Donatella Treppiedi a , Federica Mangili a , Elena Giardino a , Rosa Catalano a , b , Marco Locatelli c , d , Andrea Gerardo Lania e , Anna Spada a , f , Maura Arosio a , f , Davide Calebiro g , h , i , Giovanna Mantovani a , f , * , Erika Peverelli a
01 October 2019
The high expression of somatostatin receptor 2 (SST<sub>2</sub>) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST<sub>2</sub> signaling and expression. The aim of this study was to explore FLNA involvement in SST<sub>2</sub> intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST<sub>2</sub> internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST<sub>2</sub> internalization, control versus FLNA small interfering RNAs (siRNA) cells, respectively, p < 0.001) and human GH-secreting primary cultured cells (70.3 ± 21.1 vs. 24 ± 19.2% SST<sub>2</sub> internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST<sub>2</sub> recycling to the plasma membrane in FLNA silenced GH3 cells. Coimmunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely dependent recruited to octreotide-stimulated SST<sub>2</sub> receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST<sub>2</sub> complex in GH3 cells, it significantly impaired efficient SST<sub>2</sub> loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and 4, respectively (33.7 ± 8.9% down to 25.9 ± 6.9%, p < 0.05, and 28.4 ± 7.4% down to 17.6 ± 5.7%, p < 0.01, for SST<sub>2</sub>-Rab5 and SST<sub>2</sub>-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST<sub>2</sub> trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.