Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor

Abstract

Abstractβ1-adrenergic receptors (β1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the β1-AR (S312A) is internalized but does not recycle. We determined that WT β1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by >70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT β1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT β1-AR were colocalized by >70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT β1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the β1-AR. Next, we determined the effect of each of the rab11-intercating proteins on trafficking of the WT β1-AR. The recycling of the β1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role recycling of the human β1-AR.

A Rab11a-enriched subapical membrane compartment regulates a cytoskeleton-dependent transcytotic pathway in secretory epithelial cells of the lacrimal gland

“…Overexpression of the tail domain saturates the tail-domainbinding sites on interacting effector proteins, preventing cargo from interacting with intact myosin motors. Owing to the sensitivity of a proportion of Rab11a-enriched vesicles to actin depolymerization and the relationship between Rab11a and myosin Vb demonstrated in other cells (Lapierre et al, 2001;Volpicelli et al, 2002;Fan et al, 2004;Wakabayashi et al, 2005;Provance et al, 2008;Gardner et al, 2011), we hypothesized that expression of a myosin Vb tail construct alters the distribution or motility of Rab11a-enriched membranes in LGACs. When expressed alone, the mCherry-myosin Vb tail-enriched compartment was highly concentrated around the MTOCs in the subapical domain (Fig.…”

Sorting of β1-Adrenergic Receptors Is Mediated by Pathways That Are Either Dependent on or Independent of Type I PDZ, Protein Kinase A (PKA), and SAP97

“…In this regard, we speculate that trafficking of the ␤ 1 -AR with an active PDZ through early endosomes was distinct from that of the transferrin receptor (Tfr) because Tfr recycled by "bulk" sorting that was largely independent of specific cytoplasmic sequences (40). This concept was derived from co-localization studies that have shown early colocalization between the Tfr and ␤ 1 -or ␤ 2 -AR in Rab5a and EEA1-positive early endosomes, followed by divergent trafficking itineraries between the Tfr and these ␤-ARs in later time frames (34,36). In this view, ␤ 1 -and ␤ 2 -AR recycled in a sequence-regulated manner through specific cis-acting sequences present on the receptor's cytoplasmic surface.…”

“…We have determined the effect of prolonged agonist exposures on the fate of biotinylated WT ␤ 1 -AR and (S312A) ␤ 1 -AR in HEK-293 cells (36). These studies revealed that the recycling WT ␤ 1 -AR was not significantly degraded even after 6 h of continuous exposure to agonist, whereas the non-recycling (S312A) ␤ 1 -AR was markedly (Ͼ80%) degraded (36).…”

“…We have determined the effect of prolonged agonist exposures on the fate of biotinylated WT ␤ 1 -AR and (S312A) ␤ 1 -AR in HEK-293 cells (36). These studies revealed that the recycling WT ␤ 1 -AR was not significantly degraded even after 6 h of continuous exposure to agonist, whereas the non-recycling (S312A) ␤ 1 -AR was markedly (Ͼ80%) degraded (36). To investigate whether the recycling phenotype of the ␤ 1 -AR chimeras would also inhibit their lysosomal proteolysis, HEK-293 cells stably expressing equivalent densities of ␤ 1 -AR C-tail chimeras were tested by immunoblotting for agonist-induced proteolysis (Fig.…”

New insights in the regulation of Rab GTPases by G protein-coupled receptors

“…2 During the last decade, many studies described the involvement of Rab GTPases in the regulation of GPCR trafficking and signaling. Rab GTPases were shown to be key regulators of GPCR anterograde and retrograde transport (Rab1, Rab2, Rab6, and Rab8), [3][4][5][6] endocytosis (Rab5), [7][8][9][10][11] recycling (Rab4 and Rab11), [11][12][13][14][15][16][17][18][19] and degradation (Rab7). 20 We and others established that a direct interaction between a GPCR and a Rab GTPase appears to be necessary in the proper trafficking of the receptor.…”