Investigation of the mechanisms to effect of some pharmacological chaperones on mutants caused by nephrogenic diabetes insipidus disease

G-protein coupled reseptors (GPCRs) are a large family of integral membrane proteins involved in the management of many physiological processes in our body. Mutations in the amino acid sequences on the receptor cause conformational changes in the receptor, leading to the emergence of various diseases. Diabetes insipidus (DI) is a disease that may develop due to mutations associated with GPCR. DI is subdivided into Central DI (CDI), Nephrogenic DI (NDI), primary polydipsia, and pregnancy-induced polyuria. NDI, which is caused by mutations in the AVPR2 gene located on the X chromosome, is a rare inherited disease characterized by polydipsia and polyuria. These mutations may cause proteins to misfold and retain in ER quality control systems, and thus cause loss of function. The aim of this thesis is to investigate whether these mutant proteins can be rescued from the ER quality control system as a result of the application of YM087 and VPA985 pharmacological chaperones on R68W, V162A and T273M mutant AVPR2 proteins, which have been shown in previous studies to cause loss of function in NDI. In order to determine the doses of YM087 and VPA985 pharmacological chaperones, which performed in all experimental studies within the scope of this thesis, % cell viability was calculated by MTT and Trypan blue method. Wild-type and mutant pLV2R vectors were transfected into COS-1 cells. Cell surface ELISA and total ELISA methods were performed for the determination of the total amount of pLV2R vectors, containing wild type and mutant AVPR2 gene, on the cell surface and inside the cell. In addition, flow cytometry method was performed to measure the cell surface characteristics of mutant receptors. The results were compared with different chaperone applications by searching the literatüre. In the studies carried out within the scope of this thesis, it was observed that YM087 and VPA985 pharmacological chaperones applied on the R68W, V162A and T273M mutant receptors increased their expression on the cell surface at varying levels depending on the type of mutant proteins.