The Effect of A-kinase Anchoring Protein on G-protein-coupled Receptor Kinase 2

Objectives: Delta opioid receptors(DOR) are G-protein coupled receptors(GPCR) in the plasma membrane of sensory neurons that modulate chronic pain nociception. Proteins, such as G protein-coupled receptor kinase 2(GRK2), can inactivate GPCRs such as DOR, thereby inhibiting its potential analgesic ability. The objective of this project is to determine if A-Kinase Anchoring Protein (AKAP) influences the plasma membrane trafficking of GRK2 via Protein Kinase A (PKA) activity in peripheral sensory neurons.
Methods: There were two types of cells tested for this project, Chinese Hamster Ovary (CHO) cells and trigeminal ganglion sensory neurons from male Sprague-Dawley rats. CHO cell cultures were divided into 2 groups, wildtype/control and experimental-AKAP. The AKAP group was treated with PKA activator 8-Br-cAMP to increase PKA activity. After transfection, cell lysates were collected and quantified by the Bradford method, and aliquoted for Western blot analysis. Cultured TG sensory neurons were divided into two groups: mock-siRNA-transfected as a control and AKAP-siRNA-transfected as the experimental group. Cultures were lysed, total protein was quantified by the Bradford method, and crude plasma membrane fractions were analyzed for GRK2 expression by Western blot. One-way ANOVA statistical analysis was performed using GraphPad Prism software to determine the significance between samples means ± SEM.
Results: PKA activation did not cause a significant upregulation of overall AKAP or GRK 2 expression. However, reduced AKAP expression correlates with a decrease in PKA phosphorylation of GRK2 at the plasma membrane. Movement of GRK2 from the cytosol to the plasma membrane was decreased in cultures treated with AKAP-siRNA, suggesting that AKAP may enhance the translocation of GRK2 to the plasma membrane to be phosphorylated by PKA.
Conclusions: In conclusion, AKAP scaffolding of PKA appears to correlate with GRK2 phosphorylation and translocation to the plasma membrane of sensory neurons. This localization phenomenon likely contributes to maintain the desensitization of DOR. Additional investigations into this pathway may aid in the development of treatments to inhibit prolonged DOR desensitization in peripheral sensory neurons.