Down-Regulation of Protease-activated Receptor-1 Is Regulated by Sorting Nexin 1

Abstract

Degradation or "down-regulation" of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, is critical for termination of receptor signaling. Toward understanding the molecular mechanisms by which activated PAR1 is internalized, sorted to lysosomes, and degraded, we investigated whether PAR1 interacted with sorting nexin 1 (SNX1). SNX1 is a membrane-associated protein that functions in lysosomal sorting of the epidermal growth factor receptor. In vitro biochemical binding assays revealed a specific interaction between a glutathione S-transferase fusion of SNX1 and PAR1. In HeLa cells, activated PAR1 colocalized with endogenous SNX1 and coimmunoprecipitated SNX1. SNX1 contains a phox homology domain predicted to bind phosphatidylinositol-3-phosphate and a C-terminal coiled-coil region. To assess SNX1 function, we examined the effects of SNX1 deletion mutants on PAR1 trafficking. Neither the N terminus nor phox homology domain of SNX1 affected PAR1 trafficking. By contrast, overexpression of SNX1 C-terminal domain markedly inhibited agonist-induced degradation of PAR1, whereas internalization remained virtually intact. Immunofluorescence microscopy studies revealed substantial PAR1 accumulation in an early endosome antigen-1-positive compartment in agonist-treated cells expressing SNX1 C terminus. By contrast, lysosome-associated membrane protein-1 distribution was unperturbed. Together, these findings strongly suggest a role for SNX1 in sorting of PAR1 from early endosomes to lysosomes. Moreover, this study provides the first example of a protein involved in lysosomal sorting of a G protein-coupled receptor in mammalian cells.

A Library of 7TM Receptor C-terminal Tails

“…Human EBP50 cDNA was provided by Mark von Zastrow (UCSF). Human SNX1, GASP, and cGASP have been described previously (23,24 2 , and human ghrelin receptor were from Christian E. Elling (7TM Pharma). Human angiotensin II receptor type 1 (AT 1 ) was from Hans T. Schambye (Maxygen, Hørsholm, Denmark).…”

“…Targeting-SNX1 has been proposed to be responsible for the targeting of PAR1 to lysosomes (24). Pull-down analyses between 35 S-labeled SNX1 and the library of 7TM tail-fusion proteins showed a number of strong binders (Fig.…”

“…7TM receptors expose several intracellular loops for potential interaction with intracellular proteins. However, it is especially the C-terminal tail of the receptors that interacts with adaptor and scaffolding proteins (17)(18)(19)(20)(21)(22)(23)(24)(25). Although, for example, intracellular loop 3 is critically involved in the recognition process between the receptor and transducer/effector molecules such as the heterotrimeric G proteins and arrestins, these proteins also interact with parts of the C-terminal receptor tail (26 -30).…”

“…ERM-binding phosphoprotein 50 (EBP50, also called Na ϩ /H ϩ -exchanger regulatory factor) and N-ethylmaleimidesensitive factor (NSF) have both been suggested to be responsible for the recycling of the ␤ 2 -adrenergic receptor (21,22). In contrast, sorting nexin 1 (SNX1), which originally was demonstrated to be required for the lysosomal sorting of the epidermal growth factor receptor, was recently suggested to be involved in the lysosomal sorting of PAR1 as well (24,34). Protease-activated receptors are irreversibly activated by enzymatic digestion of the N-terminal segment of the receptor, and the sorting of activated receptors to lysosomes rather than recycling is critical for terminating signaling for these receptors.…”

Enterophilin-1, a New Partner of Sorting Nexin 1, Decreases Cell Surface Epidermal Growth Factor Receptor

“…8), characterized by the presence of a phox homology domain (9,10). They are involved in the intracellular trafficking of several membrane receptors (11)(12)(13). SNX1, the best studied member of this family, recognizes the lysosomal targeting sequence code in EGFR.…”