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

Abstract

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.

Keywords: FLNA; GPCR dimerization; SST2; SST5; in situ PLA; somatostatin analogs.

Figure 1

In situ detection of SST2/SST2 and SST5/SST5 homo-dimers. Representative in situ PLA experiment performed in GH3 (A), A7 cells (B), and M2 cells (C) showing SST₂/SST₂ (upper panels) and SST₅/SST₅ (lower panels) homo-dimers. For SST₂/SST₂ homo-dimers, mouse anti-HA and rabbit anti-SNAP antibodies were used. For SST₅/SST₅ homo-dimers, mouse and rabbit anti-SST₅ antibodies were used. PLA puncta representing homo-dimers are shown as green dots and nuclei are stained with DAPI in blue. A deconvoluted image of PLA events merged with DAPI is shown. White arrows indicated the localization of PLA puncta. Scale bars: 10 μm.

Figure 2

In situ detection of SST2/SST5 hetero-dimers. Representative in situ PLA experiment showing SST₂/SST₅ hetero-dimers in GH3 (A) and melanoma cells (B) before and after treatments with 100 nM octreotide or pasireotide for 5 min. Rabbit anti-SST₂ and mouse-anti SST₅ antibodies were used. Green dots represent PLA events and indicate close proximity between SST₂ and SST₅. Graphs resulting from the quantification of total SST₂/SST₅ puncta representing PLA events are shown for each cell line. For (B), a reduction in basal SST₂/SST₅ hetero-dimers in M2 cells compared to A7 cells and an increase in SST₂/SST₅ hetero-dimers in A7 and M2 cells treated with octreotide compared to basal are shown (n = 3, number of PLA puncta per cells was quantified for 150 cells randomly chosen from different fields per condition, *p < 0.05 vs. basal A7 cells; ^§p < 0.05 vs. corresponding basal). Scale bars: 10 μm.

Figure 3

SST2 and SST5 intracellular trafficking. Representative immunofluorescence experiment showing subcellular localization of SST₂ (green) and SST₅ (red) in GH3 cells (A), A7 cells (B), and M2 cells (C) stimulated or not with 100 nM octreotide or pasireotide for the indicated times. Nuclei are stained with DAPI in blue. Overlay of green and red channels is shown, and white arrows indicate SST₂ and SST₅ subcellular colocalization. Deconvolution algorithm was applied to further show SST₂ and SST₅ colocalization areas (yellow signals, right columns). Scale bars: 10 μm.