The cytoskeletal protein filamin A (FLNA) has been suggested to play an important role in the responsiveness of GH-secreting pituitary tumors to somatostatin receptor subtype 2 (SSTR2) agonists by regulating SSTR2 expression and signaling. However, the underlying mechanisms are unknown. In this study, we use fast multicolor single-molecule microscopy to image individual SSTR2 and FLNA molecules at the surface of living cells with unprecedented spatiotemporal resolution. We find that SSTR2 and FLNA undergo transient interactions, which occur preferentially along actin fibers and contribute to restraining SSTR2 diffusion. Agonist stimulation increases the localization of SSTR2 along actin fibers and, subsequently, SSTR2 clustering and recruitment to clathrin-coated pits (CCPs). Interfering with FLNA-SSTR2 binding with a dominant-negative FLNA fragment increases SSTR2 mobility, hampers the formation and alignment of SSTR2 clusters along actin fibers, and impairs both SSTR2 recruitment to CCPs and SSTR2 internalization. These findings indicate that dynamic SSTR2-FLNA interactions critically control the nanoscale localization of SSTR2 at the plasma membrane and are required for coupling SSTR2 clustering to internalization. These mechanisms explain the critical role of FLNA in the control of SSTR2 expression and signaling and suggest the possibility of targeting SSTR2-FLNA interactions for the therapy of pharmacologically resistant GH-secreting pituitary tumors.
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