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Jun 23, 2014

Arrestin's Muting of GPCR Signaling Visualized

Arrestin's Muting of GPCR Signaling Visualized

A structural model of the beta2 adrenergic receptor-arrestin signaling complex as deduced by electron microscopy, cross-linking, and mass spectrometry. [Duke University]

  • Capturing how G-protein coupled receptors (GPCRs) respond to extracellular stimuli is notoriously difficult. The task, for ordinary mortals, seems akin to studying the flight of a butterfly by securing the poor creature to a rack. Yet gifted scientists, such as the Nobel winners Robert J. Lefkowitz, M.D., and Brian K. Kobilka, M.D., persist in finding ways to stabilize GPCRs, the better to discern how these graceful proteins flap about their membranous perches and hold, however fleetingly, one pose or another.

    Most recently, Drs. Lefkowitz and Kobilka, affiliated with Duke University and Stanford Universty, respectively, teamed up with Georgios Skiniotis, Ph.D., a researcher at the University of Michigan, to reveal how arrestin acts to mute GPCR signaling. The scientists published their results June 22 in Nature, in an article entitled “Visualization of arrestin recruitment by a G-protein-coupled receptor.”

    “A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)–G-protein complex, has provided novel insights into the structural basis of receptor activation,” wrote the authors. “However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor–β-arrestin complexes for structural studies.”

    The researchers devised a strategy for forming and purifying a functional human β2AR–β-arrestin-1 complex that allowed them to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and chemical cross-linking.

    “High-resolution visualization of this signaling assembly is challenging because the protein complexes are transient, and highly dynamic and large amounts of the isolated proteins are required for the experiments," said co-lead author Arun K. Shukla, Ph.D., who worked with Dr. Lefkowitz at Duke and is now setting up an independent laboratory in the Department of Biological Sciences and Bioengineering at the Indian Institute of Technology, Kanpur.

    Once the authors had material available for direct structural visualization, they used electron microscopy to reveal how the individual molecules of this signaling assembly are organized with respect to each other. The researchers then combined thousands of individual images to generate a better picture of the molecular architecture. They further clarified this picture by cross-linking analysis and mass spectrometry measurements.

    “Our results suggest that arrestin probably employs a biphasic mechanism to engage the receptor. The first phase involves an interaction between the phosphorylated C-terminal tail of the receptor and the N-terminal domain of arrestin. Given the flexibility and the length of the C-terminal receptor tail, it is expected to act like a fishing line, sampling a wide interaction space at a high rate,” the authors continued. “The second point of interaction appears weak and involves primarily the insertion of the finger loop within the receptor core, resulting in a longitudinal arrangement of arrestin on the receptor. This arrangement would most certainly preclude GPCR engagement of G-protein heterotrimers, thereby blocking classical GPCR signalling and inducing desensitization.”

    “It is crucial to visualize how these receptors work to fully appreciate how our bodies respond to a wide array of stimuli, including light, hormones and various chemicals,” added Dr. Lefkowitz.

    The authors next aim to obtain greater detail about this assembly using X-ray crystallography, a technology that should reveal atomic level insights into this architecture. Such atomic details could then be used in experiments to design novel drugs and develop a better understanding of fundamental concepts in GPCR biology.

    “This is just a start and there is a long way to go,” Dr. Shukla said. “We have to visualize similar complexes of other GPCRs to develop a comprehensive understanding of this family of receptors.”


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