G protein-coupled receptors, short GPCRs, are a family of 800 different membrane proteins in the human genome. The adhesion GPCR subgroup contains 33 family members, which are outstanding due to their very large extracellular domain as well as their ability to sense mechanical stimuli.
Surprisingly the length of the intracellular loops and C-terminus is very versatile and does not follow any obvious pattern. Inexplicable is why some receptors do have very short intracellular C-termini and others excessively long C-termini. It is evident that their intracellular architecture has an impact on the downstream signaling through different effectors. A mechanistical understanding of this structural regulation of signaling biased could provide a completely new insight into GPCR signaling transduction.
In the past, I was working with class A GPCRs as well as arrestins and their downstream effectors JNK and Src kinases. Arrestins were found to have several switch regions which rearrange during the activation by GPCRs. It is unknown how arrestins bind different downstream effectors and how the active structure of arrestin influences the binding. The aim was to understand the regulatory mechanism of arrestin on these different effectors. How structural rearrangements within the protein cause an activation of different kinases. We found specific binding motifs of Src family kinases within arrestin using techniques like X-ray crystallography, NMR spectroscopy, and classical biochemical approaches like peptide arrays and pull-down assays. Now I would like to use this knowledge and apply it to the field of adhesion GPCRs.

 

Fig 1. Schematic view of intracellular effector binding. The BAI2 is shown in black, arrestin is colored in green (PDB: 3P2D), Calmodulin in blue (PDB: 3IQL) and Endophilin A1 in red (PDB: 1ZWW) and downstream effectors like ERK2 in yellow (PDB: 6D5Y) and Src in tinte colors (PDB: 2SRC).

 

Downstream effectors of adhesion GPCRs
Our group focuses on the elucidation of signaling pathways through different effectors of adhesion GPCRs. We will target predominantly arrestin, calmodulin as well as Src family kinases in combination with ERK. So far, only little is known about the downstream signaling of adhesion GPCRs.
We will use X-ray crystallography as well as NMR spectroscopy to identify structural mechanisms of downstream effector activation. Furthermore, we will use classical biochemical approaches to characterize the protein-protein interaction. These findings will be compiled with different functional cell assays which will give further insight into the mechanistic and even physiological impact of these interactions.

  

• Investigating the interaction of the BAI2 C-terminus with Calmodulin
• Regulation of Src family kinases by BAI2

 

Lisa Kupsch (Master Student)
Melina Hötger (Master Student)

 

Peer-reviewed Publications

1. Berndt S, Liebscher I. New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation. Int J Mol Sci. 2021 Jun 17;22(12):6489.
2. Perez I, Berndt S, Agarwal R, Castro MA, Vishnivetskiy SA, Smith JC, Sanders CR, Gurevich VV, Iverson TM: A model for the signal initiation complex between Arrestin-3 and the Src family kinase Fgr. J Mol Biol. 2021 Dec 10;167400
3. Chen Q, Zhuo Y, Sharma P, Perez I, Francis DJ, Chakravarthy S, Vishnivetskiy SA, Berndt S, Hanson SM, Zhan X, Brooks EK, Altenbach C, Hubbell WL, Klug CS, Iverson TM, Gurevich VV. An eight amino acid segment controls oligomerization and preferred conformation of the two non-visual arrestins. J Mol Biol. 2020; 166790.
4. Berndt S, Gurevich VV, Iverson TM: Crystal structure of the SH3 domain of human Lyn non-receptor tyrosine kinase. PLoS One. 2019; 14(4):e0215140.
5. Perry NA, Kaoud TS, Ortega OO, Kaya AI, Marcus DJ, Pleinis JM, Berndt S, Chen Q, Zhan X, Dalby KN, Lopez CF, Iverson TM, Gurevich VV. Arrestin-3 scaffolding of the JNK3 cascade suggests a mechanism for signal amplification. Proc Natl Acad Sci USA. 2019; 116(3):810-815.
6. Starbird CA, Perry NA, Chen Q, Berndt S, Yamakawa I, Loukachevitch LV, Limbrick EM, Bachmann BO, Iverson TM, McCulloch KM. The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains. Biochemistry. 2018;57(50):6827-6837.
7. Chen Q, Perry NA, Vishnivetskiy SA, Berndt S, Gilbert NC, Zhuo Y, Singh PK, Tholen J, Ohi MD, Gurevich EV, Brautigam CA, Klug CS, Gurevich VV, Iverson TM: Structural basis of arrestin-3 activation and signaling. Nat Commun. 2017;8(1):1427.
8. Berndt S, Gurevich VV, Gurevich EV. Arrestins in Cell Death. In: Gurevich V. (eds) The Structural Basis of Arrestin Functions. Springer, Cham. 2017.
9. Witte K, Kaiser A, Schmidt P, Splith V, Thomas L, Berndt S, Huster D, Beck-Sickinger AG. Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration.Biol Chem. 2013;394(8):1045-56.
10. Berger C, Berndt S, Pichert A, Theisgen S, Huster D. Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct. Biotechnol Bioeng. 2013;110(6):1681-90.
11. Berger C, Montag C, Berndt S, Huster D. Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production. Protein Expr Purif. 2011;76(1):25-35.
12. Schmidt P, Berger C, Scheidt HA, Berndt S, Bunge A, Beck-Sickinger AG, Huster D. A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment. Biophys Chem. 2010;150(1-3):29-36.
13. Schmidt P, Lindner D, Montag C, Berndt S, Beck-Sickinger AG, Rudolph R, Huster D. Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2.Biotechnol Prog. 2009;25(6):1732-9.

 

 

01/2021 – present	Rudolf-Schönheimer-Institute of Biochemistry, University of Leipzig, Leipzig, Germany  Group leader with Prof. Ines Liebscher Research: Adhesion GPCRs, cell signaling, X-ray crystallography, EPR and NMR spectroscopy, Src kinases, Arrestins
05/2015 – 12/2020	Department of Pharmacology, Vanderbilt University, Nashville, TN, USA  Postdoctoral Fellow with Prof. Vsevolod Gurevich and Prof. Tina Iverson. Research: Cell signaling, Arrestin, Src family kinases, G protein-coupled receptor, cell culture, recombinant protein expression, X-ray crystallography, NMR spectroscopy
08/2013 – 04/2015	Department of Biochemistry, University of Cambridge, Cambridge, UK  Postdoctoral Fellow with Dr. Daniel Nietlispach. Research: G protein-coupled receptor, ß1-adrenergic receptor, insect cell expression, NMR-spectroscopy
06/2009 – 08/2013	Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany  Graduate Student with Prof. Daniel Huster. Thesis: The extracellular lysine residues of in vitro folded neuropeptide Y receptor type 2 interacting with its ligand observed by NMR-spectroscopy
01/2004 – 06/2009	Institute of Biotechnology, Martin-Luther University of Halle-Wittenberg, Germany  Diploma Student (Diploma in Biochemistry). Thesis: Optimization of the in vitro-preparation of prokaryotic expressed Y2 Receptor for structural analysis by NMR-spectroscopy