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Development of a High Throughput Flow Cytometry Protein Interaction Assay to Identify Inhibitors of G Protein‐Coupled Receptor Kinase 2
Author(s) -
Thal David Michael,
Tesmer Valerie M.,
Tesmer John J.G.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb283
Subject(s) - g protein coupled receptor kinase , beta adrenergic receptor kinase , g protein coupled receptor , heterotrimeric g protein , kinase , receptor , microbiology and biotechnology , chemistry , phosphorylation , flow cytometry , protein kinase a , biology , pharmacology , biochemistry , g protein
G protein‐coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from glucose homeostasis to contractility of the heart. Phosphorylation of these receptors by GPCR kinases (GRKs) is a major regulatory mechanism for the desensitization of activated receptors. Our lab is particularly interested in GRK2, which plays an essential role in the regulation of myocardial signaling. Small molecule inhibitors of GRK2 will not only provide a novel mechanism for understanding GRK2's regulatory mechanisms, but may ultimately serve as potential therapeutics for the treatment of heart disease. Our lab has utilized a high throughput flow cytometry protein interaction assay allowing us to study the binding interactions of GRK2. The first interaction we have studied is between GRK2 and the heterotrimeric G protein Gα q . In the presence of GDP·AlF 4 − GRK2 has 3.2 ± 1.7 nM binding affinity for Gα q . So far we have screened nearly 40,000 compounds obtaining Z' values ranging from 0.5‐0.9 in a 384‐well format. We are currently in the process of confirming the activity of approximately 20 compounds that showed activity in follow up dose‐response titrations. Preparations are also underway to screen GRK2 against a novel RNA aptamer that selectivity binds and inhibits the kinase activity of GRK2. Z' values for this assay also range from 0.5‐0.9. Supported by grants from the NIH and AHA.