The effect of aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations on the expression and activity of vasopressin V2 receptor gene.
BACKGROUND
Vasopressin type 2 receptor (V2R) plays an important role in the water reabsorption in the kidney collecting ducts. V2R is a G protein coupled receptor (GPCR) and the triplet of amino acids aspartate-arginine-histidine (DRH) in this receptor might significantly influence its activity similar to other GPCR. However, the role of this motif has not been fully confirmed. Therefore, the present study attempted to shed some more light on the role of DRH motif in G protein coupling and V2R function with the use of site-directed mutagenesis.


METHODS
Nested PCR using specific primers was used to produce DNA fragments containing aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations with replacements of the arginine to lysine and histidine to tyrosine, respectively. After digestion, these inserts were ligated into the pcDNA3 vector and transformation into E. coli HB101 was performed using heat shock method. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using diethylaminoethyl-dextran method, the adenylyl cyclase activity assay was performed for functional study. The cell surface expression was analyzed by indirect ELISA method.


RESULTS
The functional assay indicated that none of these mutations significantly altered cAMP production and cell surface expression of V2R in these cells.


CONCLUSIONS
Since some substitutions in arginine residue have shown to lead to the inactive V2 receptor, further studies are required to define the role of this residue more precisely. However, it seems that the role of the histidine residue is not critical in the V2 receptor function.
Najafzadeh
Hossein
H
Dept. of Pharmacology and Toxicology, Shahid Chamran University, Ahvaz, Iran.
Safaeian
Leila
L
Mirmohammad Sadeghi
Hamid
H
Rabbani
Mohammad
M
Jafarian
Abbas
A
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000235:genetics; D007685:Kidney Tubules, Collecting / Q000502:physiology*; D008239:Lysine / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017483:Receptors, Vasopressin / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000235:genetics; D014867:Water / Q000378:metabolism*
D008565:Membrane Proteins; D017483:Receptors, Vasopressin; D007532:Isoleucine; D014867:Water; D001224:Aspartic Acid; D014443:Tyrosine; D001120:Arginine; D000242:Cyclic AMP; D008239:Lysine