title: Biochemical Large-Scale Interaction Analysis of Murine Olfactory Receptors and Associated Signaling Proteins with Post-Synaptic Density 95, Drosophila Discs Large, Zona-Occludens 1 (PDZ) Domains.
abstract: G protein-coupled receptors (GPCRs) constitute the largest family among mammalian membrane proteins and are capable of initiating numerous essential signaling cascades. Various GPCR-mediated pathways are organized into protein microdomains that can be orchestrated and regulated through scaffolding proteins, such as PSD-95/discs-large/ZO1 (PDZ) domain proteins. However, detailed binding characteristics of PDZ-GPCR interactions remain elusive because these interactions seem to be more complex than previously thought. To address this issue, we analyzed binding modalities using our established model system. This system includes the 13 individual PDZ domains of the multiple PDZ domain protein 1 (MUPP1; the largest PDZ protein), a broad range of murine olfactory receptors (a multifaceted gene cluster within the family of GPCRs), and associated olfactory signaling proteins. These proteins were analyzed in a large-scale peptide microarray approach and continuative interaction studies. As a result, we demonstrate that canonical binding motifs were not overrepresented among the interaction partners of MUPP1. Furthermore, C-terminal phosphorylation and distinct amino acid replacements abolished PDZ binding promiscuity. In addition to the described in vitro experiments, we identified new interaction partners within the murine olfactory epithelium using pull-down-based interactomics and could verify the partners through co-immunoprecipitation. In summary, the present study provides important insight into the complexity of the binding characteristics of PDZ-GPCR interactions based on olfactory signaling proteins, which could identify novel clinical targets for GPCR-associated diseases in the future.
authors.affiliation: From the ‡Department of Cell Physiology, Faculty for Biology and Biotechnology.
authors.forename: Fabian
authors.initials: F
authors.lastname: Jansen
authors.affiliation: From the ‡Department of Cell Physiology, Faculty for Biology and Biotechnology.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Kalbe
authors.affiliation: From the ‡Department of Cell Physiology, Faculty for Biology and Biotechnology.
authors.forename: Paul
authors.initials: P
authors.lastname: Scholz
authors.affiliation: §Department of Analytical Chemistry, Faculty for Chemistry and Biochemistry, Ruhr-University Bochum, Germany;
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fränzel
authors.affiliation: From the ‡Department of Cell Physiology, Faculty for Biology and Biotechnology.
authors.forename: Markus
authors.initials: M
authors.lastname: Osterloh
authors.affiliation: §Department of Analytical Chemistry, Faculty for Chemistry and Biochemistry, Ruhr-University Bochum, Germany;
authors.forename: Dirk
authors.initials: D
authors.lastname: Wolters
authors.affiliation: From the ‡Department of Cell Physiology, Faculty for Biology and Biotechnology.
authors.forename: Hanns
authors.initials: H
authors.lastname: Hatt
authors.affiliation: ¶Institute for Pharmacology and Toxicology, University Clinic Jena.
authors.forename: Eva Maria
authors.initials: EM
authors.lastname: Neuhaus
authors.affiliation: From the ‡Department of Cell Physiology, Faculty for Biology and Biotechnology, Sabrina.Baumgart@rub.de.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Osterloh
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008822:Mice, Transgenic; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D018035:Receptors, Odorant / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C491750:Mpdz protein, mouse; D010455:Peptides; D018035:Receptors, Odorant