1998 Global Conference Proceeding

March 02 - 04, 1998

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Conference: 1998 Global Conference
Title: Biochemical Characterization of Partial Nephrogenic Diabetes Insipidus (NDI) Phenotypes
Authors: Sadeghi, Hamid; Robertson, Gary; Bichet, Daniel G.; Innamorati, Giulio; Birnbaumer, Mariel
Institutions: University of Isfahan, Northwestern University Medical School, Hopital du Sacre-Coeur de Montreal, Vita Salute University School of Medicine, National Institute of Environmental Health Sciences
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Two mutant V2 vasopressin receptors (V2R) detected in patients exhibiting partial NDI phenotype were studied in transiently transfected HEK293 cells. Substitution of Asp85 to Asn in the second transmembrane region of the V2R produced a decreased stimulation of adenylyl cyclase activity (EC50 and maximal response) in response to addition of vasopressin. Cell surface expression of this mutant, however, was not different from the wild type receptor as shown by binding assays and immunoprecipitation of the receptor. These results along with the calculation of coupling efficiency demonstrated that the major contributing factor in the attenuation of Asp85Asn V2R mutant function was alteration of receptor coupling to Gs. The second mutant V2R examined was one containing Asp instead of Gly201 in the second extracellualr loop of this protein. Stimulation of adenylyl cyclase activity but not coupling efficiency to Gs was reduced. Binding assays demonstrated a decreased cell surface expression of the mutant as compared to the wild type receptor with a slight decrease in ligand binding affinity. These results suggested that Gly201Asp mutation affects receptor function mainly by alteration in the number of receptors present in the plasma membrane. Furthermore, Gly201 seems to play a role in the V2R selectivity for different ligands since a significant defference in the stimulation of adenylyl cyclase activity was observed when desmopressin was used instead of vasopressin.
Two mutant V2 vasopressin receptors (V2R) detected in nephrogenic diabetes insipidus (NDI) patients were studied. In the first V2R, the mutation occurred in its second transmembrane region where a section of the genetic sequence, Asp85, that should have been there was replaced by Asn. This mutant V2R showed a decreased ability to stimulate an enzyme called adenylyl cyclase in response to vasopressin (AVP), though it could travel from inside the cell to the cell membrane like non-mutated V2Rs can. The main factor contributing to this mutant's dysfunction was its reduced ability to stimulate this enzyme via decreased coupling to a protein which is essential to the kidneys' processes. The second mutant was characterized by another substitution of one segment of a genetic sequence with another (Asp substituting for Gly2O1). This substitution occurred in the second extracellular loop of V2R. This mutation decreased stimulation of enzyme activity and was unable to get to the cell surface as much as normal V2R. The inability to reach the cell surface was the main reason this mutant V2R could not function as effectively as normal V2R.