Abstract
G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-Gβγ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-Gβγ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.
Footnotes
This work was supported by an American Heart Association Predoctoral Fellowship from the Midwest Affiliate (to D.M.T.) and the National Institutes of Health National Heart Lung and Blood Institute [Grants HL071818, HL086865] (to J.J.G.T.). This research used the Cell and Molecular Biology Core of the Michigan Diabetes Research and Training Center supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK20572]. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor [Grant 085P1000817]. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [Contract DE-AC02-06CH11357].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.071522.
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ABBREVIATIONS:
- GRK
- G protein-coupled receptor kinase
- GPCR
- G protein-coupled receptor
- CMPD103A
- N-(2,6-difluorobenzyl)-3-(((4-propyl-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-yl)methyl)amino)benzamide
- CMPD101
- 3-(((4-methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)methyl)amino)-N-(2-(trifluoromethyl)benzyl)benzamide
- ANS
- anilinonaphthalene-8-sulfonic acid
- PK
- protein kinase
- P-loop
- phosphate-binding loop
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- PAGE
- polyacrylamide gel electrophoresis
- DMSO
- dimethyl sulfoxide
- MES
- 4-morpholineethanesulfonic acid
- PDB
- Protein Data Bank
- bROS
- bovine rod outer segment(s)
- KT5720
- (9S,10S,12R)-2,3,9,10,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]-benzo-diazocine-10-carboxylic acid hexyl ester.
- Received February 1, 2011.
- Accepted May 19, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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