|Titel:||REGULATION OF EPITHELIAL TIGHT JUNCTIONS BY HUMAN SOMATOSTATIN RECEPTOR SUBTYPE 3||Sonstige Titel:||REGULATION DER EPITHELIALEN TIGHT JUNCTIONS DURCH DEN HUMANEN SOMATOSTATINREZEPTOR-SUBTYP 3||Sprache:||Englisch||Autor*in:||LIEW, CHONG WEE||Schlagwörter:||epithelial cell; tight junction; somatostatin receptor||Erscheinungsdatum:||2004||Tag der mündlichen Prüfung:||2004-11-26||Zusammenfassung:||
The regulatory peptide, somatostatin, acts as an inhibitory regulator of various cellular functions throughout the brain and periphery. These actions are mediated by the somatostatin receptor (SSTR) family, which comprises five distinct subtypes (SSTR1-5). In a yeast two-hybrid screen, I identified the tight junction multi-PDZ domain protein MUPP1 as an interaction partner for the C-terminus of human SSTR3. This interaction was shown to be mediated by the PDZ binding motif at the extreme C-terminus of hSSTR3 and the PDZ domain 10 of MUPP1. Subsequently, the interaction was confirmed biochemically in both over-expressing as well as native systems. Interestingly, the rodent homolog of SSTR3 was demonstrated not to interact with MUPP1 due to a subtle difference in its PDZ binding motif. MUPP1 was found to be part of a large protein complex at the synaptic and tight junction. Wild type hSSTR3 was shown to co-localize with MUPP1 at the cell-cell junction in fully polarized MCF-7 and MDCK II cells stably expressing the receptor. Due to the pro-apoptotic properties of the wild type hSSTR3, a fusion receptor was constructed which consisted of mouse SSTR5 and the entire C-terminus of hSSTR3. The fusion receptor was shown by confocal microscopy, to co-localize with MUPP1 at the tight junction. As a fusion receptor lacking the PDZ binding motif was found to be diffusely distributed and did not co-localize with MUPP1, I concluded that the receptor is targeted to a large protein complex at the tight junction through its PDZ-mediated interaction with MUPP1. Functional studies in the breast carcinoma MCF-7 cell line revealed that the receptor was able to disrupt and slow down formation of the tight junction in an agonist-dependent manner. This effect requires a G-protein mediated signaling pathway, as it could be prevented by pretreatment of cells with pertussis toxin (PTX), an inhibitor of GƒÑi/o. However, activation of the receptor in MDCK II stable cell lines yielded a contrasting picture. Agonist treatment was shown to slow down the tight junction formation initially and facilitate the formation later in a calcium-switch paradigm. The agonist-induced effects could be partially reversed by PTX both at the early and late phase of tight junction formation. The 26S proteasome inhibitor, MG-132, was shown to only block the late phase of tight junction formation. Biochemical analyses showed that upon agonist treatment, the tight junction transmembrane protein claudin-2, a determinant for leaky junctions, was selectively targeted for degradation by the 26S proteasome. The PDZ-domain dependent localization to and regulation of tight junctions by the hSSTR3 add a new aspect to the signaling repertoire of G-protein coupled receptors.
|URL:||https://ediss.sub.uni-hamburg.de/handle/ediss/806||URN:||urn:nbn:de:gbv:18-23189||Dokumenttyp:||Dissertation||Betreuer*in:||Richter, Dietmar (Prof. Dr.)|
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|