Characterization of G protein-coupled receptor kinase 4 and measuring its constitutive activity in vivo.

G protein-coupled receptor kinase 4 (GRK4) was originally identified in the brain and was initially thought to have a limited expression pattern and functionality; however, more recent studies have found that GRK4 is expressed in multiple tissues and cell types and that it contributes to cardiovascular disease. Additionally, human GRK4 exists as four splice variants and each variant can harbor at least three functionally relevant polymorphisms. The primary role of GRK4 is to phosphorylate G protein-coupled receptors (GPCR), which leads to desensitization of the G protein signaling mechanism while simultaneously recruiting β-arrestins and initializing the internalization of the receptor. Interestingly, GRK4 has been shown to be constitutively active in some, but not all, cases. A constitutive active GRK could lead to increased β-arrestin-mediated signaling while inhibiting traditional/canonical GPCR-mediated signaling mechanisms. Therefore, it is important to determine if GRK4 is constitutively active in a system. Measuring agonist-mediated activity of GRK4 is relatively straightforward since it inhibits second messenger signaling; however, only a few studies have directly examined the constitutive activity of GRK4 which requires techniques without an agonist. Since GRK4 has significant biological effects, identifying the mechanism underlying GRK4's constitutive activity and ligand-stimulated activity becomes increasingly important. Therefore, the methods provided here are designed to aid researchers in determining if GRK4 is expressed, and if so which GRK4 species is expressed, followed by procedures to identify if GRK4 is constitutively active in its model system. Last, procedures are explained for identifying if GRK4 is involved in its system in a nonconstitutive manner. The protocols described here are designed to be accessible to a wide range of scientists, which should allow for more laboratories to examine GRK4 constitutive activity as well as agonist-mediated activity.