ECE2016 Guided Posters Neuroendocrinology (10 abstracts)
High-resolution spatiotemporal analysis of Somatostatin Receptor Type 2 (SSTR2) – Filamin A (FLNA) interaction by single-molecule imaging
Donatella Treppiedi 1 , Giovanna Mantovani 1 , Erika Peverelli 1 , Titiwat Sungkaworn 2, , Ulrike Zabel 2, , Elena Giardino 1 , Martin J Lohse 2, , Anna Spada 1 & Davide Calebiro 2,
1Endocrine Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy; 2Institute of Pharmacology and Toxicology and 3 Rudolf Virchow Center, Deutsche Forschungsgemeinschaft–Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; 3Deutsche Forschungsgemeinschaft–Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
SSTR2 is one of the main pharmacological target for GH-secreting pituitary adenomas treatment and the cytoskeletal protein FLNA plays an important role in tumor responsiveness by regulating SSTR2 expression and signaling. Single-Molecule Imaging was used to investigate the spatial distribution of FLNA-SSTR2 at the plasma membrane level and the involvement of FLNA in SSTR2 mobility, receptor clustering organization/internalization. This method is based on labeling SNAP/CLIP-tagged proteins (SNAP-tagged SSTR2, CLIP-tagged FLNA) and on total internal reflection fluorescence (TIRF) microscopy to visualize single particles on the surface of living cells. First, we observed dynamic and transient FLNA-SSTR2 interactions along actin fibers in transfected CHO cells. As far as SSTR2 mobility was concerned, no differences were found in SSTR2 average speed between basal and 100 nM BIM23120 stimulated CHO cells. However, an increase of the immobile receptors fraction (2.17%±0.21% vs 6.05%±0.69%) has been observed after SSTR2 agonist stimulation (this population of static receptor is the one that likely undergo internalization). To evaluate the contribution of FLNA in the enrichment of the immobile SSTR2 population, we assessed experiments with the FLNA dominant negative mutant that prevents SSTR2-FLNA binding (FLNA 19-20), but no differences were found with respect to negative control (FLNA 17-18). This result was confirmed in human melanoma cell lines M2 (FLNA-deficient) and A7 (FLNA-expressing). In conclusion, the spatiotemporal behavior of SSTR2 and SSTR2-FLNA interactions have been characterized for the first time by means high-resolution strategy. The interaction between FLNA and SSTR2 resulted to be extremely dynamic along actin filaments, but FLNA does not seem to be involved in SSTR2 receptor mobility. Since SSTR2 tended to cluster on FLNA fibers in stimulated cells, further experiments are required to better investigate the possible role of FLNA in the regulation of SSTR2 clusterization/internalization processes.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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