|GRK2 Enzyme Helps Keep G Proteins at Bay|
|Wednesday, 28 January 2004 00:00|
G proteins in the cell serve as molecular switches for important signaling cascades, including those that control heart rate, blood pressure, and glucose metabolism and those that mediate the senses of taste, smell, and vision. The G proteins themselves are activated by G-protein-coupled receptors (GPCRs), which reside in the cell membrane and react to specific external signals, such as light or adrenaline. In order for cells to adapt to changes in their external (extracellular) environments, activated GPCRs must be rapidly desensitized. This process is initiated by G-protein-coupled receptor kinases (GRKs), enzymes that phosphorylate (add phosphate groups to) the portions of activated GPCRs that project into the cell. To study this mechanism at the molecular level, a collaboration from the University of Texas and the Duke University Medical Center has determined the crystal structure of a form of GRK in complex with portions of its target molecule.
The GRK family has seven members (GRK1–7), the best characterized of which is GRK2. GRK2 is responsible for the desensitization of adrenaline receptors in the heart and is essential for proper heart development. However, abnormal levels of GRK2 have also been linked to congestive heart failure, high blood pressure, and opiate addiction. GRK2 is a cytoplasmic protein that targets activated GPCRs via the interaction of its pleckstrin homology (PH) domain with the heterotrimeric G-protein subunits β and γ (Gβγ). Free Gβγ subunits are released from Gα subunits only after GPCR activation.
The researchers purified bovine GRK2 and Gβγ subunits as a 1:1 complex and determined their structure by x-ray crystallography. Diffraction maxima from the crystals of 125-kDa GRK2–Gβγ complex were collected at ALS Beamline 8.2.1, and the crystal structure was solved using molecular-replacement and density-modification techniques. Given the small size of the GRK2–Gβγ crystals, the intense, highly collimated source at Beamline 8.2.1 allowed collection of diffraction data at much higher resolution than achieved with a laboratory x-ray source (2.4 Å compared with 3.5 Å, respectively).
The structure of the GRK2–Gβγ complex is the first that has been determined of any member of the GRK family and the first of any effector enzyme targeted by Gβγ. The three GRK2 domains [the RGS homology (RH), protein kinase, and PH domains] are arranged such that they form a triangular shape approximately 80 Å on a side. The "membrane proximal" surface of the triangular GRK2–Gβγ complex is positively charged and flat, suggesting that it is this surface that interacts with the negatively charged plasma membrane. This surface also includes the phospholipid binding loop in the PH domain and the geranylgeranyl group of Gγ that tethers Gβγ to the plasma membrane in cells.
The interdomain contacts of GRK2 fix the orientations of its three domains at the membrane surface such that each can perform a discrete desensitizing task. The kinase active site cleft faces the cell membrane where GPCRs are found; the PH domain is oriented to allow its interaction with Gβγ and phospholipid head groups; and the RH domain is positioned such that it can sequester an activated Gα subunit. Furthermore, Gβγ, Gα, and GPCR binding sites are found at unique vertices of the GRK2 triangle, suggesting that GRK2 can bind all three proteins at the same time. This configuration allows for the rapid attenuation of the signal that was propagated by the activated GPCR; thus the GPCR can be desensitized by phosphorylation while the free Gβγ and Gα subunits are blocked from activating their downstream signaling cascades.
Research conducted by D.T. Lodowski and J.J.G. Tesmer (University of Texas at Austin), J. A. Pitcher (University College London), and W.D. Capel R.J. Lefkowitz (Howard Hughes Medical Institute and Duke University Medical Center).
Research funding: American Heart Association (Texas and National affiliates), Welch Foundation, Research Corporation, the National Institutes of Health, and the Howard Hughes Medical Institute. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.
Publication about this research: D.T. Lodowski, J.A. Pitcher, W.D. Capel, R.J. Lefkowitz, J.J.G. Tesmer, "Keeping G proteins at bay: A complex between G protein-coupled receptor kinase 2 and Gβγ," Science 300, 1256 (2003).
ALS Science Highlight #71