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D(2)-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 D(2)-dopamine receptors (D(2)R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D(2)R 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 D(2)R. 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 and delta opioid receptors are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of mu opioid receptor. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D(2)R and that DRM is involved in the formation of a D(2)R signaling complex. D(2)R-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.

SUBMITTER: Celver J

PROVIDER: S-EPMC3240708 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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D(2)-Dopamine receptors target regulator of G protein signaling 9-2 to detergent-resistant membrane fractions.

Celver Jeremy J Sharma Meenakshi M Kovoor Abraham A

Journal of neurochemistry 20111124 1

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 D(2)-dopamine receptors (D(2)R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D(2)R 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 ...[more]

PMID: 22035199

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Project description:Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry.DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles.We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Dataset Information

D(2)-Dopamine receptors target regulator of G protein signaling 9-2 to detergent-resistant membrane fractions.

Publications

D(2)-Dopamine receptors target regulator of G protein signaling 9-2 to detergent-resistant membrane fractions.

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