Filamin A is required for somatostatin receptor type 5 expression and pasireotide-mediated signaling in pituitary corticotroph tumor cells

Treppiedi, Donatella; Muro, Genesio Di; Mangili, Federica; Catalano, Rosa; Giardino, Elena; Barbieri, Anna Maria; Locatelli, Marco; Arosio, Maura; Spada, Anna; Peverelli, Erika; Mantovani, Giovanna

doi:10.1016/j.mce.2021.111159

Cited by 7 publications

(15 citation statements)

References 51 publications

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“…For in situ Proximity Ligation Assay (PLA) a commercially available kit was used (Duolink; Sigma-Aldrich, St. Louis, MO, USA), accordingly to previous protocols [27]. For both applications, coverslips were mounted on glass slides with Duolink In Situ Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) for subsequent observation at fluorescence microscope.…”

Section: Immunofluorescence Analysis and In Situ Proximity Ligation Assaymentioning

confidence: 99%

“…For both applications, coverslips were mounted on glass slides with Duolink In Situ Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) for subsequent observation at fluorescence microscope. Proximity ligation events were quantified with NIH ImageJ software [27]. Briefly, cells were first searched using an excitation light of 405 nm (DAPI signal) and fine focus adjustment was performed switching the filter of the excitation light to FITC (488 nm).…”

Section: Immunofluorescence Analysis and In Situ Proximity Ligation Assaymentioning

confidence: 99%

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Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

Treppiedi

Barbieri

Muro

et al. 2021

Cancers

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Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, p < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, p < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, p < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.

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Section: Immunofluorescence Analysis and In Situ Proximity Ligation Assaymentioning

confidence: 99%

Section: Immunofluorescence Analysis and In Situ Proximity Ligation Assaymentioning

confidence: 99%

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

Treppiedi

Barbieri

Muro

et al. 2021

Cancers

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“…Proximity ligation events were quantified with NIH ImageJ software after image deconvolution. The average number of PLA puncta per cell are related to images acquired from randomly chosen fields per condition from three independent experiments and quantified as previously described (34).…”

Section: Plasmid Transfectionmentioning

confidence: 99%

“…By means of single-molecule imaging and in situ proximity ligation assay (PLA), we have previously demonstrated that SST 2 and SST 5 directly interact with FLNA (33, 34). In pituitary tumoral cell lines, the association of FLNA with both SST 2 and SST 5 is crucial for an efficient SSA-induced signal transduction (34,35). In addition, FLNA expression is required for SST 2 internalization, recycling to the plasma membrane and protein stability after prolonged agonist stimulation (35), and to maintain a stable amount of SST 5 in basal conditions by preventing both lysosomal and proteosomal degradation (34).…”

mentioning

confidence: 99%

“…In pituitary tumoral cell lines, the association of FLNA with both SST 2 and SST 5 is crucial for an efficient SSA-induced signal transduction (34,35). In addition, FLNA expression is required for SST 2 internalization, recycling to the plasma membrane and protein stability after prolonged agonist stimulation (35), and to maintain a stable amount of SST 5 in basal conditions by preventing both lysosomal and proteosomal degradation (34). However, whether FLNA may participate to SST dimeric assembly remains to be assessed.…”

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confidence: 99%

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Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation

Treppiedi

Marra²,

Muro³

et al. 2022

Front. Endocrinol.

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The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)–based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P < 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P < 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulates SST2 and SST5 intracellular trafficking induced by octreotide and pasireotide.

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Predictors of biochemical response to somatostatin receptor ligands in acromegaly

Marazuela,

Martínez-Hernandez,

Marques-Pamies

et al. 2024

Best Practice & Research Clinical Endocrinology & Metab

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Filamin A is required for somatostatin receptor type 5 expression and pasireotide-mediated signaling in pituitary corticotroph tumor cells

Cited by 7 publications

References 51 publications

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation

Predictors of biochemical response to somatostatin receptor ligands in acromegaly

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