D₂‐Dopamine receptors target regulator of G protein signaling 9‐2 to detergent‐resistant membrane fractions

Abstract

Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts that are involved in compartmentalizing cell membranes. We report that the majority of D2-dopamine receptors (D2R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D2R in a cell line shifted the expression of regulator of G protein signaling 9-2 (RGS9-2) into DRM. RGS9-2 is a protein that is highly enriched in the striatum and specifically regulates striatal D2R. In the striatum, RGS9-2 is mostly associated with DRMs but when expressed in cell lines, RGS9-2 is present in the soluble cytoplasmic fraction. In contrast, the majority of mu opioid receptors (MOR) and delta opioid receptors (DOR) are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of MOR. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D2R and that DRM is involved in the formation of a D2R signaling complex. D2R-mediated targeting of RGS9-2 to DRM was blocked by the deletion of the RGS9-2 DEP domain or by a point mutation that abolishes the GTPase accelerating protein function of RGS9-2.

D1-D2Dopamine Receptor Synergy Promotes Calcium Signaling via Multiple Mechanisms

“…Lipid rafts are a well-known but poorly understood platform for modulating certain protein-protein interactions in neurons as well as affecting GPCR ligand sensitivity, membrane trafficking, and signaling (Allen et al, 2007;Korade and Kenworthy, 2008;Björk and Svenningsson, 2011;Kong et al, 2011;Sebastião et al, 2011;Celver et al, 2012). Lipid rafts would readily enable cross-talk between the D 1 R and D 2 R and could assist in the multifaceted signaling profile of the D 1 -D 2 receptor complex.…”

Plasma Membrane Compartmentalization of D2 Dopamine Receptors

“…Previously, we had reported that D2R co-expression alters the subcellular distribution of G␣ i1 in HEK293T cells (19). In the absence of D2R, the expression of the G␣ i1 is spread out uniformly through the cytoplasm, and upon D2R co-expression, it moves toward the cell boundaries (19).…”

“…We previously showed that the vast majority of D2R expressed in the brain is uniquely resistant to detergent solubilization (19) and wanted to explore the biological significance of our observation. We utilized a novel in-cell biotin transfer assay involving the Escherichia coli biotin ligase enzyme, BirA, that specifically attaches biotin to a unique acceptor peptide (AP) (20).…”

Psychostimulants, Brain Membrane Lipids and Dopamine Transmission

“…For instance, exogenously increasing membrane cholesterol level in striatal synaptosomes and neuronal-like MN9D cells increases the binding B max values for cocaine analogs but had no effect of DAT surface level [51]. Moreover, cholesterol depletion with MβCD significantly reduced the ability of DAT to take up dopamine without altering DAT surface expression in LLC-PK1, HEK293 and N2A cells [40,41,44].…”

“…D1Rs and caveolae can form a functional complex in lipid rafts and regulate D1R-mediated G protein signaling. Similar to D1Rs, D2Rs are also primarily localized in lipid raft microdomains in HEK293 cells and mouse striatum tissues [51,52] although it is unknown whether D2Rs physically interact with certain raft-associated proteins. Moreover, dopamine-induced D2R internalization is largely restricted to D2Rs localized in lipid rafts [53].…”