Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation
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), than...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2022-08-01
|
| Series: | Frontiers in Endocrinology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fendo.2022.892668/full |
| _version_ | 1811220889126043648 |
|---|---|
| author | Donatella Treppiedi Giusy Marra Genesio Di Muro Genesio Di Muro Rosa Catalano Federica Mangili Emanuela Esposito Davide Calebiro Davide Calebiro Maura Arosio Maura Arosio Erika Peverelli Giovanna Mantovani Giovanna Mantovani |
| author_facet | Donatella Treppiedi Giusy Marra Genesio Di Muro Genesio Di Muro Rosa Catalano Federica Mangili Emanuela Esposito Davide Calebiro Davide Calebiro Maura Arosio Maura Arosio Erika Peverelli Giovanna Mantovani Giovanna Mantovani |
| author_sort | Donatella Treppiedi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T07:50:38Z |
| format | Article |
| id | doaj.art-ea7432ff91a648e5afe643811db35589 |
| institution | Directory Open Access Journal |
| issn | 1664-2392 |
| language | English |
| last_indexed | 2024-04-12T07:50:38Z |
| publishDate | 2022-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Endocrinology |
| spelling | doaj.art-ea7432ff91a648e5afe643811db355892022-12-22T03:41:37ZengFrontiers Media S.A.Frontiers in Endocrinology1664-23922022-08-011310.3389/fendo.2022.892668892668Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formationDonatella Treppiedi0Giusy Marra1Genesio Di Muro2Genesio Di Muro3Rosa Catalano4Federica Mangili5Emanuela Esposito6Davide Calebiro7Davide Calebiro8Maura Arosio9Maura Arosio10Erika Peverelli11Giovanna Mantovani12Giovanna Mantovani13Department of Clinical Sciences and Community Health, University of Milan, Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyUniversity Sapienza of Rome, Rome, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyInstitute of Metabolism and Systems Research, University of Birmingham, Birmingham, United KingdomCentre of Membrane Proteins and Receptors, Universities of Birmingham and Nottingham, Birmingham, United KingdomDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyEndocrinology Unit, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca’ Granda Ospedale Maggiore Policlinico, Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, Milan, ItalyEndocrinology Unit, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca’ Granda Ospedale Maggiore Policlinico, Milan, ItalyThe 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.https://www.frontiersin.org/articles/10.3389/fendo.2022.892668/fullGPCR dimerizationin situ PLAsomatostatin analogsFLNASST2SST5 |
| spellingShingle | Donatella Treppiedi Giusy Marra Genesio Di Muro Genesio Di Muro Rosa Catalano Federica Mangili Emanuela Esposito Davide Calebiro Davide Calebiro Maura Arosio Maura Arosio Erika Peverelli Giovanna Mantovani Giovanna Mantovani Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation Frontiers in Endocrinology GPCR dimerization in situ PLA somatostatin analogs FLNA SST2 SST5 |
| title | Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation |
| title_full | Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation |
| title_fullStr | Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation |
| title_full_unstemmed | Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation |
| title_short | Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation |
| title_sort | dimerization of gpcrs novel insight into the role of flna and ssas regulating sst2 and sst5 homo and hetero dimer formation |
| topic | GPCR dimerization in situ PLA somatostatin analogs FLNA SST2 SST5 |
| url | https://www.frontiersin.org/articles/10.3389/fendo.2022.892668/full |
| work_keys_str_mv | AT donatellatreppiedi dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT giusymarra dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT genesiodimuro dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT genesiodimuro dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT rosacatalano dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT federicamangili dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT emanuelaesposito dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT davidecalebiro dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT davidecalebiro dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT mauraarosio dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT mauraarosio dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT erikapeverelli dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT giovannamantovani dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation AT giovannamantovani dimerizationofgpcrsnovelinsightintotheroleofflnaandssasregulatingsst2andsst5homoandheterodimerformation |