Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A

Abstract

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pulldown and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton. I mbalances in dopaminergic signaling are implicated in many neuropsychiatric and motor disorders, including schizophrenia and Parkinson's disease (1). In mammalian brain, dopaminergic signaling is mediated via a cohort of dopamine receptors. Among the cloned dopamine receptor subtypes, the D2-like receptors (D2, D3, and D4) are the major target of antipsychotics, both typical and atypical, as well as anti-Parkinson's drugs (1). These receptors mediate intracellular signaling cascades by coupling to inhibitory subsets of heterotrimeric GTP-binding (G) proteins. In a variety of cell types, D2-like receptor signaling modulates calcium, potassium, and sodium currents through specific regulation of ion channel activities (2, 3). The activation of D2-like receptors also has been implicated in the regulation of cellular morphogenesis (4) and in the maintenance of neuronal structure in adult brain (5-7). Although the D2-like receptors appear to activate discrete signal transduction pathways, the question of whether individual D2-like receptors subserve distinct functional roles is an issue that has not yet been satisfactorily addressed.To better understand the regulation of dopamine receptor signaling, we are interested in identifying dopamine receptor-interacting proteins. Identification of dopamine receptor-interacting proteins unique to specific receptor subtypes may provide important clues to how functional differences between dopamine receptor subtypes are manifested. We conducted yeast two-hybrid screens of a human brain cDNA library, with the third intracellular loop of the D2S and D3 receptors as bait. Using this approach, we identified filamin A (FLN-A) as a dopamine rece...

Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking filamin A

“…Filamin A is known to bind to various membrane and signaling molecules, see reviews (Stossel et al, 2001;van der Flier and Sonnenberg, 2001)}. More recently different laboratories, including our own, demonstrated that filamin A associates with various GPCRs, such as the D2 and D3 dopamine receptors (Li et al, 2000;Lin et al, 2001), the calcium-sensing receptor (CaR) (Awata et al, 2001;Hjalm et al, 2001), the metabotropic glutamate receptor type 7 (Enz, 2002), alpha1-adrenergic receptors (Zhang et al, 2004), calcitonin receptor (Seck et al, 2003) and the mu opioid receptor (MOP) (Onoprishvili et al, 2003). In some cases, such as the calcium sensing receptor and the MOP, the binding of filamin A is to the carboxyl tail, while for other receptors, such as the D2 and D3 dopamine receptors, the binding is to the third cytoplasmic loop.…”

The Adenosine A2A Receptor Interacts with the Actin-binding Protein α-Actinin

“…Recent studies have identified filamin A, another actin-cross-linking protein similar to ␣-actinin, as an intracellular binding partner for other heptaspanning membrane receptors, viz. the dopamine D 2 and D 3 receptors (20,21), the calcium-sensing receptor (22), and metabotropic glutamate receptors (23). A role for these interactions in the proper membrane targeting and synaptic localization of all these receptors has been proposed.…”

Direct Interaction between the Actin-binding Protein Filamin-A and the Inwardly Rectifying Potassium Channel, Kir2.1

“…Filamin-deficient cells have been reported to have a reduced surface expression of many receptors and ion channels, suggesting that organization of the submembranous cytoskeleton may be important for the appropriate insertion or removal of proteins from the membrane. As an extreme example of this, expression of the D2 dopamine receptor in filamin-deficient cells results in an almost total loss of receptor expression in the membrane (7). To test if filamin had a similar influence on the trafficking of Kir2.1 we expressed eGFP-tagged Kir2.1 (24) in filamin-deficient and filamin-replenished cell lines, filamin ϩ and filamin Ϫ , respectively.…”

“…Indeed to date, more than 20 different protein partners have been identified for filamin. These include not only transmembrane proteins such as ␤-integrins (5, 6), D2 and D3 dopamine receptors (7), and K v 4.2 potassium channels (8) but also intracellular signaling molecules such as the Rho family of GTPases (9), the stressactivated kinase SEK-1 (also known as MKK-4 (10)), and protein kinase C␣ (11). This ability to aggregate cytoskeletal elements, transmembrane receptors, and cytoplasmic signaling proteins is potentially important not only in the stabilization of receptors at the cell surface but also in cell signal integration (1,2).…”