The scientists teamed up with Gabriel Vargas, MD, PhD, a UCSF assistant adjunct professor of psychiatry conducting research in the laboratory of Mark von Zastrow, PhD, on how cell surface receptors carry messages into the cell that activate specific events. The V2 receptor has been widely studied as an example of a large family of proteins called G protein-coupled receptors (GPCR), which are involved in signal transduction and are responsible for many physiological processes.
Normally, when vasopressin binds to the V2R, it stimulates a chain of events inside the cell. Vesicles containing the water channel Aquaporin 2 (AQP-2) are shuttled from the cytoplasm to the apical membrane of the collecting duct kidney cells. This process increases water permeability and diminishes urine output from the kidneys.
The group set out to prove that changing the amino acid at position 137, from arginine to either cysteine or leucine, caused the AVPR2 to produce a mutated V2 receptor that is always "on," inappropriately signaling the kidneys to retain water. The opposite condition, diabetes insipidus, is caused when the arginine at R137 is changed to histidine, and the resulting V2 receptor signals the kidneys to excrete water excessively.
To test the hypothesis that V2 receptors were active in their patients even though vasopressin was absent, Feldman developed an assay for receptor activity. One marker of this activity is an increase in the amount of cyclic AMP (cAMP) within the cell. He worked in the laboratory of pediatric endocrinologist Walter Miller, MD, using a method to determine cAMP levels in cells by using a luciferase plasmid that responds to cAMP.
Luciferase is the substance that lights up in fireflies. Feldman showed that wild-type cells contain low levels of light from luciferase -- and thu
Source:University of California - San Francisco