassembling of the luciferase fragments fused to the receptors and inhibitor. These results provide novel insight into an approach that is cheap and easy to apply to determine protein-protein interactions and highlight the need of further investigation of the interaction of the FOP mutant mAlk2R206H and its interaction partner FKBP12.
doi:10.25365/thesis.40068 fatcat:bqwkph2lvreona2xkj6ik33nla