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Cell Signaling: G-Protein Coupled Receptors and the β2-Adrenergic Receptor Dianna Amasino, Axel Glaubitz, Susan Huang, Joy Li, Hsien-Yu Shih, Junyao Song, Xiao Zhu Madison West High School, Madison, WI 53726 Advisor: Basudeb Bhattacharyya, University of Wisconsin, Madison, WI 53706 Mentors: Dr. Dave Nelson and Peter Vander Velden, University of Wisconsin, Madison, WI 53706 Introduction G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins coded by the human genome. GPCRs are important for signal transduction with the general structural characteristic of a plasma membrane receptor with seven transmembrane segments (Figure 1). One example of a GPCR targeted by pharmaceutical companies is the β2-adrenergic receptor. Adrenergic receptors are found through out the body and are triggered by the hormone epinephrine (also known as adrenaline, hence the name adrenergic). When epinephrine binds to the receptors, it causes a slight conformational change within the receptor. This change then triggers activation of a G-protein, which induces a response within the cell (for example, muscle contraction). When this signal transduction event functions normally in the body, it helps regulate heart rate and blood pressure and is important for the “fight or flight” response. Beta blockers are medically used to bind to adrenergic receptors, manipulating the hormone’s concentration. We have used rapid prototyping technology to model the interaction of the human β2adrenergic receptor with the beta blocker, carazolol. By modeling the β2-adrenergic receptor, we hope to better understand GPCRs as well as understand the mechanism of hormone/drug binding, which will aid in developing better drug treatments. Mechanism of the β adrenergic receptor: an example of GPCR signal transduction A conserved structure and mechanism Cherezov, et al., 2007 Figure 4. Overlay of bovine rhodopsin (cyan) and the human β2-adrenergic receptor (orange) Figure 4 above demonstrates how conserved the structure, and hence the mechannsm, of signal transduction using GPCRs really is. Rhodopsin, one of the most thoroughly studied GPCRs, binds retinal and is needed for vision. The two proteins’ active sites line up almost perfectly three dimensionally, even though the two proteins have seemingly completely different purposes. A Pharmaceutical Target Nelson and Cox, 2007 Figure 2. Mode of Action of the β2 adrenergic receptor Cherezov, et al., 2007 Figure 1. General Structure and Location of GPCRs Like all GCPRs, adrenergic receptor has a structure of seven alpha-helical transmembrane architecture. The external signal—the ligand—binds to the receptor and causes it to undergo a conformational change. The mechanism, which involves the transfer of G proteins, is universally adopted by all GCPRs. The picture above illustrates the structure of β2adrenergic receptor cross cell membrane. Cholesterol is also required for proper functioning of the receptor. ¾ Upon binding of the ligand (epinephrine) to the cytosolic face, a conformational change occurs to both the receptor and the G protein (guanine nucleotide binding protein). ¾ The conformational change leads to the exchange of GDP for GTP on the α-subunit of the G protein. ¾ Upon binding of GTP, the α-subunit dissociates and binds to its effector protein. The beta and gamma subunits facilitate the G-protein transfer and help specify the enzyme. ¾ After some random amount of time, the GTPase activity that is built into the α-subunit cleaves GTP to GDP. ¾ Cleavage of GTP causes the α-subunit to revert to its inactive conformation, to leave its target protein, and to re-associate with the β- and γ-subunits. The system thus returns to its resting state. ¾ In the case of epinephrine, epinephrine binds to its receptor, the conformational change of the receptor allows it to be coupled to the G protein, which binds to adenylate cyclase as its effector protein and activates it. Nelson and Cox, 2007 Figure 3. Nobel Prize Winning Research Alfred Gilman and Martin Rodbell won the 1994 Nobel Prize in Medicine for understanding signal transduction through GPCR mechanisms. Figure 5. β2-adrenergic receptor bound to the beta blocker drug carazolol (orange) Many human diseases, such as diabetes and hypertension (high blood pressure) are linked to GPCRs. Determining the structure of GPCRs such as the β2-adrenergic receptor will lead to the development of more effective drug treatments. We have modeled the interaction between one such drugGPCR interaction. References Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, and Stevens RC. (2007) High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein-Coupled Receptor. Science. 318: 1258-1265. Nelson DL and Cox MM. (2007) Lehninger Principles of Biochemistry. 5th Ed. Worth Publishers. New York.