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).

Turning Off the Spigot

Sometimes it's easier to start something than to stop it. Just ask the Sorcerer's Apprentice. A similar dilemma occurs in the cells of our bodies as they respond to various chemical signals. The initiating event is the binding of a signaling molecule to a receptor in the cell membrane. The receptor then activates a molecular switch inside the cell that triggers a cascade of signaling processes. G proteins within the cell are an important example of such a switch because they control a host of bodily functions including heart rate, blood pressure, and glucose metabolism and mediate the senses of taste, smell, and vision. They also act as amplifiers, because as long as the G-protein switches are on, the signals continue. The problem then becomes how to turn the switches off when the external signal changes. The cell solves the problem with another protein, an enzyme called GRK.

By determining the atomic structure of a complex consisting of a GRK and two of the three subunits comprising a "heterotrimeric" G protein, Lodowski et al. have provided a molecular-level look at how the interaction between a G-protein-coupled receptor (GPCR), the GRK, and the G protein simultaneously desensitizes the GPCR so it stops activating G proteins and sequesters the G proteins that are already activated. Abnormal levels of the GRK studied have been linked to congestive heart failure, high blood pressure, and opiate addiction, so the mechanism is of more than academic interest.

Domain structure of GRK2. The Gβγ subunits have been omitted from the figure for clarity.

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.

Model of GRK2 in complex with G proteins and a GPCR. A GPCR (red) and Gαq (teal) have been docked with the GRK2–Gβγ complex. This configuration would efficiently shut down GPCR-mediated signaling by inactivating the receptor at the same time as it sequesters the G proteins that the receptor itself activated.

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).