Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway

Abstract

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo-and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (Ϸtwofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo. INTRODUCTIONMembrane trafficking pathways play a fundamental role in shaping cellular responses to receptor-mediated signals. This relationship is clear when one examines endocytic trafficking of transmembrane signaling receptors, such as G protein-coupled receptors (GPCRs), for which the cellular response depends on the number of functional receptors available at the cell surface (Lefkowitz et al., 1998;Moore et al., 2007;von Zastrow and Sorkin, 2007). Removal of surface receptors by endocytosis is one mechanism by which cells can terminate or rapidly attenuate cellular responses to an activating ligand. Subsequent trafficking of internalized receptors to lysosomes promotes receptor proteolysis, typically resulting in a prolonged attenuation of cellular signaling responsiveness (Marchese et al., 2008). Recycling of internalized receptors to the plasma membrane, in contrast, restores the complement of surface receptors and can contribute to rapid recovery of functional signaling (Carman and Benovic, 1998;Ferguson, 2001;Hanyaloglu and von Zastrow, 2008). Despite the established importance of endocytic trafficking in controlling receptor-mediated signaling, relatively little is known about whether or how signaling receptors regulate the endocytic pathway.The beta-2 adrenergic receptor (B2AR) is a seven-transmembrane G protein-coupled receptor, thus representing the largest known family o...

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

“…SpH-B2AR clustered and internalized rapidly in response to agonist (Fig. 3A), as described previously (20,29). After 5 min, serial acquisition of images at 10-30 frames per second (fps) revealed transient bursts of SpH-B2AR fluorescence in the plasma membrane (Fig.…”

“…Downstream activation of PKA has a homeostatic effect on B2AR delivery to the surface, making it a good candidate (20). We first optimized a live-cell imaging assay to directly visualize and resolve B2AR endosomal sorting at the level of discrete events mediating B2AR recycling.…”

“…We first optimized a live-cell imaging assay to directly visualize and resolve B2AR endosomal sorting at the level of discrete events mediating B2AR recycling. HEK293 cells expressing tagged B2AR were generated, and cells expressing a moderate level (∼1-2 pmol/mg) were chosen, because this level of expression has been shown to preserve the signaling and trafficking characteristics of B2AR (14,20). When imaged under high-resolution confocal microscopy, fluorescently labeled B2AR was readily detected on the cell surface before activation (Fig.…”

“…1D). Furthermore, in an assay detecting surface fusion of recycling vesicles (20), KT increased the number of B2AR recycling vesicles fusing with the surface (Fig. 3E).…”

“…Interestingly, B2AR itself contains two cytoplasmic consensus PKA sites that are phosphorylated in response to an agonist (2,23,24). One of these two sites, Ser-261/262, located in the third cytoplasmic loop, is not involved in endocytic trafficking (24)(25)(26)(27); thus, we focused on Ser-345/346 in the cytoplasmic tail, which has been implicated in surface delivery of B2AR (20). We first detected iso-induced B2AR phosphorylation at this site, using a phospho-specific Ser346 antibody.…”