New Insights into Arrestin Recruitment to GPCRs.
- Spillmann M Paul Scherrer Institute, Laboratory of Nanoscale Biology, PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- Thurner L Paul Scherrer Institute, Laboratory of Nanoscale Biology, PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- Romantini N Paul Scherrer Institute, Center for Radiopharmaceutical Sciences (CRS), PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- Zimmermann M InterAx Biotech AG, PARK innovAARE, 5234 Villigen, Switzerland.
- Meger B Paul Scherrer Institute, Laboratory of Nanoscale Biology, PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- Behe M Paul Scherrer Institute, Center for Radiopharmaceutical Sciences (CRS), PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- Waldhoer M InterAx Biotech AG, PARK innovAARE, 5234 Villigen, Switzerland.
- Schertler GFX Paul Scherrer Institute, Division of Biology and Chemistry (BIO), PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- Berger P Paul Scherrer Institute, Laboratory of Nanoscale Biology, PSI, Forschungsstrasse 111, 5232 Villigen, Switzerland.
- 2020-07-17
Published in:
- International journal of molecular sciences. - 2020
English
G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G proteins or arrestins, modulating intracellular signaling cascades. Here, we use a protein-fragment complementation approach based on nanoluciferase (split luciferase assay) to assess interaction of all four known human arrestins with four different GPCRs (two class A and two class B receptors) in live cells. Besides directly tagging the 11S split-luciferase subunit to the receptor, we also could demonstrate that membrane localization of the 11S subunit with a CAAX-tag allowed us to probe arrestin recruitment by endogenously expressed GPCRs. Varying the expression levels of our reporter constructs changed the dynamic behavior of our assay, which we addressed with an advanced baculovirus-based multigene expression system. Our detection assay allowed us to probe the relevance of each of the two arrestin binding sites in the different GPCRs for arrestin binding. We observed remarkable differences between the roles of each arresting binding site in the tested GPCRs and propose that the distinct advantages of our system for probing receptor interaction with effector proteins will help elucidate the molecular basis of GPCR signaling efficacy and specificity in different cell types.
- Language
-
- English
- Open access status
- gold
- Identifiers
-
- DOI 10.3390/ijms21144949
- PMID 32668755
- Persistent URL
- https://sonar.ch/global/documents/126049
Statistics
Document views: 17
File downloads:
- Full-text: 0