A method for the determination of activated receptor phosphorylation state following in vivo drug treatment

Abstract

Reversible phosphorylation events can alter many critical cellular functions and has been hypothesized to play a role in the development of tolerance to chronically administered agonists. The technique described here was developed to assess the phosphorylation state of μ-opioid receptor protein isolated from rodent brain tissue following 4 days of continual, subcutaneously released morphine. We have adapted and combined two techniques: back-phosphorylation reactions, which make use of tissue from animal models, and phosphorylation followed by immunoprecipitation, an approach used in cell culture models. μ-receptor protein is precipitated using a receptor-specific antibody, and the protein phosphorylation state is preserved by the use of phosphatase inhibitors. Phosphorylation sites that are dephosphorylated on the isolated protein are then radiolabelled by the addition of purified cAMP-dependent protein kinase (PKA) catalytic subunit and [ 32 P ]ATP. The samples are separated by gel electrophoresis, and the resulting bands are quantified using a phosphoimager. The amount of 32 P incorporated into the protein is assumed to be inversely related to the degree of phosphorylation prior to the reaction (i.e., the in vivo state of the protein). These estimates of μ-opioid receptor phosphorylation can then be correlated with a behavioral endpoint such as antinociception, to better understand the role of phosphorylation events in tolerance development. Themes: Neurotransmitters, modulators, transporters and receptors Topics: Second messengers and phosphorylation, receptor modulation, up- and down-regulation