engleski

Towards structural studies of ligand-induced conformational changes in arginine-vasopressin V2 receptor

Human arginine-vasopressin V2 receptor (V2R) is primarily expressed in kidney tubules and is involved in mechanisms that concentrate urine and keep water homeostasis. Several biased ligands stabilize distinct conformational states of V2R leading to biased signaling pathways [1]. In order to elucidate conformational changes in V2R induced by ligand binding, we devised a research strategy based on conformational phi-value analysis. This protein-engineering method was originally applied to obtain structural information about transition states during protein folding [2]. In its modified form and combined with alanine scanning mutagenesis, conformational phi-value analysis becomes a valuable tool in the study of ligand-induced conformational changes in GPCRs. For this purpose, we measure thermal stabilities of each alanine mutant in presence and absence of a particular ligand. By comparing thermal stabilities of the mutants with those for the wild type, we can quantify the ligand-induced structural changes at a single-residue level. In addition, thermostability data could be used to engineer conformationally stabilized GPCRs [3] that are better suited for crystallographic and NMR studies. In designing V2R constructs for crystallographic studies, we are also investigating applicability of transferring some of the thermostabilizing point mutations and truncations from other GPCRs as well as replacing the intracellular loop 3 with different fusion partners [4]. [1] Rahmeh, R. ; Damian, M. ; Cottet, M. ; Orcel, H. ; Mendre, C. ; Durroux, T. ; Sharma, K. S. ; Durand, G. ; Pucci, B. ; Trinquet, E. ; Zwier, J. M. ; Deupi, X. ; Bron, P. ; Banères, J.-L. ; Mouillac, B. ; Granier, S. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 6733-6738. [2] Fersht, A. R. ; Matouschek, A. ; Serrano, L. J. Mol. Biol. 1992, 224, 771-782. [3] Serrano-Vega, M. J. ; Magnani, F. ; Shibata, Y. ; Tate, C. G. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 877-882. [4] Tate, C. G. Trends Biochem. Sci. 2012, 37, 343-352.

V2 receptor; GPCR

nije evidentirano