Real-time imaging of µ-opioid receptor phosphorylation and dephosphorylation
G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors. Phosphorylation is a major mode of regulation of GPCR activity mainly by modifying their ability to interact with various intracellular partners. Agonists can induce specific phosphorylation patterns that establish a barcode to stimulate functional-selective signaling pathways. Agonist-induced phosphorylation of GPCRs is mediated by a family of G protein-coupled receptor kinase (GRKs) that specifically detect the activated state of these receptors.
We have recently demonstrated that phosphorylation of the μ-opioid receptor (MOR) in mouse brain in vivo is regulated by drug-selective engagement of distinct GRKs. In fact, the preferential recruitment of GRK3 or GRK5 leads to distinct MOR phosphorylation patterns that influence different opioid-related behaviors such as tolerance and addiction. These results indicate that GRKs can act as unique sensors that detect different active receptor conformations stabilized by chemically diverse ligands. After phosphorylation, βarrestins bind to the receptor and initiate a second wave of G protein-independent signaling. Hence, dephosphorylation is a critical step in the GPCR live cycle that is required for termination βarrestin signaling and receptor resensitization.
We have recently identified protein phosphatase 1γ (PP1γ) as first G protein-coupled receptor phosphatase (GRP) for MOR and PP1β as GRP for the somatostatin receptor 2 (sst2). Whereas receptor-arrestin-interactions have been studied in great detail, little is known about the molecular interaction of individual GRKs or GRPs with pharmacological relevant GPCRs. In this proposal, we will establish novel FRET-based measurements of the interaction of GRK2, GRK3, GRK5 and GRK6 with MOR and other model GPCRs and analyze the agonist-selective regulation of MOR by different GRKs. We will then develop FRET-based measurements of the interaction of different GRPs such as PP1α, PP1β and PP1γ with a variety of receptors and evaluate the selectivity of GPCR-GRP interactions. Using these novel tools, we will elucidate the temporal dynamics of MOR activation, phosphorylation, arrestin binding and dephosphorylation.
We will combine such measurements with mutational analyses to provide molecular insight into the mechanisms of the selective engagement of different GRKs and GRPs with MOR activated by high- or low-efficacy agonists.
We will also utilize newly generated phosphosite-specific single-domain antibodies (nanobodies pT370, pS375, and pT379) to directly visualize agonist-selective MOR phosphorylation and dephosphorylation in native neurons in vivo and in vitro.
Finally, we will use super-resolution microscopy (dSTORM) and single-molecule microscopy combined with single particle tracking to investigate in which subcellular compartments and microdomains MOR phosphorylation and dephosphorylation is occurring as well as to characterize the interactions between individual MORs and GRPs.
- Friedrich Schiller University Jena
Stefan Schulz will coordinate and supervise the project together with other members of the team.
|Loss of Morphine Reward and Dependence in Mice Lacking G Protein-Coupled Receptor Kinase 5||2014||Gluck, L., Loktev, A., Mouledous, L., Mollereau, C., Law, P. Y., and Schulz, S.||Biol Psychiatry||More|
|Heterologous regulation of agonist-independent mu-opioid receptor phosphorylation by protein kinase C||2014||Illing, S., Mann, A., and Schulz, S.||Brit J Pharmacol||More|
|Differentiation of opioid drug effects by hierarchical multi-site phosphorylation||2013||Just, S., Illing, S., Trester-Zedlitz, M., Lau, E. K., Kotowski, S. J., Miess, E., Mann, A., Doll, C., Trinidad, J. C., Burlingame, A. L., von Zastrow, M., and Schulz, S.||Mol Pharmacol||More|
|Regulation of mu-opioid receptors: desensitization, phosphorylation, internalization, and tolerance||2013||Williams, J. T., Ingram, S. L., Henderson, G., Chavkin, C., von Zastrow, M., Schulz, S., Koch, T., Evans, C. J., and Christie, M. J.||Pharmacol Rev||More|
|Rapid dephosphorylation of G protein-coupled receptors by protein phosphatase 1beta is required for termination of beta-arrestin-dependent signaling||2012||Poll, F., Doll, C., and Schulz, S.||J Biol Chem||More|
|Deciphering mu-opioid receptor phosphorylation and dephosphorylation in HEK293 cells||2012||Doll, C., Poll, F., Peuker, K., Loktev, A., Gluck, L., and Schulz, S.||Brit J Pharmacol||More|
|Analgesic tolerance to high-efficacy agonists but not to morphine is diminished in phosphorylation-deficient S375A mu-opioid receptor knock-in mice||2011||Grecksch, G., Just, S., Pierstorff, C., Imhof, A. K., Gluck, L., Doll, C., Lupp, A., Becker, A., Koch, T., Stumm, R., Hollt, V., and Schulz, S.||J Neurosci||More|
|Agonist-selective patterns of mu-opioid receptor phosphorylation revealed by phosphosite-specific antibodies||2011||Doll, C., Konietzko, J., Poll, F., Koch, T., Hollt, V., and Schulz, S.||Brit J Pharmacol||More|
|Real-time monitoring of somatostatin receptor-cAMP signaling in live pituitary||2010||Jacobs, S., Calebiro, D., Nikolaev, V. O., Lohse, M. J., and Schulz, S.||Endocrinology||More|
|Morphine induces terminal mu-opioid receptor desensitization by sustained phosphorylation of serine-375||2004||Schulz, S., Mayer, D., Pfeiffer, M., Stumm, R., Koch, T., and Hollt, V.||EMBO J||More|