A Novel Sorting Sequence in the β2-Adrenergic Receptor Switches Recycling from Default to the Hrs-dependent Mechanism

Abstract

Plasma membrane recycling of G protein-coupled receptors can occur by at least two distinct mechanisms as follows: a "default" mechanism that occurs nonselectively, and a specifically sorted mechanism that requires the endosome-associated protein Hrs. In this study we have defined a sequence in the ␤ 2 -adrenergic receptor cytoplasmic tail that confers Hrs dependence on receptor recycling. This sequence resembles acidic dileucine class motifs found in other membrane proteins but is structurally and functionally distinct from previously identified sorting sequences. Mutation of the novel sorting sequence rendered plasma membrane recycling independent of Hrs and independent of a distal PDZ ligand required for Hrs-dependent recycling. We propose that the novel sorting sequence functions to "switch" endocytic trafficking between mechanistically distinct recycling modes, thereby explaining failure of the wild type ␤ 2 -adrenergic receptor to recycle efficiently by default.

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

“…3 G-I). A downstream sorting step, based on affinity interactions of sequence elements (e.g., a PDZ domain), then sorts cargo into physically distinct domains that mediate sequence-dependent recycling or degradation (17).…”

“…Recent work has identified physically and biochemically distinct microdomains on early endosomes that mediate B2AR recycling independent of TfR (14)(15)(16). Although the exact mechanisms of B2AR sorting into these domains remain under investigation, this sorting clearly requires specific sequence elements on B2AR (1,10,11,17). Importantly, why signaling receptor sorting is subject to such specialized requirements, considering that cargo like TfR apparently can recycle without specific sequence requirements, is not clear (1,(12)(13)(14)(15)(16).…”

“…Importantly, why signaling receptor sorting is subject to such specialized requirements, considering that cargo like TfR apparently can recycle without specific sequence requirements, is not clear (1,(12)(13)(14)(15)(16). One possibility is that these requirements allow signaling pathways to regulate and redirect receptor trafficking between different pathways as needed (17)(18)(19). Although this is an attractive idea, whether and how physiological signals regulate receptor sorting remain poorly understood (7,19).…”

The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis

“…HeLa cells were cotransfected with Flag-tagged V2R and either GFP, GFP-PLIC-1, or GFP-PLIC-2. Fluorescence flow cytometry has been used previously to accurately quantify changes in surface receptor fluorescence in response to agonist in a large population of cells (10,000) (Tanowitz and von Zastrow, 2003;Gage et al, 2005;Hanyaloglu et al, 2005;Hanyaloglu and von Zastrow, 2007), and it allows the specific advantage of analyzing only those cells coexpressing Flag-tagged receptors and GFPtagged proteins. In GFP and Flag-V2R-expressing cells, the addition of agonist (0 -45 min) resulted in a loss of surface receptor, with a t 1/2 of ϳ 7 min, similar to rates previously shown for the V2R (Pfeiffer et al, 1998), and it was not significantly different from cells expressing only Flag-V2R (data not shown).…”

“…Quantification of receptor internalization was obtained by measuring cell surface receptor expression with and without agonist treatment by flow cytometry, as described previously (Hanyaloglu et al, 2005;Hanyaloglu and von Zastrow, 2007). Briefly, transfected HeLa cells were incubated with M1 anti-FLAG antibody followed by agonist treatment for 0 -45 min, then they were detached with trypsin, incubated with a PE-coupled anti-mouse secondary antibody (Sigma-Aldrich), and analyzed by flow cytometry (FACSCalibur; BD Biosciences).…”