Constitutive Internalization of the Leucine-rich G Protein-coupled Receptor-5 (LGR5) to the Trans-Golgi Network

Abstract

Background: Expression of the G protein-coupled receptor LGR5 demarcates adult tissue stem cells in the intestine, stomach, hair follicle, and mammary epithelium. Results:LGR5 is rapidly and constitutively internalized to the trans-Golgi network at steady state. Conclusion: Internalization occurs through a potential phosphorylation domain within the C-terminal tail. Significance: An understanding of LGR5 trafficking dynamics is expected to clarify its role in signaling and stem cell biology.

Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions

“…1A-C) (Snyder et al, 2013b). However, when the internalization of Lgr5 was blocked either by overexpression of dominant-negative dynamin-1 (K44A), by C-terminal tail truncation at position 834 (834del) or by exchanging the C-terminal tail for that of the human vasopressin V2 receptor (V2R; Lgr5-V2Rtail) (Fig.…”

“…2). Relative to Lgr5, receptors like the b2-AR (ADRB2) do not possess considerable constitutive internalization properties, illustrating that simply preventing GPCR internalization does not cause the formation of membrane protrusions (Snyder et al, 2013b). However, to completely discount the off-target effects of blocking endocytosis, the b2-AR was co-expressed with dynamin K44A.…”

“…Clones encoding GPCRs were either previously described [Lgr5 and Lgr4 (pcDNA3.1 backbone and CMV promoter) and ADRA1A-opto] or available in our laboratory (Airan et al, 2009;Snyder et al, 2013b). Clones containing bovine GFP-MYO10 (Berg and Cheney, 2002), GFPfascin-1 (Adams and Schwartz, 2000) and GFP-VASP (Svitkina et al, 2003) were gifts of Dr Richard Cheney, and a clone for rat dynamin-1 K44A was available in our laboratory.…”

“…Internalization assays were performed as described previously (Snyder et al, 2013b) with a few changes. Briefly, Nunc tissue-culture-treated, clear 96-well plates (167008; Nunc; Rochester NY) were incubated in 50 ml of poly-D-lysine (P0899; Sigma) diluted in H 2 O to 100 mg/ml for 6 hours at room temperature or overnight at 4˚C.…”

“…In fact, GPCR signaling is multifaceted in nature and is tightly regulated by endocytosis of activated receptors (Rajagopal et al, 2010). We have reported on the cellular trafficking properties of Lgr5 using EGFP-tagged forms of the receptor and found that the C-terminal tail regulates its unique constitutive internalization and retrograde trafficking to the trans-Golgi network in the apparent absence of ligand (Snyder et al, 2013b). Therefore, we hypothesized that inhibiting this constitutive internalization would provide new insight into the cellular function of Lgr5.…”