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High-Order Membrane Complexes from Activated G-Protein Subunits Print
Wednesday, 28 June 2006 00:00

Many physiological processes initiated in response to external (extracellular) signals such as hormones, neurotransmitters, or light are regulated by a complex dance involving GTP-binding (G) proteins: G-protein-coupled receptors (GPCRs), proteins integral to the cell membrane, sense the signal and activate G proteins in the cellular cytoplasm, but enzymes such as G-protein-coupled receptor kinase 2 (GRK2) inhibit the activity of the G proteins. A joint University of Michigan–University of Illinois, Chicago, team has determined the first structure of a particular G-protein–GRK2 complex. The structure in combination with previous structures of related G-protein complexes shows how Nature has evolved the G-protein structure to not only propagate activation signals but at the same time also directly respond to regulatory proteins that control the duration of the signal.

A Snapshot of
Molecules at Work

G proteins are molecular switches that both initiate and inhibit a vast array of biochemical reactions in cells. The switches are turned on by G-protein-coupled receptors (GPCRs) located in the cell membrane that sense external chemical signals like hormones or neurotransmitters or detect light. The switches are turned off by other proteins known as G-protein-coupled receptor kinases (GRKs). Understanding the molecular basis for signal passage from activated receptors through G proteins and then to downstream targets has been aided by protein crystallography, which yields atomic structures of the respective players, either alone or in concert. Previous atomic models provided the first and last frames, respectively, of a molecular movie that describes the course of the signaling, but the events between the first and last frames of this movie have remained foggy. By solving the structure of a complex formed by subunits of a G protein and GRK2, Tesmer et al. have revealed the configuration of the subunits as they engage a single protein target, thereby providing another snapshot of the events that unfold after G-protein activation by a GPCR.

The G proteins are heterotrimers consisting of three subunits (α, β, and γ). GPCRs activated by the extracellular signal catalyze nucleotide exchange on G proteins, thereby converting the inactive (GDP-bound) Gαβγ heterotrimer into activated Gα·GTP and Gβγ proteins. These subunits then interact with downstream proteins called effectors to elicit an appropriate intracellular response. For example, activation of receptors coupled to Gq leads to formation of Gαq·GTP and Gβγ, which in turn bind to and stimulate phospholipase Cβ (PLCβ). Gq represents one of the four subfamilies of the Gα subunit (Gαs, Gαt, Gαq, and Gα12/13).

Heterotrimeric G proteins (ribbon structures above) undergo a dramatic change in configuration (below) as they become activated and bind GRK2 (solid spheres). The view is from the perspective of the membrane surface. The Gαq and Gβγ subunits separate by about 80 Å and the Gα subunit rotates more than 100° with respect to Gβγ after GRK2 binding. The so-called “switch” regions of Gαq that undergo conformational change upon binding GTP are shown in red.

In order to adapt to new extracellular conditions, Gq-coupled receptors are desensitized by enzymes such as GRK2, which not only add phosphate groups to (phosphorylate) activated GPCRs, initiating their down-regulation, but also sequester activated Gαq and Gβγ subunits from PLCβ and presumably other effectors. In order to better understand the arrangement of heterotrimeric G proteins in complex with GRK2, the Michigan–Illinois team crystallized the Gαq-GRK2-Gβγ complex. After visiting three different beamlines, the team obtained the best diffraction data at ALS Beamline 8.3.1, which allowed them to solve the structure at a resolution of 3.1 Å by the molecular-replacement method.

The Gαq-GRK2-Gβγ structure is the first structure obtained to date of Gαq; it also reveals for the first time the configuration of activated Gα and Gγβ subunits at the cell membrane. In the Gαq-GRK2-Gβγ complex, activated Gαq is completely dissociated from Gβγ and undergoes a dramatic, approximately 105° rotation with respect to its position in the Gαβγ heterotrimer. Because GPCRs also interact with GRK2, the Gαq-GRK2-Gβγ structure supports the hypothesis that high-order complexes consisting of receptors, G proteins, and effectors assemble at the cell membrane in response to extracellular stimuli, in this case with GRK2 serving as the scaffold.

GRK2 interacts with Gαq through its regulator of G-protein-signaling (RGS) homology (RH) domain. Many other proteins with RH domains (e.g., RGS4) function as GTPase-activating proteins (GAPs) for Gα subunits, thereby terminating signal transduction by converting the GTP back to GDP. However, the RH domain of GRK2 interacts with Gαq in a manner that is inconsistent with that of a GAP. Instead, the GRK2 RH domain binds to an analogous site on Gαq used by the enzymes adenylyl cyclase or cGMP phosphodiesterase to bind Gαs or Gαt, respectively. This binding site could mean that GRK2, like PLCβ, is in fact a downstream effector target of Gαq. In support of this hypothesis, GRK2 does not appear to impede the binding of RGS4 to Gαq. Therefore, RGS proteins such as RGS4 potentially also participate in the high-order membrane complexes assembled by GRK2.

With the Gαq-GRK2-Gβγ structure, each of the four Gα subunit families has been structurally characterized. Moreover, structures of effector complexes involving each of these subfamilies have been determined. Comparison of these complexes reveals remarkable similarities in how structurally unrelated effectors bind to Gα subunits. Remarkably, each of these effector complexes leaves room for the simultaneous binding of a GAP (such as RGS4). This feature explains how Gα subunits can propagate signals, while remaining responsive to regulatory proteins that control the duration of the signal.


Research conducted by V.M. Tesmer, A. Shankaranarayanan, and J.J.G. Tesmer (University of Michigan, Ann Arbor) and T. Kawano and T. Kozasa (University of Illinois, Chicago).

Research funding: American Heart Association, U.S. National Institutes of Health, and the American Cancer Society. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: V.M. Tesmer, T. Kawano, A. Shankaranarayanan, T. Kozasa, and J.J.G. Tesmer, “Snapshot of activated G proteins at the membrane: The Gαq-GRK2-Gβγ complex,” Science 310, 1686 (2005).