Association of the D2 Dopamine Receptor Third Cytoplasmic Loop with Spinophilin, a Protein Phosphatase-1-interacting Protein

Abstract

Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia. To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library. Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors. A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins. The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil. This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time. The interaction is not mediated via the unique 29-amino acid insert in D2 long ; both D2 long and D2 short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays. Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag. We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton.Dopamine receptors are members of the G protein-coupled receptor (GPCR) 1 superfamily that mediate their actions by coupling to intracellular signal transduction cascades via heterotrimeric G proteins. Five different dopamine receptors have been described belonging to two classes that are defined based on structure, function, and pharmacology (1). D2-like receptors, D2, D3, and D4, couple to G proteins of the G i/o class and have diverse downstream effectors, including adenylyl cyclase and G protein-coupled inward rectifier K ϩ channels (GIRKs). In contrast, D1-like receptors D1 and D5 are coupled to increases in adenylyl cyclase activity through G s . D1-like receptors have short third cytoplasmic loops (i3) and very long carboxyl-terminal cytoplasmic tails. Conversely, D2-like receptors are characterized by long third cytoplasmic loops and short carboxylterminal tails. D2-like receptors are more polymorphic than D1-like receptors; for example, D2 exists as two isoforms, D2 long and D2 short , which differ by 29 amino acids within the third cytoplasmic loop (2). In many GPCRs the third cytoplasmic loop is important for the coupling to G proteins. Typically, short peptide sequences near the amino and carboxyl termini of the third loop participate in...

Dopaminergic signaling in dendritic spines

“…Besides short peptide sequences involved in G protein and arrestin binding, the remaining portions of these intracellular domains are presumably free to interact with other proteins. Using approaches such as co-immunoprecipitation and the yeast two-hybrid system, an increasing number of DRIPs are being identified [41][42][43]53,[68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83]. The confirmed DRIPs and their suggested functions are summarized in Table 1.…”

“…This interaction appears to modulate cocaine-elicited striatal NR2 phosphorylation and motor activities. Finally, the D2 receptor interacts with spinophilin/neurabin-II (neuronal actin binding protein II), an F-actin-associating postsynaptic scaffolding protein in the PSD [68]. Spinophilin itself interacts with PP-1, the major protein phosphatase in spines.…”

Isoform Specificity among Ankyrins

“…Assays were performed as described (Ref. 15; Clontech). The bait plasmid was transformed into the yeast strain AH109 (ADE2, HIS3, lacZ selection) using lithium acetate.…”

Structural Basis for Spinophilin−Neurabin Receptor Interaction

“…Both neurabin and spinophilin contain an F-actin binding, a PP1-binding, a PDZ, and a C-terminal coiled-coil domain. In addition, neurabin, but not spinophilin [in vertebrates (7)], contains a sterile R motif (SAM) domain in its C-terminus, while spinophilin, but not neurabin, is proposed to have a dopamine receptor-R-adrenergic interacting domain in its N-terminus, possibly between spinophilin residues 200 and 400 (8,9). The highest level of primary sequence identity between neurabin and spinophilin is found in the PDZ domains (86%), the protein phosphatase 1 (PP1) binding domains (81%), and the coiled-coil domains (63%).…”