Abstract
RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/Gq signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/Gq signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/Gq signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function.
Highlights
Intracellular trafficking of regulator of G-protein signaling (RGS) proteins determines their function as G-protein inhibitors
Previous work using immunogold labeling and electron microscopic analysis of RGS4 in the macaque cerebral cortex showed that it can be localized to intracellular endosomal structures [32]. Consistent with this observation, our data showed that exogenously expressed RGS4 is localized to different endosomal pools and that its trafficking through this compartment is highly dependent on its palmitoylation status [16]
Under basal conditions, when RGS4-YFP is co-expressed with the pECFP control vector, it localizes efficiently to the plasma membrane and to a variety of endosomal structures of different size, speed, and fluorescence intensity that are visible against the background fluorescence of RGS4-YFP in the cytosol (Fig. 1, A and B, and supplemental movie 1)
Summary
Intracellular trafficking of RGS proteins determines their function as G-protein inhibitors. Both Rab activation and Rab inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/Gq signaling complex This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. The Rab superfamily of small GTPase proteins contains a large number of key regulators of endosome trafficking and remodeling In their GTP-bound (activated) state, Rabs have a tor; M1R, M1 muscarinic receptor; Lamp, lysosomal associated membrane protein 1; MVB, multivesicular body; CFP, cyan fluorescent protein; RFP, red fluorescent; TGN, trans-Golgi network; HEK, human embryonic kidney; PM, plasma membrane; G-protein, guanine nucleotide-binding protein; ANOVA, analysis of variance; FR, fluorescence ratio. Our data show for the first time that Rab-mediated internalization and intracellular trafficking of an RGS protein are important components of the intracellular mechanisms that regulate its plasma membrane targeting and function as an inhibitor of GPCR signaling
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