Thomas Jefferson University

Main menu:

Department of Biochemistry & Molecular Biology

Biochemistry

The Department of Biochemistry and Molecular Biology is an established basic science department that plays a major role in the research and education missions of Thomas Jefferson University. The overall goal of the Department is to make basic and translational discoveries that impact our understanding of the biological sciences and human health and to train the researchers, educators and health care professionals of the future.

The Department’s research programs cover a diverse range of areas from cell signaling, transcription and programmed cell death to understanding diseases such as cancer, neurodegeneration and AIDS. Areas of particular strength include cell signaling and receptor biology, protein trafficking, DNA replication and repair, transcription and translation, protein and nucleic acid structure and function, and mechanisms involved in disease. The Department also coordinates several major areas of education including the Biochemistry and Molecular Pharmacology PhD program in the Jefferson College of Biomedical Sciences and the training of first-year medical students in the Sidney Kimmel Medical College and first-year pharmacy students in the Jefferson College of Pharmacy


Recent Departmental Highlights

A collaborative study by the laboratories of Jeffrey L. Benovic, PhD, in the Department of Biochemistry and Molecular Biology at Thomas Jefferson University and the Sidney Kimmel Cancer Center at Jefferson, and Dr. Brian K. Kobilka in the Department of Molecular and Cellular Physiology at the Stanford University School of Medicine provides new insights into a general mechanism regulating signaling from G protein-coupled receptors (GPCRs). Two senior postdoctoral researchers, Dr. Konstantin E. Komolov of the Benovic lab and Dr. Yang Du of the Kobilka lab, share co-first authorship of the new study, which is published in the April 20th issue of Cell.

GPCRs physically span the cell membrane. There they play a central role in enabling cells to respond to extracellular stimuli, including various hormones, neurotransmitters, peptides, and proteins. Stimulation of the GPCR results in activation of cellular G proteins, which in turn modulate the activity of downstream effectors that ultimately control numerous cellular functions, such as cell growth and motility. These signaling events are tightly regulated.  One such regulator is a family of proteins called GPCR kinases (GRKs). GRKs bind to and phosphorylate the stimulated receptor, after which a different protein called arrestin then specifically binds to the phosphorylated GPCR.  Through this process, GRKs deactivate G protein signaling and activate arrestin-mediated signaling. As Benovic explains, “GRKs play a central role in switching cells from G protein signaling to arrestin-mediated signaling, which is critical in maintaining normal cellular homeostasis.”

In the Cell study, the Benovic and Kobilka groups studied the interaction of a particular GPCR kinase, GRK5, with the β2-adrenergic receptor (β2AR), a cell membrane bound GPCR that is activated (stimulated) by binding to catecholamines such as adrenaline. Their findings reveal key mechanistic features of how these two proteins interact, and how this interaction leads to conformational changes in the GRK that are essential for mediating receptor phosphorylation.

In order to study these interactions within the GRK5/β2AR complex, the researchers first had to replicate the formation of the stable complex, which in cells is associated with the cell membrane. They found that acidic lipids, like the naturally occurring ones found in cell membranes, greatly enhanced the binding of GRK5 to β2AR.  They further found that although GRK5 would bind to an inactive form of β2AR, it was binding of GRK5 to an activated (agonist-bound) β2AR that produced a functional complex, one in which GRK5 could phosphorylate β2AR. This suggested that agonist binding created conformational changes in β2AR structure, making it the preferred GRK5 binding partner and enabling formation of a functional GRK5/β2AR complex. 

With an active, functional complex in hand, the team used a comprehensive integrated approach to analyze the molecular interactions of GRK5 with β2AR.  They demonstrated that GRK5 binding to β2AR involved interactions at multiple sites to produce a functional complex.  In addition, just as the binding of an agonist caused changes in β2AR structure and shape, the binding of β2AR was shown to induce conformational changes in GRK5 – by disrupting key internal contacts between two major GRK5 domains, causing them to separate which in turn caused the kinase domain of GRK5 to adopt an active conformation. 

Finally, using multiple cross linking-Mass Spectroscopy (MS) strategies, the Jefferson and Stanford researchers mapped the GRK5/β2AR interface, identifying the protein regions that directly participated in the interactions. Those data guided computational modeling and docking studies, permitting the investigators to generate a 3-D model of the GRK5/β2AR complex – one that shows a possible progression of the conformational changes associated with three potential stages of complex formation.  Data from additional studies, using hydrogen-deuterium exchange MS, supported the team’s model of the active GRK5/β2AR complex.

“The molecular model derived from these studies provides important insights into a common mechanism of GRK-GPCR interaction, raising the exciting possibility of exploiting this mechanism to control GPCR signaling,” adds Benovic.

The new findings hold promise for many clinical applications in the future. GPCRs are the target of ~30% of drugs currently on the market, including drugs for the treatment of cancer, cardiovascular and airway disease, as well as various neurological and metabolic disorders. Because GRKs play a central role in regulating GPCR function, a better understanding of the mechanisms involved in this process provides an opportunity to manipulate this pathway in treating various diseases. Another illustration of the importance of understanding the physiological roles of GRK-GPCR interactions in human physiology comes from the report, by another group, that a naturally occurring difference in a single amino acid of the GRK5 protein may enhance phosphorylation of β2AR. Especially intriguing, it has been proposed that this amino acid difference may confer some protection against the development of congestive heart failure for the large percentage of the African American population that have this amino acid difference.    

Future plans include dissecting further the interaction of GRK5 with β2AR  using high-resolution structural and imaging approaches such as X-ray crystallography and cryo-electron microscopy, and also analyzing the dynamics of the interaction using approaches such as radiolytic footprinting and double electron electron resonance (DEER) spectroscopy. Studies are also envisioned to explore the broader significance of the newly reported results by examining other interacting pairs of GPCRs and GRKs. Commenting on the long-term goals of this project, Benovic notes that “understanding the structure of a GRK-GPCR complex should help us develop small molecules that enable us to either enhance or inhibit GRK regulation of the receptor, which should have tremendous implications for treating a wide variety of diseases.”

This work was supported by NIH awards R01GM068857 and P01HL114471 (to J.L.B.) and R01GM083118 (to B.K.K.), the Mathers Foundation (to B.K.K.) a Stanford University Terman Faculty Fellowship (to R.O.D.) and the National Research Foundation of Korea funded by the Korean government (NFR-2015R1A1A1A05027473 and NRF-2012R1A5A2A28671860) (to K.Y.C.).

Article Reference: Konstantin E. Komolov, Yang Du, Nguyen Minh Duc, Robin M. Betz, João P. G. L. M. Rodrigues, Ryan D. Leib, Dhabaleswar Patra, Georgios Skiniotis, Christopher M. Adams, Ron O. Dror, Ka Young Chung, Brian K. Kobilka, and Jeffrey L. Benovic, “Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5,” 2017, Cell 169, 407-421.  DOI: 10.1016/j.cell.2017.03.047.