Investigation of the effects of H80Y, V88L, V215M and L219P mutations on v2 receptor function

Diabetes insipidus (DI) is a rare disease that is characterized by significantly high urine production (polyuria) and excessive water consumption (polydypsia). This disease generally prevents concentration mechanism of urine. Dehydration tests are applied for confirming the diagnosis of DI. The purpose of dehydration test is to observe the patient's ability to concentrate urine and to evaluate the patient's reaction to arginine vasopressin (vasopressin, antidiuretic hormone, ADH, AVP). In case of late diagnosis, DI can result in serious hypernatremia and dehydration. DI is treated with desmopressin (DDAVP). Variations of DI can occur either as a hereditary or an acquired disease. Mutations of arginine vasopressin receptor 2 (V2 receptor, V2R, AVPR2) and its coding genes as well as mutations of aquaporin 2 channels (AQP2) and its coding genes cause congenital (hereditary) type Nephrogenic Diabetes Insipidus (NDI). In cases of congenital NDI, urine cannot be concentrated despite normal secreted and synthesized AVP levels or high plasma concentrations. Mutations on V2R gene that cause congenital NDI, can also result in the creation of different phenotypes of receptors. The course and the severity of disease can vary due to incompatible receptor functions caused by different phenotypes of receptors. The aim of this thesis is to examine the relation between H80Y, V88L, V215M and L219P mutations identified in V2R gene, which is known to cause congenital NDI, and V2 receptor functions. The purposes of this examination are to better illuminate the course of disease and to provide information for further research on the treatment. In accordance with the purpose of this study following procedure is conducted as an experimental research. Relevant mutations mentioned above were produced utilizing sitedirected mutagenesis method to mammalian expression vectors containing wild type V2R gene. These mammalian expression vectors were transfected to COS-7 cells. Intracellular and cell surface expressions of both wild and mutant types of V2 receptors were determined with the application of enzyme-linked immunosorbent assay (ELISA) experiments. Levels of cyclic adenosine monophosphate (cyclic AMP, cAMP) synthesized as a response to stimulation of transfected cells with AVP were scanned to observe mutant V2 receptor activity. Positioning of mutant V2 receptors in the cell, and their locations in the endoplasmic reticulum (ER) were observed with the fluorescent imaging method. As a result, all mutant type V2 receptors studied within the scope of this thesis are observed to demonstrate varying degrees of function loss compared to wild type V2 receptors.