Spatiotemporal and structural aspects of G protein-mediated signal transduction
Prof. Dr. Moritz Bünemann
Department of Pharmacology and Clinical Pharmacy
Philipps-Universtitaet Marburg
Karl-von-Frisch-Strasse 1
D-35033 Marburg
Phone: +49 6421 2825702
Fax: +49 6421 2825720
moritz.buenemann@staff.uni-marburg.de
Homepage: http://www.uni-marburg.de/fb16/ipkp
Project description:
This project aims to understand the spatiotemporal dynamics of G protein signaling. A detailed analysis of how, when and where proteins within this signaling cascade interact may be a key towards understanding the specificity of receptor and drug induced actions. We aim to spatially and temporally resolve interactions of GPCRs with G proteins and interactions of G protein subunits with downstream effectors and regulators in the absence and presence of agonists in order to delineate the underlying coupling mechanisms. Special attention will be given to the role of spatiotemporal compartmentalization of selected signaling pathways in respect to generation of signal specificity. The main objects of this project are G protein pathways regulating ion channels, adenylyl cyclases and phospholipases C β. We will study these pathways in the context of physiologically and pharmacologically relevant receptors such as muscarinic, adrenergic, and serotonergic receptor subtypes. The proposed studies are based on fluorescence microscopy, specifically on fluorescence resonance energy transfer (FRET) and protein mobility assays by means of fluorescence recovery after photobleaching (FRAP). Goals of the project are to determine the role of membrane micro-compartments such as caveolae on the spatiotemporal dynamics of G protein signaling and to resolve their impact on generation of signal specificity important for disease and therapy.
1. Dynamics of Gq protein mediated signal transduction in and outside of caveolae
Goals:
• Mapping the distribution of Gq protein-interacting proteins into membrane micro-compartments
–Pharmacologically relevant receptors such as M1,3-AChR and angiotensin AT1-R, Gq proteins, PLCb and RhoGEF
• How fast do these proteins move in and out of lipid
micro-compartments?
–Immobilize micro-compartments by means of antibody-mediated crosslinking
–Determine the effect of caveolae
on the lateral mobility of individual members of the Gq protein
signaling pathway.
-FRAP experiments on micropatterned surfaces
• How do caveolae affect dynamics of Gq signaling?
–Determination of receptor / G protein interaction
–Gq protein activation
–Gaq and Gbg interactions with PLCb and RhoGEF proteins in dependence of caveolar integrity
2. Project: Receptor mediated regulation of adenylyl cyclases: micro-compartments and dynamics
Goals:
• Identification of membrane micro-compartments for b1- and b2-adrenergic receptors and downstream members of the signaling cascade
–Analysis of sorting and trafficking in polarized MDCK cells
–Role of caveolae
–Cholesterol dependence
–Dynamics of residence
• How do caveolae and raft-like domains affect dynamics of receptor mediated signaling via adenylyl cyclases?
–Analysis of dynamics of individual steps of the signaling cascade in dependence of targeting to caveolae or raft-like domains
–Micropatterning of membrane subdomains to microarrays
Staff:
Dr. Andreas Rinne, Postdoc
Sandra Engel, Technician
Markus Milde, PhD student
Thomas Pollinger, MD student
Dr. Cornelius Krasel, research associate (Akadem. Rat)
Publications since 2007
Werthmann R, Lohse, MJ, and Bünemann M. Temporally resolved cAMP monitoring in endothelial cells uncovers a thrombin-induced [cAMP] elevation mediated via the Ca2+-dependent production of prostacyclin. Journal of Physiology 589(1):181-93. (2011)
Hommers LG, Dees C and Bünemann M. G proteins in reverse mode – receptor-mediated GTP release inhibits G protein and effector function. Journal of Biological Chemistry 285(11):8227-33. (2010)
Dorsch S, Klotz KN, Engelhardt S, Lohse MJ and Bünemann M. Analysis of receptor oligomerization by FRAP microscopy. Nat Methods. 6, 225-30. (2009)
Rochais F, Vilardaga J-P, Viacheslav O Nikolaev, Bünemann M, Lohse MJ Engelhardt S. Real-time optical recording of β1- adrenergic receptor activation and signaling reveals supersensitivity of the Arg-389 variant to carvedilol. Journal of Clinical Investigation. 117(1):229-35. (2007)
Nikolaev VO, Boettcher C, Dees C, Bünemann M, Lohse MJ, Zenk MH. Live cell monitoring of µ-opioid receptor-mediated G-protein activation reveals strong biological activity of close morphine biosynthetic precursors. Journal of Biological Chemistry 282(37):27126-32. (2007)
