Cytoskeleton Protein Filamin A Is Required for Efficient Somatostatin Receptor Type 2 Internalization and Recycling through Rab5 and Rab4 Sorting Endosomes in Tumor Somatotroph Cells

Abstract

The high expression of somatostatin receptor 2 (SST₂) 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 SST₂ signaling and expression. The aim of this study was to explore FLNA involvement in SST₂ intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST₂ internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST₂ 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% SST₂ internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST₂ 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 SST₂ receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST₂ complex in GH3 cells, it significantly impaired efficient SST₂ 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 SST₂-Rab5 and SST₂-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST₂ trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.

Pituitary Tumors: Genetic and Molecular Factors Underlying Pathogenesis and Clinical Behavior

“…A protective effect of FLNA from receptor degradation was also demonstrated for other receptors, including calcium-sensing receptor [125], calcitonin receptor [126], cystic fibrosis transmembrane conductance regulator [127], and the high-affinity IgG receptor FcgammaRI [128]. FLNA was required for efficient recycling of SST2 in GH-secreting PitNETs [129] and chemokine receptor CCR2 and β2-adrenergic receptor [130].…”

“…In GH-secreting PitNET cells, FLNA was essential for SST2 internalization upon agonist incubation (Fig. 3) [129]. Despite FLNA being a binding partner of β-arrestin-2, FLNA knockdown did not prevent the formation of β-arrestin-2-SST2 complexes in GH3 cells but significantly impaired SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers, Rab5 and 4, respectively, and prevented SST2 recycling to the cell membrane [129].…”

The Actin Bundling Protein Fascin-1 as an ACE2-Accessory Protein

“…Rearrangement of actin cytoskeleton has been shown to play important roles in intracellular traffic (Gallop 2019 ) and various proteins have been shown to specifically regulate the transport of receptors like tubulin for AT 1 R and α 2B -adrenergic receptors (Duvernay et al 2011 ; Zhang et al 2013 ), filamin A for somatostatin receptor type 2 (Treppiedi et al 2019 ), and ERM proteins for tyrosine kinase receptors and viruses (Neisch and Fehon 2011 ). Our data reveal that beside the previously reported functions of fascin-1 (Adams 2004 ; Gallop 2019 ; Jayo and Parsons 2010 ), this actin bundling protein also mediates the traffic from plasma membrane to endo-lysosomal system.…”

Epithelial–Mesenchymal Transition in the Resistance to Somatostatin Receptor Ligands in Acromegaly

“…Additionally, it has been proved that FLNA mediates octreotide-induced SSTR2 trafficking through endosomal proteins in acromegaly. Moreover, FLNA influences the number of available SSTR2 at the surface of the cell (73).…”

“…Additionally, it has been proved that FLNA mediates octreotide-induced SSTR2 trafficking through endosomal proteins in acromegaly. Moreover, FLNA influences the number of available SSTR2 at the surface of the cell ( 73 ). For more detailed explanation of the cytoskeleton involvement in SRLs resistance, we recommend the review by Peverelli et al.…”