Role of filamin-A in the regulation of dopamine D2 receptor localization and signaling in a lactotroph cell model
Giovanna Mantovani1, Erika Peverelli1, Stefano Ferrero2, Eleonora Vitali1, Paolo Beck-Peccoz1, Anna Spada1 & Andrea Lania1
Dopamine agonists (DA) inhibit prolactin (PRL) secretion and expression on the pituitary lactotrophs by binding dopamine D2 receptor. Prolactinomas are the most common secreting pituitary adenomas, and DA are the first choice for their treatment because they reduce PRL levels and tumor size. However, a subset of patients is resistant to DA. The mechanisms involved in DA resistance are not fully understood, although decreased expression of D2 receptor or altered signal transduction have been suggested. Recent studies identified specific proteinprotein interactions as determinant in the regulation of receptor anchoring and signaling. D2 receptor was found to associate with filamin-A (FLNA), a widely expressed cytoskeleton protein that, through its scaffolding properties, affects the intracellular signaling and trafficking of a number of receptors.
The aim of our study was to investigate the role of FLNA in signaling and targeting of D2 receptor in prolactinomas. We first evaluated the expression of this protein in normal and tumoral human pituitaries by immunohistochemistry: prolactinomas showed a strong reduction of FLNA expression when compared to the normal pituitary, this effect being generally more pronounced in DA resistant adenomas. To study the effects of the reduced FLNA expression on D2 receptor signaling and localization we silenced the expression of FLNA by siRNA technique in MMQ cells, a rat lactotroph cell line, and we evaluated the effects on D2 membrane expression as well as on the most relevant intracellular D2-mediated responses. We found that FLNA silencing reduced D2 localization at the plasma membrane; moreover, a prolonged absence of FLNA induced the down-regulation of receptor expression. Finally, in silenced cells a reduced inhibition of intracellular cAMP (46% inhibition versus 64% of control at 10 nM) and a reduced anti-proliferative effect (7% inhibition versus 23% of control at 100 nM) were observed, suggesting that FLNA is crucial for D2 receptor targeting and signaling in a lactotroph cell model. We suggest that the strong reduction of FLNA expression observed in prolactinomas may be associated with an impaired response to inhibitory effects of D2 on PRL secretion and cell proliferation.